(en)The present invention relates to compositions comprising factor VIII coagulation factors linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of making and using such compositions in treatment of factor VIII-related diseases, disorders, and conditions.

1.ApplicationNumber: US-201214379192-A
1.PublishNumber: US-2015158929-A1
2.Date Publish: 20150611
3.Inventor: SCHELLENBERGER VOLKER
CHANG PEI-YUN
VARFAJ FATBARDHA
DING SHENG
SILVERMAN JOSHUA
WANG CHIA-WEI
SPINK BENJAMIN
STEMMER WILLEM P.
GEETHING NATHAN
KULMAN JOHN
LIU TONGYAO
TOBY GARABET G.
JIANG HAIYAN
PETERS ROBERT
WANG DEPING
MEI BAISONG

4.Inventor Harmonized: SCHELLENBERGER VOLKER(US)
CHANG PEI-YUN(US)
VARFAJ FATBARDHA(US)
DING SHENG(US)
SILVERMAN JOSHUA(US)
WANG CHIA-WEI(US)
SPINK BENJAMIN(US)
STEMMER WILLEM P(US)
GEETHING NATHAN(US)
KULMAN JOHN(US)
LIU TONGYAO(US)
TOBY GARABET G(US)
JIANG HAIYAN(US)
PETERS ROBERT(US)
WANG DEPING(US)
MEI BAISONG(US)

5.Country: US
6.Claims:
(en)The present invention relates to compositions comprising factor VIII coagulation factors linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of making and using such compositions in treatment of factor VIII-related diseases, disorders, and conditions.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority benefit to U.S. Provisional Application Ser. No. 61/599,400 filed Feb. 15, 2012, which application is incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 11, 2012, is named 32887346.txt and is 13,344,768 Bytes in size.


BACKGROUND OF THE INVENTION
Factor VIII is an important component of the intrinsic pathway of the blood coagulation cascade. In the circulation, factor VIII is mainly complexed to von Willebrand factor. Upon activation by thrombin, (Factor IIa), it dissociates from the complex to interact with factor IXa in the intrinsic coagulation cascade, which, in turn, activates factor X. Once removed from the von Willebrand factor complex, activated factor VIII is proteolytically inactivated by activated Protein C (APC), factor Xa, and factor IXa, and is quickly cleared from the blood stream. When complexed with normal von Willebrand factor protein, the half-life of factor VIII is approximately 12 hours, whereas in the absence of von Willebrand factor, the half-life of factor VIII is reduced to 2 hours (Tuddenham E G, et al., Br J Haematol. (1982) 52(2):259-267).
In hemophilia, the clotting of blood is disturbed by a lack of certain plasma blood clotting factors. Hemophilia A is a deficiency of factor VIII, and is a recessive sex-linked, X chromosome disorder that represents 80% of hemophilia cases. The standard of care for the management of hemophilia A is replacement therapy with recombinant factor VIII concentrates. Subjects with severe hemophilia A have circulating procoagulant factor VIII levels below 1-2% of normal, and are generally on prophylactic therapy with the aim of keeping factor VIII above 1% between doses, which can usually be achieved by giving factor VIII two to three times a week. Persons with moderately severe hemophilia (factor VIII levels of 2-5% of normal) constitute 25-30% hemophilia incidents and manifest bleeding after minor trauma. Persons with mild hemophilia A (factor VIII levels of 5-40% of normal) comprise 15-20% of all hemophilia incidents, and develop bleeding only after significant trauma or surgery.
The in vivo activity of exogenously supplied factor VIII is limited both by a short protein half-life and inhibitors that bind to the factor VIII and diminish or destroy hemostatic function.
Up to 30% of hemophilia A patients receiving exogenously-supplied factor VIII mount an IgG immune response towards factor VIII (Towfighi, F., et al. Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity. Acta Haematol (2005) 114:84-90), which can result in the complete inhibition of its procoagulant activity and/or promote more rapid clearance of the factor VIII (Briet E et al. High titer inhibitors in severe haemophilia A. A meta-analysis based on eight long-term follow-up studies concerning inhibitors associated with crude or intermediate purity factor VIII products. Throm. Haemost. (1994) 72: 162-164). The IgG antibodies, called FVIII inhibitors, are primarily directed towards the A2, A3 and C2 domains (Scandella D et al. Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization. Blood (1989) 74:1618-1626), but can arise against the A1, B and C1 domains, as well. As such, treatment options for patients with FVIII inhibitors are limited.
Large proteins such as factor VIII are normally given intravenously so that the medicament is directly available in the blood stream. It has been previously demonstrated that an unmodified factor VIII injected intramuscularly yielded a maximum circulating level of only 1.4% of the normal plasma level (Pool et al, Ineffectiveness of Intramuscularly Injected Factor VIII Concentrate in Two Hemophilic Patients. New England J. Medicine (1966) 275(10):547-548). Formulations that could be administered other than by the intravenous route would greatly simplify their use, increase safety, and result in substantial cost savings.
Chemical modifications to a therapeutic protein can modify its in vivo clearance rate and subsequent serum half-life. One example of a common modification is the addition of a polyethylene glycol (PEG) moiety, typically coupled to the protein via an aldehyde or N-hydroxysuccinimide (NHS) group on the PEG reacting with an amine group (e.g. lysine side chain or the N-terminus). However, the conjugation step can result in the formation of heterogeneous product mixtures that require extraction, purification and/or other further processes, all of which inevitably affect product yield and quality control. Also, the pharmacologic function of coagulation factors may be hampered if amino acid side chains in the vicinity of its binding site become modified by the PEGylation process. Other approaches include the genetic fusion of an Fc domain to the therapeutic protein, which increases the size of the therapeutic protein, hence reducing the rate of clearance through the kidney. In some cases, the Fc domain confers the ability to bind to, and be recycled from lysosomes by the FcRn receptor, resulting in increased pharmacokinetic half-life. Unfortunately, the Fc domain does not fold efficiently during recombinant expression, and tends to form insoluble precipitates known as inclusion bodies. These inclusion bodies must be solubilized and functional protein must be renatured from the misfolded aggregate, which is a time-consuming, inefficient, and expensive process.
SUMMARY OF THE INVENTION
The present invention relates to novel coagulation factor VIII fusion protein compositions and the uses thereof. Specifically, the compositions provided herein are particularly used for the treatment or improvement of a condition associated with hemophilia A, deficiencies of factor VIII, bleeding disorders and coagulopathies. In one aspect, the present invention provides compositions of isolated fusion proteins comprising a factor VIII (FVIII) and one or more extended recombinant polypeptides (XTEN) wherein the fusion protein exhibits procoagulant activity. A subject XTEN useful for constructing such fusion proteins is typically a polypeptide with a non-repetitive sequence and unstructured conformation. In one embodiment, one or more XTEN is linked to a coagulation factor FVIII (“CF”) selected from native human factor VIII, factor VIII B-domain deleted sequences (“FVIII BDD”), and sequence variants thereof (all the foregoing collectively “FVIII” or “CF”), resulting in a recombinant factor VIII-XTEN fusion protein (“CFXTEN”). The factor VIII polypeptide component of the CFXTEN comprises an A1 domain, an A2 domain, a C1 domain, a C2 domain, and optionally a B domain or a portion thereof. In some embodiments, the FVIII is further characterized by delineation of the aforementioned domains to comprise an acidic a1, a2 and a3 spacer. In another embodiment, the present disclosure is directed to pharmaceutical compositions comprising the fusion proteins and the uses thereof in methods and regimens for treating factor VIII-related conditions. The CFXTEN compositions have enhanced pharmacokinetic and pharmacologic properties compared to FVIII not linked to XTEN, which may permit more convenient dosing and improved efficacy.
In a first aspect, the invention relates to recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and one or more extended recombinant polypeptide (XTEN) linked to the factor VIII. In some embodiments, the invention provides recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least one extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain including an a1 acidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3 acidic spacer region, C1 domain, C2 domain and optionally all or a portion of B domain, and wherein said at least one XTEN is linked to said factor VIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) within B domain of said factor VIII polypeptide if all or a portion of B domain is present; (iii) within the A1 domain of said factor VIII polypeptide; (iv) within the A2 domain of said factor VIII polypeptide; (v) within the A3 domain of said factor VIII polypeptide; (vi) within the C1 domain of said factor VIII polypeptide; (vii) within the C2 domain of said factor VIII polypeptide; (viii) at the N-terminus of said factor VIII polypeptide, or (ix) between two domains of said factor VIII polypeptide, wherein the fusion protein retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% of the procoagulant activity, when measured by an in vitro coagulation assay, compared to a corresponding factor VIII not linked to XTEN. In one embodiment, in the foregoing recombinant factor VIII fusion protein the at least one XTEN is linked to said factor VIII polypeptide at a site at or within 1 to 6 amino acids of a site selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In other embodiments, the invention provides recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least a first extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain including an a1 acidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3 acidic spacer region, a C1 domain, a C2 domain and optionally all or a portion of a B domain, and wherein said first XTEN is linked to said factor VIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) within the B domain of said factor VIII polypeptide if all or a portion of the B domain is present; (iii) within the A1 domain of said factor VIII polypeptide; (iv) within the A2 domain of said factor VIII polypeptide; (v) within the A3 domain of said factor VIII polypeptide; (vi) within the C1 domain of said factor VIII polypeptide; or (vii) within the C2 domain of said factor VIII polypeptide; and when compared to a corresponding factor VIII protein not linked to XTEN, the fusion protein (a) retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 200%, 300%, 400%, or 500% of the procoagulant activity in an in vitro coagulation assay described herein or other such assays known in the art, and/or (b) exhibits reduced binding to an anti-factor VIII antibody in an in vitro binding assay described herein or other such assays known in the art. In one embodiment, in the foregoing recombinant factor VIII fusion protein the at least one XTEN is linked to said factor VIII polypeptide at a site at or within 1 to 6 amino acids of a site selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In other embodiments, the invention provides recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least a first extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain including an a1 acidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3 acidic spacer region, a C1 domain, a C2 domain and optionally all or a portion of a B domain, and wherein said first XTEN is linked to said factor VIII polypeptide at an insertion site selected from Table 6 and Table 7 and wherein the fusion protein retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% of the procoagulant activity, when measured by an in vitro coagulation assay described herein or other such assays known in the art, compared to a corresponding factor VIII protein not linked to XTEN. Non-limiting examples of the factor VIII protein not linked to XTEN includes native FVIII, BDD FVIII, pBC100 and sequences from Table 1. In another embodiment of the recombinant factor VIII fusion protein, the factor VIII polypeptide has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity to a sequence selected from the group consisting of the sequences of Table 1, the sequence depicted in FIG. 3 , and the sequence depicted in FIG. 4 , when optimally aligned. In yet another embodiment, the fusion protein comprises at least another XTEN linked to said factor VIII polypeptide at the C-terminus of said factor VIII polypeptideor within or optionally replacing the B domain of said factor VIII polypeptide. In a specific embodiment, the fusion protein comprises at least one XTEN sequence located within or optionally replacing the B domain of said factor VIII polypeptide. In another specific embodiment, the fusion protein comprises at least one XTEN sequence linked to said factor VIII polypeptide at the C-terminus of said factor VIII polypeptide. In one embodiment, the recombinant factor VIII fusion protein comprises a B-domain deleted variant of human factor VIII, wherein the B-domain deletion starts from a first position at about amino acid residue number 741 to about 750 and ending at a second position at amino acid residue number 1635 to about 1648 with reference to full-length human factor VIII sequence as set forth in FIG. 3 . In another embodiment, the recombinant factor VIII fusion protein comprises a first XTEN sequence linked to said factor VIII polypeptide at the C-terminus of said factor VIII polypeptide, and at least a second XTEN within or replacing the B domain of said factor VIII polypeptide, wherein the second XTEN is linked to the C-terminal end of about amino acid residue number 741 to about 750 and to the N-terminal end of amino acid residue numbers 1635 to about 1648 with reference to full-length human factor VIII sequence as set forth in FIG. 3 , wherein the cumulative length of the XTEN is at least about 100 amino acid residues. In one embodiment, in the foregoing fusion protein, the second XTEN links the factor VIII amino acids between N745 to P1640 or between S743 to Q1638 or between P747 to V1642 or between N745 and Q1656 or between N745 and S1657 or between N745 and T1667 or between N745 and Q1686 or between R747 and V1642 or between T751 and T1667. In one embodiment, the recombinant factor VIII fusion protein comprises a sequence having at least about 80% sequence identity, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, to about 100% sequence identity compared to a sequence of comparable length selected from Table 21, when optimally aligned. In another embodiment, the recombinant factor VIII fusion protein comprises at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, wherein each of the second, third, fourth, fifth, or sixth XTEN is linked to said factor VIII polypeptide at a second, third, fourth, fifth, or sixth site selected from the group consisting of an insertion site from Table 5, Table 6, Table 7 Table 8, and Table 9; a location within 6 amino acids of amino acid residue 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 of mature factor VIII; a location between any two adjacent domains of said factor VIII polypeptide, wherein said two adjacent domains are selected from the group consisting of A1 and A2 domains, A2 and B domains, B and A3 domains, A3 and C1 domains, and C1 and C2 domains; a location within the B domain of said factor VIII polypeptide, wherein the second XTEN is linked to the C-terminal end of about amino acid residue number 741 to about 750 and to the N-terminal end of amino acid residue numbers 1635 to about 1648 of a native factor VIII sequence; and the C-terminus of said factor VIII polypeptide. In one embodiment, the first XTEN is separated from the second XTEN by at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, or at least 400 amino acids. In one embodiment of the recombinant factor VIII fusion protein that comprises at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, each XTEN has at least about 80% sequence identity, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity compared to an XTEN of comparable length selected from the group consisting of the sequences in Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned. In yet another embodiment of the recombinant factor VIII fusion protein that comprises at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, In preferred embodiments, the recombinant factor VIII fusion protein exhibits a terminal half-life at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administered to a subject, wherein said subject is selected from human and factor VIII/von Willebrand factor double knock-out mouse. Further, in the embodiments of this paragraph, the fusion protein exhibits reduced binding to anti-factor VIII antibody or greater retained procoagulant activity, or both as compared to a corresponding factor VIII not linked to XTEN. In one embodiment, the procoagulant activity of the recombinant factor VIII fusion protein is at least 30%, or 40%, 50%, 80%, 100%, 200%, 300%, 400%, or 500% greater procoagulant activity in the presence of the anti-FVIII antibody compared to a corresponding factor VIII not linked to XTEN when each are assayed by an in vitro coagulation assay. In one embodiment, the reduced binding of the fusion protein to anti-factor VIII antibody is determined using a Bethesda assay using anti-factor VIII antibody selected from the group consisting of the antibodies of Table 10 and polyclonal antibody from a hemophilia A patient with factor VIII inhibitors, wherein the reduced binding and retained procoagulant activity of the fusion protein is evidenced by a lower Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 Bethesda units for the fusion protein compared to that for the factor VIII not linked to XTEN.
In one embodiment, the recombinant factor VIII fusion protein can, for example, comprise one or more XTEN wherein the XTEN has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN of comparable length selected from Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned.
In another aspect, the invention relates to recombinant factor VIII fusion proteins comprising FVIII and one or more XTEN in specific N- to C-terminus configurations. In one embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula I:
(XTEN) x -CF-(XTEN) y I

wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced from Table 1; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. Accordingly, the CFXTEN fusion composition can have XTEN-CF, XTEN-CF-XTEN, or CF-XTEN configurations.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula II:
(XTEN) x -(S) x -(CF)-(XTEN) y II

wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein, wherein the fusion protein is of formula III:
(XTEN) x -(S) x -(CF)-(S) y -(XTEN) y III

wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequence set for in Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula IV:
(A1)-(XTEN) u -(A2)-(XTEN) v -(B)-(XTEN) w -(A3)-(XTEN) x -(C1)-(XTEN) y -(C2)-(XTEN) z IV

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; v is either 0 or 1; w is either 0 or 1; x is either 0 or 1; y is either 0 or 1; y is either 0 or 1 with the proviso that u+v+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula V:
(XTEN) t -(S) a -(A1)-(S) b -(XTEN) u -(S) b -(A2)-(S) c -(XTEN) v -(S) c -(B)-(S) d -(XTEN) w -(S) d -(A3)-(S) e -(XTEN) x -(S) e -(C1)-(S) f -(XTEN) y -(S)-(C2)-(S) g -(XTEN) z V

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t+u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VI:
(XTEN) u -(S) a -(A1)-(S) b -(XTEN) v -(S) b -(A2)-(S) c -(XTEN) w -(S) c -(A3)-(S) d -(XTEN) x -(S) d -(C1)-(S) e -(XTEN) y -(S) e -(C2)-(S) f -(XTEN) z VI

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VII:
(SP)-(XTEN) x -(CS) x -(S) x -(FVIII — 1-745)-(S)-(XTEN)-(S)-(FVIII — 1640-2332)-(S) z -(CS) z -(XTEN) z VII

wherein independently for each occurrence, SP is a signal peptide, preferably with sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is a cleavage sequence listed in Table 12, S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include amino acids compatible with restrictions sites, “FVIII — 1-745” is residues 1-745 of Factor FVIII and “FVIII — 1640-2332” is residues 1640-2332 of FVIII, x is either 0 or 1, y is either 0 or 1, and z is either 0 or 1, wherein x+y+z>2; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity sequences set forth in Table 4. In one embodiment of formula VII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VIII:
(A1)-(S) a -(XTEN) v -(S) a -(A2)-(B1)-(S) b -(XTEN) w -(S) b -(B2)-(A3)-(S)-(XTEN) x -(S) c -(C1)-(S) d -(XTEN) y -(S) d -(C2)-(S) e -(XTEN) z VIII

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; B1 is a fragment of the B domain that can have from residue 741 to 743-750 of FVIII or alternatively from about residue 741 to about residues 745 of FVIII; B2 is a fragment of the B domain that can have from residues 1635-1686 to 1689 of FVIII or alternatively from about residue 1640 to about residues 1689 of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In one embodiment of formula VIII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula IX:
(A1 N )-(S) a -(XTEN) t -(S) b -(A1 C )-(A2 N )-(S) c -(XTEN) u -(S) d -(A2 C )-(B N )-(S) e -(XTEN) v -(S) f (Bc)-(A3 N )-(S) g -(XTEN) w (S) h -(A3 C )-(C1 N )-(S) i -(XTEN) x -(S) j -(C1 C )-(C2 N )-(S) k -(XTEN) y (S)-(C2 C )-(S) m -(XTEN) z IX

wherein independently for each occurrence, A1 N is a fragment of the A1 domain from at least residue number 1 (numbered relative to native, mature FVIII) to no more than residue number 371, A1 c is a fragment of the A1 domain from at least residue number 2 to no more than residue number 372, with the priviso that no sequence of the A1 N fragment is duplicated in the A1 o is a fragment; A2 N is a fragment of the A2 domain from at least residue number 373 to no more than residue number 739, A2 e is a fragment of the A2 domain from at least residue number 374 to no more than residue number 740, with the priviso that no sequence of the A2 N fragment is duplicated in the A2 e is a fragment; B N is a fragment of the B domain from at least residue number 741 to no more than residue number 1647, Be is a fragment of the B domain from at least residue number 742 to no more than residue number 1648, with the priviso that no sequence of the B N fragment is duplicated in the Be is a fragment; A3 N is a fragment of the A3 domain from at least residue number 1649 to no more than residue number 2019, A3 e is a fragment of the A3 domain from at least residue number 1650 to no more than residue number 2019, with the priviso that no sequence of the A3 N fragment is duplicated in the A3 e is a fragment; C1 N is a fragment of the C1 domain from at least residue number 2020 to no more than residue number 2171, C1, is a fragment of the C1 domain from at least residue number 2021 to no more than residue number 2172, with the priviso that no sequence of the C1 N fragment is duplicated in the C1, is a fragment; C2 N is a fragment of the C2 domain from at least residue number 2173 to no more than residue number 2331, C2, is a fragment of the C2 domain from at least residue number 2174 to no more than residue number 2332, with the priviso that no sequence of the C2 N fragment is duplicated in the C2, is a fragment; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0 or 1; k is either 0 or 1; 1 is either 0 or 1; m is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t+u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN of comparable length selected from Table 4. In one embodiment of formula IX, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12. In another embodiment of formula IX, Z is 1. In another embodiment of the fusion protein of formula IX V is 1 and the XTEN is linked to the C-terminal end of about amino acid residue number 741 to about 750 and to the N-terminal end of amino acid residue numbers 1635 to about 1648 with reference to full-length human factor VIII sequence as set forth in FIG. 3 . In another embodiment of the fusion protein of formula IX, the sum of t, u, v, w, x, y, and z equals 2, 3, 4, 5, or 6. In another embodiment of formula IX, the sum of t, u, v, w, x, y, and z equals 2, and v is 1 and z is 1. In another embodiment of the fusion protein of formula IX, the sum of t, u, v, w, x, y, and z equals 3, v and z each equal 1, and either t, u, w, x or y is 1. In another embodiment of formula IX, the sum of t, u, v, w, x, y, and z equals 4, v and w and z each equal 1, and two of t, u, x or y is 1. In another embodiment of the fusion protein of formula IX, the cumulative length of the XTENs is between about 84 to about 3000 amino acid residues. In another embodiment of formula IX, at least one XTEN is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human factor VIII selected from the group consisting of amino acid residue number 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In another embodiment of the fusion protein formula IX, each XTEN is linked to said fusion protein at sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In another embodiment of the fusion protein formula IX, each XTEN has at least about 80%, or about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity compared to an XTEN of comparable length selected from the group consisting of the sequences in Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned.
In another embodiment of the CFXTEN composition, the invention provides a first recombinant factor VIII polypeptide of formula X:
(A1)-a1-(A2)-a2-[B] X

and a second polypeptide comprising Formula XI:
a3-(A3)-(C1)-(C2) XI

wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, A1 is an A1 domain of factor VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of factor VIII, a fragment thereof, or is deleted; A3 is an A3 domain of factor VIII; C1 is a C1 domain of factor VIII; C2 is a C2 domain of factor VIII; a1, a2, and a3 are acidic spacer regions; wherein the A1 domain comprises an XTEN permissive loop-1 (A1-1) region and an XTEN permissive loop-2 (A1-2) region; wherein the A2 domain comprises an XTEN permissive loop-1 (A2-1) region and an XTEN permissive loop-2 (A2-2) region; wherein the A3 domain comprises an XTEN permissive loop-1 (A3-1) region and an XTEN permissive loop-2 (A3-2) region; wherein an XTEN sequence is inserted into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2; and wherein the recombinant factor VIII protein exhibits procoagulant activity. In one embodiment of the heterodimer, the first polypeptide and the second polypeptide form a single polypeptide chain comprising the formula (A1)-a1-(A2)-a2-[B]-[a3]-(A3)-(C1)-(C2). In one embodiment of the foregoing, “fused” means a peptidic bond.
In another embodiment of the CFXTEN composition, the invention provides a first recombinant factor VIII polypeptide of formula X:
(A1)-a1-(A2)-a2-[B]X
and a second polypeptide comprising Formula XI:
a3-(A3)-(C1)-(C2) XI

wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, A1 is an A1 domain of factor VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of factor VIII, a fragment thereof, or is deleted; A3 is an A3 domain of factor VIII; C1 is a C1 domain of factor VIII; C2 is a C2 domain of factor VIII; a1, a2, and a3 are acidic spacer regions; wherein an XTEN sequence is inserted into a3; and wherein the recombinant factor VIII protein exhibits procoagulant activity. In one embodiment of the heterodimer, the first polypeptide and the second polypeptide form a single polypeptide chain comprising the formula (A1)-a1-(A2)-a2-[B]-[a3]-(A3)-(C1)-(C2). In one embodiment of the foregoing, “fused” means a peptidic bond.
In embodiments of the foregoing formulae X and XI polypeptides, the XTEN permissive loops are contained within surface-exposed, flexible loop structures, and wherein A1-1 is located between beta strand 1 and beta strand 2, A1-2 is located between beta strand 11 and beta strand 12, A2-1 is located between beta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33, A3-1 is located between beta strand 38 and beta strand 39 and A3-2 is located between beta strand 45 and beta strand 46, according to the secondary structure of mature factor VIII stored as Accession Number 2R7E of the DSSP database. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A1-1 corresponds to a region in native mature human factor VIII from about amino acid 15 to about amino acid 45. In other embodiments of the foregoing formulae X and XI polypeptides the A1-1 corresponds to a region in native mature human factor VIII from about amino acid 18 to about amino acid 41. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A1-2 corresponds to a region in native mature human factor VIII from about amino acid 201 to about amino acid 232. In other embodiments of the foregoing formulae X and XI polypeptides the A1-2 corresponds to a region in native mature human factor VIII from about amino acid 218 to about amino acid 229. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A2-1 corresponds to a region in native mature human factor VIII from about amino acid 395 to about amino acid 421. In other embodiments of the foregoing formulae X and XI poly peptides, the A2-1 corresponds to a region in native mature human factor VIII from about amino acid 397 to about amino acid 418. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A2-2 corresponds to a region in native mature human factor VIII from about amino acid 577 to about amino acid 635. In other embodiments of the foregoing formulae X and XI polypeptides, the A2-2 corresponds to a region in native mature human factor VIII from about amino acid 595 to about amino acid 607. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A3-1 corresponds to a region in native mature human factor VIII from about amino acid 1705 to about amino acid 1732. In other embodiments of the foregoing formulae X and XI poly peptides, the A3-1 corresponds to a region in native mature human factor VIII from about amino acid 1711 to about amino acid 1725. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A3-2 corresponds to a region in native mature human factor VIII from about amino acid 1884 to about amino acid 1917. In other embodiments of the foregoing formulae X and XI polypeptides, the A3-2 corresponds to a region in native mature human factor VIII from about amino acid 1899 to about amino acid 1911. In other embodiments of the foregoing formulae X and XI polypeptides, an XTEN sequence is inserted into at least two of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2. In other embodiments of the foregoing formulae X and XI polypeptides, an XTEN sequence is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human factor VIII selected from the group consisting of amino acid residue number 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In other embodiments of the foregoing formulae X and XI polypeptides, an additional XTEN sequence is inserted into the a3 acidic spacer region. In other embodiments of the foregoing formulae X and XI polypeptides, an additional XTEN sequence is inserted into the a3 acide spacer immediately downstream of an amino acid which corresponds to amino acid 1656. In other embodiments of the foregoing formulae X and XI polypeptides, the A1 domain comprises an XTEN permissive loop-1 (A1-1) region and an XTEN permissive loop-2 (A1-2) region wherein the A2 domain comprises an XTEN permissive loop-1 (A2-1) region and an XTEN permissive loop-2 (A2-2) region, and wherein the A3 domain comprises an XTEN permissive loop-1 (A3-1) region and an XTEN permissive loop-2 (A3-2) region, and wherein an additional XTEN sequence is inserted into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2. In other embodiments of the foregoing formulae X and XI polypeptides, an additional XTEN sequence is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human factor VIII selected from the group consisting of amino acid residue number 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In the foregoing embodiments of formulae X and XI polypeptides, the fusion protein exhibits at least about 30%, 40%, 50%, 60%, 70%, or 80%, or 90% of the procoagulant activity of the corresponding factor VIII not linked to XTEN, wherein the procoagulant activity is assayed by an in vitro coagulation assay.
In all embodiments, the polypeptide can, for example, exhibit an in vitro procoagulant activity exceeding 0.5 IU/ml, or 1.0, or 1.5, or 2.0 IU/ml when expressed in cell-culture medium and assayed by an in vitro coagulation assay. The procoagulant activity can be measured by a chromogenic assay, a one stage clotting assay (e.g., a aPTT) or both.
In some embodiments, wherein the recombinant factor VIII fusion protein comprises a factor VIII and at least a first and a second XTEN, the at least first XTEN is separated from the at least second XTEN by at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, or at least 400 amino acids.
In preferred embodiments, the recombinant factor VIII fusion protein comprising a factor VIII and at least a first XTEN and, optionally, at least a second, or optionally at least a third, or optionally at least a fourth XTEN, the fusion protein exhibits reduced binding to an anti-factor VIII antibody as compared to the corresponding factor VIII not linked to XTEN. The reduced binding can be assessed either in vivo or by an in vitro assay. In one embodiment, the in vitro assay is an ELISA assay, wherein the binding of an anti-FVIII antibody to the fusion protein is reduced at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or at least about 40% or more compared to a FVIII not linked to XTEN. In another embodiment, the in vitro assay is a Bethesda assay wherein the reduced binding of the fusion protein is evidenced by a lower Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 Bethesda units for the fusion protein compared to that for a factor VIII not linked to XTEN. In the in vitro assays, the anti-factor VIII antibody is selected from an antibody of Table 10 and polyclonal antibody from a hemophilia A patient with factor VIII inhibitors. In particular embodiments of a recombinant factor VIII fusion protein comprising a factor VIII and at least a first and a second XTEN exhibiting reduced binding to a factor VIII inhibitor antibody, the first XTEN is linked to said factor VIII polypeptide within a C2 domain of said factor VIII polypeptide, and the second XTEN is linked to said factor VIII polypeptide within an A1 or A2 domain of said factor VIII polypeptide, wherein said fusion protein exhibits reduced binding to a factor VIII inhibitor antibody as compared to the corresponding factor VIII not linked to XTEN, wherein the factor VIII inhibitor antibody is capable of binding to an epitope located within the A1, A2 or C2 domain, and further wherein the fusion protein exhibits procoagulant activity. In one embodiment of the foregoing fusion protein, the second XTEN is linked to said factor VIII polypeptide within the A2 domain of the factor VIII polypeptide and the factor VIII inhibitor antibody binds to the A2 domain of the factor VIII polypeptide. In another embodiment of the foregoing fusion protein, the second XTEN is linked to said factor VIII polypeptide within the C2 domain of the factor VIII polypeptide and the factor VIII inhibitor antibody binds to the C2 domain of the factor VIII polypeptide. The binding of an anti-factor VIII antibody to the fusion protein is reduced by at least about 5%, 10%, 5%, 20%, 25%, 30%, 35% or 40% compared to the corresponding factor VIII not linked to XTEN when assayed by an ELISA assay, wherein the anti-factor VIII antibody is selected from the group consisting of the antibodies in Table 10 and a polyclonal antibody from a hemophilia A subject with factor VIII inhibitors. The foregoing fusion proteins can further comprise at least three XTENs, wherein the at least third XTEN is linked to the factor VIII at a site selected from within or replacing the B domain, at the C-terminus, and at or within 1, 2, 3, 4, 5, or 6 amino acids of an insertion site selected from Table 7 or Table 9. In the embodiments with reduced binding to anti-factor VIII antibodies, the fusion protein has greater procoagulant activity in the presence of the anti-FVIII antibody of at least 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300%, 400%, or 500% or more compared to a corresponding factor VIII not linked to XTEN when assayed by an in vitro coagulation assay (e.g., a chromogenic or one-stage clotting assay).
In all embodiments, the XTEN of the fusion protein can, for example, be characterized in that the XTEN comprise at least 36, or at least 42, or at least 72, or at least 96, or at least 144, or at least 288, or at least 400, or at least 500, or at least 576, or at least 600, or at least 700, or at least 800, or at least 864, or at least 900, or at least 1000, or at least 2000, to about 3000 amino acid residues or even more residues; the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the total amino acid residues of the XTEN; the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are identical unless the amino acids are serine; (ii) at least about 80% of the XTEN sequence consists of non-overlapping sequence motifs, each of the sequence motifs comprising about 9 to about 14, or about 12 amino acid residues consisting of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN sequence has a subsequence score of less than 10; the XTEN has greater than 90%, or greater than 95%, or greater than 99% random coil formation as determined by GOR algorithm; the XTEN has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm; the XTEN lacks a predicted T-cell epitope when analyzed by TEPITOPE algorithm, wherein the TEPITOPE threshold score for said prediction by said algorithm has a threshold of −9, and wherein said fusion protein exhibits a terminal half-life that is longer than at least about 12 h, or at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, or at least about 21 days or greater. In one embodiment, the recombinant factor VIII fusion protein comprises at least a second, or at least a third, or at least a fourth XTEN, which can be identical or different to the other XTEN. According to a different approach, the at least one, at least a second, or at least a third, or at least a fourth XTEN of the CFXTEN fusion protein each have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN of comparable length selected from Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned. In yet another different approach, the at least one, at least a second, or at least a third, or at least a fourth XTEN of the CFXTEN fusion protein each have at least 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a sequence selected from AE42 — 1, AE42 — 2, AE42 — 3, AG42 — 1, AG42 — 2, AG42 — 3, AG42 — 4, AE144 — 1A, AE144 — 2A, AE144 — 2B, AE144 — 3A, AE144 — 3B, AE144 — 4A, AE144 — 4B, AE144 — 5A, AE144 — 6B, AG144 — 1, AG144 — 2, AG144_A, AG144_B, AG144_C, AG144_F, AG144 — 3, AG144 — 4, AE288 — 1, AE288 — 2, AG288 — 1, and AG288 — 2.
In one embodiment, the factor VIII component of the CFXTEN recombinant factor VIII fusion protein comprises one, two or three amino acid substitutions selected from residues R1648, Y1680, and R1689, numbered relative to mature human factor VIII, wherein the substitutions are selected from alanine, glycine, and phenylalanine. Non-limiting examples of said substitutions include R1648A, Y1680F, and R1689A.
In another embodiment, the CFXTEN fusion protein exhibits an apparent molecular weight factor of at least about 1.3, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about 10, when measured by size exclusion chromatography or comparable method.
In some embodiments of the CFXTEN fusion proteins, one or more of the XTEN is to the FVIII via one or two cleavage sequences that each is cleavable by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20, wherein cleavage at the cleavage sequence by the mammalian protease releases the factor VIII sequence from the XTEN sequence, and wherein the released factor VIII sequence exhibits an increase in procoagulant activity compared to the uncleaved fusion protein. In one embodiment, the cleavage sequence(s) are cleavable by factor XIa.
According to a different approach, the CFXTEN fusion proteins comprise at least three XTENs located at different locations of the factor VIII polypeptide, wherein said different locations are selected from: an insertion location at or within 1 to 6 amino acids from a site selected from Table 5, Table 6, Table 7 Table 8, and Table 9; a location at or within 1 to 6 amino acids of amino acid residue 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 of mature factor VIII; a location between any two adjacent domains in the factor VIII sequence, wherein said two adjacent domains are selected from the group consisting of A1 and A2, A2 and B, B and A3, A3 and C1, and C1 and C2; a location within an internal B domain deletion starting from a first position at about amino acid residue number 741 to about 750 and ending at a second position at amino acid residue number 1635 to about 1648 with reference to full-length human factor VIII sequence as set forth in FIG. 3 and the C-terminus of the factor VIII sequence, wherein the cumulative length of the multiple XTENs is at least about 100 to about 3000 amino acid residues and wherein the fusion protein retains at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulant activity compared to the corresponding factor VIII not linked to XTEN, wherein the procoagulant activity is assayed by an in vitro coagulation assay. In one embodiment of the foregoing, the fusion protein exhibits a prolonged terminal half-life when administered to a subject as compared to a corresponding factor VIII polypeptide lacking said XTEN, wherein said fusion protein exhibits a terminal half-life at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administered to a subject. In one embodiment, the subject is selected from the group consisting of human and a factor VIII/von Willebrand factor double knock-out mouse. In one embodiment of the foregoing, the fusion protein does not comprise a sequence selected from GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34), and GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing, the fusion protein does not contain an XTEN sequence consisting of GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59), PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSS (SEQ ID NO: 71), or PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGS (SEQ ID NO: 80).
In a further aspect, the invention concerns CFXTEN fusion proteins with enhanced pharmacokinetic properties, including enhanced parameters compared to FVIII not linked to XTEN, wherein the enhanced properties include but are not limited to longer terminal half-life, larger area under the curve, increased time in which the blood concentration remains within the therapeutic window, increased time between consecutive doses results in blood concentrations within the therapeutic window, and decreased dose in IU over time that can be administered compared to a FVIII not linked to XTEN, yet still result in a blood concentration above a threshold concentration needed for a procoagulant effect. In some embodiments, a CFXTEN fusion proteins exhibit a prolonged terminal half-life when administered to a subject as compared to a corresponding factor VIII polypeptide lacking said XTEN. The subject can be a human or a mouse, such as a factor VIII/von Willebrand factor double knock-out mouse. In one embodiment of the foregoing, the CFXTEN exhibits a terminal half-life that is at least about two-fold, or about three fold, or about four-fold, or about five-fold, or about 10-fold, or about 20-fold longer when administered to a subject compared to the corresponding factor VIII not linked to XTEN. In one embodiment, the CFXTEN fusion protein exhibits a terminal half-life at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administered to the subject. In other embodiments, the enhanced pharmacokinetic property of the fusion proteins of the embodiments is the property of maintaining a circulating blood concentration of procoagulant fusion protein in a subject in need thereof above a threshold concentration of 0.01 IU/ml, or 0.05 IU/ml, or 0.1 IU/ml, or 0.2 IU/ml, or 0.3 IU/ml, or 0.4 IU/ml or 0.5 IU/ml for a period that is at least about two fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold longer compared to the corresponding FVIII not linked to XTEN and administered to a subject at a comparable dose. The increase in half-life and time spent above the threshold concentration permits less frequent dosing and decreased amounts of the fusion protein (in moles equivalent) that are administered to a subject, compared to the corresponding FVIII not linked to XTEN.
In one embodiment, administration of a subject fusion protein to a subject using a therapeutically-effective dose regimen results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold, or at least six-fold, or at least eight-fold, or at least 10-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold or higher between at least two consecutive Cmax peaks and/or Cmin troughs for blood levels of the fusion protein compared to the corresponding FVIII not linked to the XTEN and administered using a comparable dose regimen to a subject.
In preferred embodiments, the CFXTEN fusion proteins retain at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulant activity compared to the corresponding factor VIII not linked to XTEN, wherein the procoagulant activity is assayed by an in vitro coagulation assay such as, but not limited to a chromogenic assay or a one- or two-stage clotting assay.
According to a different approach, the invention provides recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least one extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises A1 domain, A2 domain, A3 domain, C1 domain, C2 domain and optionally all or a portion of B domain, and wherein said at least one XTEN is linked to said factor VIII polypeptide at an insertion site selected form residue numbers 18-32, or 40, or 211-224, or 336-403, or 599, or 745-1640, or 1656-1728, or 1796-1804, or 1900-1912, or 2171-2332; and wherein the fusion protein retains at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulant activity compared to the corresponding factor VIII not linked to XTEN. In one embodiment of the foregoing, the fusion protein comprises at least a second XTEN, or at least a third, or at least a fourth XTEN wherein the XTEN are linked to the factor VIII at a site at or within 1 to 6 amino acids of a site selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In another embodiment, the invention provides an recombinant factor VIII fusion protein further comprising at least a second XTEN, or at least a third, or at least a fourth XTEN linked to said FVIII polypeptide at an insertion site selected from Table 5, Table 6, Table 7, Table 8, Table 9, at or within 6 amino acids to the N- or C-terminus side of an insertion location at one or more insertion locations from FIG. 8 and within one or more insertion ranges from FIG. 9 wherein at least two XTEN are separated by an amino acid sequence of at least 100 to about 400 amino acids.
The invention provides CFXTEN wherein the XTEN have a Ratio XTEN Radii of at least 2.3 or at least 2.5, and are separated by an amino acid sequence of at least about 20 amino acid residues, or at least about 50, or at least about 100, or at least about 200, or at least about 300, or at least about 400 amino acid residues. In other embodiments, the CFXTEN comprise at least four XTEN wherein the XTEN have a Ratio XTEN Radii of at least 2.3, or at least 2.5, or at least 2.8, and wherein at least three of the four of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least about 20 amino acid residues, or at least about 50, or at least about 100, or at least about 200, or at least about 300, or at least about 400 amino acid residues, and the fourth XTEN is linked within the B domain (or a fragment thereof) or within the C domain (or the terminus thereof).
In some embodiments, the subject compositions are configured to have reduced binding affinity for a clearance receptor in a subject as compared to the corresponding FVIII not linked to the XTEN. In one embodiment, the CFXTEN fusion protein exhibits binding affinity for a clearance receptor of the FVIII in the range of about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or about 2% to about 10% of the binding affinity of the corresponding FVIII not linked to the XTEN. In another embodiment, a fusion protein with reduced affinity for a clearance receptor has reduced active clearance and a corresponding increase in half-life of at least about 2-fold, or 3-fold, or at least 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 12-fold, or at least about 15-fold, or at least about 17-fold, or at least about 20-fold longer compared to the corresponding FVIII that is not linked to the XTEN.
In an embodiment, the invention provides a recombinant factor VIII fusion protein comprising FVIII and one or more XTEN wherein the fusion protein exhibits increased solubility of at least three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at least 40-fold, or at least 60-fold at physiologic conditions compared to the FVIII not linked to XTEN.
In a further aspect, the invention provides a pharmaceutical composition comprising the fusion protein of any of the embodiments described herein and a pharmaceutically acceptable carrier.
In another embodiment, the invention provides a method of treating a coagulopathy in a subject, comprising administering to said subject a composition comprising a clotting effective amount of the pharmaceutical composition. In one embodiment of the method, after said administration, a blood concentration of procoagulant factor VIII is maintained at about 0.05, or 1, or 1.5 IU/ml or more for at least 48 hours after said administration. In another embodiment, the invention provides a method of clotting blood in a subject, comprising contacting a clotting effective amount of the pharmaceutical composition with the blood.
In another embodiment, the invention provides a method of treating a coagulopathy in a subject with circulating inhibitors of factor VIII, comprising administering to said subject a composition comprising a therapeutically effective amount of the pharmaceutical composition of CFXTEN, wherein the composition exhibits greater procoagulant activity in said subject compared to a composition comprising the corresponding factor VIII not linked to XTEN and administered using a comparable amount. In one embodiment of the method, the coagulopathy is hemophilia A. In another embodiment, the coagulopathy is the result of trauma or surgery or infection.
The invention provides a method of treating a bleeding episode in a subject, comprising administering to said subject a composition comprising a clotting effective amount of the CFXTEN pharmaceutical composition, wherein the clotting effective amount of the fusion protein arrests a bleeding episode for a period that is at least three-fold, or at least four-fold, or at least five-fold longer compared to a corresponding factor VIII not linked to XTEN and administered using a comparable amount to said subject. Non-limiting examples of a corresponding factor VIII not linked to XTEN include native FVIII, the sequences of Table 1, BDD-FVIII, and the pCB0114 FVIII.
In another embodiment, the invention provides a CFXTEN recombinant factor VIII fusion protein for use in a pharmaceutical regimen for treating a hemophilia A patient, said regimen comprising a pharmaceutical composition comprising a CFXTEN fusion protein. In one embodiment of the pharmaceutical regimen, the regimen further comprises the step of determining the amount of pharmaceutical composition comprising the CFXTEN needed to achieve hemostasis in the hemophilia A patient. In another embodiment, the pharmaceutical regimen for treating a hemophilia A subject comprises administering the pharmaceutical composition in two or more successive doses to the subject at an effective amount, wherein the administration results in at least a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the hemophilia A disease compared to the factor VIII not linked to XTEN and administered using a comparable dose. Non-limited examples of parameters improved include blood concentration of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic assay time, a reduced bleeding assay time, resolution of a bleeding event, or a reduced Bethesda titer to native FVIII.
In another aspect, the invention provides isolated nucleic acid sequences encoding the fusion proteins of any one of the embodiments of the CFXTEN fusion protein. In one embodiment, the isolated nucleic acid is the complement of a sequence encoding a CFXTEN fusion protein of the embodiments. In one embodiment, the isolated nucleic acid further comprises a sequence encoding a signal peptide, wherein said sequence is ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613), or the complement thereof. In another embodiment, the invention provides an expression vector comprising the nucleic acid encoding the fusion protein, or the complement thereof. In another embodiment, the invention provides an isolated host cell comprising the foregoing expression vector. In another embodiment, the invention provides a method of producing the fusion protein of any of the embodiments, comprising providing a host cell comprising the expression vector; culturing the host cell to effect production of the fusion protein; and recovering the fusion protein.
In one embodiment, the invention provides an isolated fusion protein comprising a polypeptide having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a sequence of comparable length selected from Table 21, when optimally aligned.
In another embodiment, the invention provides an isolated nucleic acid comprising a polynucleotide sequence selected from (a) a sequence having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a sequence of comparable length selected from Table 21, when optimally aligned, or (b) the complement of the polynucleotide of (a). In another embodiment, the isolated nucleic acid comprises the sequence ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613) linked to the 5′ end of the nucleic acid of (a) or the complement of the sequence linked to the 3′ end of (b).
It is specifically contemplated that the recombinant factor VIII fusion proteins can exhibit one or more or any combination of the properties disclosed herein.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
The features and advantages of the invention may be further explained by reference to the following detailed description and accompanying drawings that sets forth illustrative embodiments.
FIG. 1 shows a schematic representation of the FVIII architecture and spatial arrangement of the domains during processing and clotting, and is intended to represent both native FVIII and B domain deleted variants. The A1 domain ranges from residue 1 to 372 (numbering relative to the mature form of FVIII sequence NCBI Protein RefSeq NP — 000123 and encompassing a1 residues), A2 domain ranges from residue 373 to 740, B domain ranges from residue 741 to 1648, A3 domain ranges from residue 1649 to 2019 (encompassing a3 acidic region), C1 domain ranges from 2020 to 2172, and the C2 domain ranges from residue 2173 to 2332. BDD variants include deletions between the range 741 to 1648, leaving some or no remnant residues, with a non-limiting BDD remnant sequence being SFSQNPPVLKRHQR (SEQ ID NO: 1614). FIG. 1A shows the domain architecture of a single chain FVIII prior to processing. Arrows indicate the sites at residues R372, R740, R1648, and R1689 that are cleaved in the processing and conversion of FVIII to FVIIIa. FIG. 1B shows the FVIII molecule that has been processed into the heterodimer by the cleavage at the R1648 residue, with the a3 acidic region of the A3 domain indicated on the N-terminus of the A3. FIG. 1C shows the FVIII molecule processed into the FVIIIa heterotrimer by the cleavage at the R372, R740, and R1689 residues.
FIG. 2 is a schematic of the coagulation cascade, showing the intrinsic and extrinsic arms leading to the common pathway.
FIG. 3 depicts the amino acid sequence of mature human factor V111 (SEQ ID NO: 1592).
FIG. 4 depicts a factor VIII sequence with a deletion of a portion of the B domain (SEQ ID NO: 1593).
FIG. 5 illustrates several examples of CFXTEN configurations of FVIII linked to XTEN (the latter shown as thick, wavy lines). In all cases, the FVIII can be either native or a BDD form of FVIII, or a single chain form in which the entire B domain, including the native cleavage sites are removed. FIG. 5A shows, left to right, three variations of single chain factor VIII with XTEN linked to the N-terminus, the C-terminus, and two XTEN linked to the N- and C-terminus. FIG. 5B shows six variations of mature heterodimer FVIII with, left to right, an XTEN linked to the N-terminus of the A1 domain; an XTEN linked to the C-terminus of the C2 domain; an XTEN linked to the N-terminus of the A1 domain and the C-terminus of the C2 domain; an XTEN linked to the N-terminus of the A1 domain and to the N-terminus of the A3 domain; an XTEN linked to the C-terminus of the C2 domain and to the N-terminus of the A3 domain via residual B domain amino acids; and an XTEN linked to the N-terminus of the A1 domain, the C-terminus of the A2 domain via residual B domain amino acids, and to the C-terminus of the C2 domain. FIG. 5C shows, left to right, three variations of single chain factor VIII: an XTEN linked to the N-terminus of the A1 domain, an XTEN linked within a surface loop of the A1 domain and an XTEN linked within a surface loop of the A3 domain; an XTEN linked within a surface loop of the A2 domain, an XTEN linked within a surface loop of the C2 domain and an XTEN linked to the C terminus of the C2 domain; an XTEN linked to the N-terminus of the A1 domain and within a surface loop of the C1 domain and to the C-terminus of the C domain. FIG. 5D shows six variations of mature heterodimer FVIII with, left to right, an XTEN linked to the N-terminus of the A1 domain, an XTEN linked within a surface loop of the A1 domain, and an XTEN linked within a surface loop of the A3 domain; an XTEN linked within a surface loop of the A2 domain, and an XTEN linked within a surface loop of the C1 domain, and an XTEN linked to the C-terminus of the C2 domain; an XTEN linked to the N-terminus of the A1 domain, an XTEN linked within a surface loop of the A1 domain, an XTEN linked within a surface loop of the A3 domain, and an XTEN linked to the C-terminus of the C2 domain; an XTEN linked to the N-terminus of the A1 domain, an XTEN linked to the N-terminus of the A3 domain via residual amino acids of the B domain, and an XTEN linked within a surface loop of the C2 domain; an XTEN linked within a surface loop of the A2 domain, an XTEN linked to the N-terminus of the A3 domain via residual amino acids of the B domain, an XTEN linked within a surface loop of the C1 domain, and an XTEN linked to the C-terminus of the C2 domain; and an XTEN linked within the B domain or between the residual B domain residues of the BDD variant (and the invention also contemplates a variation in which the XTEN replaces the entirety of the B domain, including all native cleavage sites, linking the A2 and A3 domains, resulting in a single chain form of factor VIII). This figure also embodies all variations in which one or more XTEN sequences are inserted within the B domain and the resulting fusions are cleaved at one or more sites (e.g., at R1648 site) during intracellular processing.
FIG. 6 is a graphic portrayal of a CFXTEN construct with an XTEN inserted within the B domain and linked to the C-terminus of the C2 domain illustrating the unstructured characteristic of the XTEN leading to random coil formation that can cover portions of the factor VIII proximal to the XTEN. In the lower panel, the drawing depicts that when XTEN is in random coil, it can adopt a conformation resulting in steric hindrance that blocks binding of factor VIII inhibitor antibodies that would otherwise have affinity for epitopes proximal to the XTEN site of insertion.
FIG. 7 is a graphic portrayal of the various analyses performed on a FVIII B-domain deleted sequence to identify insertion sites for XTEN within the FVIII sequence. Each of lines A-H are on an arbitrary scale of Y axis values across the FVIII BDD sequence such that low values represent areas with a high predicted tolerance for XTEN insertion, with the residue numbers on the X axis. Line A shows the domain boundaries; all discontinuities in this line represent boundaries that are likely to accept XTEN. Line B shows exon boundaries; i.e., each step in the line represents a new exon. Line C shown regions that were not visible in the X-ray structure due to a lack of order in the crystal. Lines labeled D represents multiple predictions of order that were calculated using the respective programs FoldIndex found on the World-Wide web site bip.weizmann.ac.il/fldbin/findex (last accessed Feb. 23, 2011) (see Jaime Prilusky, Clifford E. Felder, Tzviya Zeev-Ben-Mordehai, Edwin Rydberg, Orna Man, Jacques S. Beckmann, Israel Silman, and Joel L. Sussman, 2005, Bioinformatics based on the Kyte & Doolitlle algorithm, as well as RONN found on the World-Wide web site strubi.ox.ac.uk/RONN (last accessed Feb. 23, 2011) (see Yang, Z. R., Thomson, R., McMeil, P. and Esnouf, R. M. (2005) RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins Bioinformatics 21: 3369-3376. Lines E and F were calculated based on multiple sequence alignments of FVIII genes from 11 mammals available in GenBank. Line E represents the conservation of individual residues. Line F represent the conservation of 3 amino acid segments of FVIII. Lines G and H represent gaps and insertions observed in the multiple sequence alignment of 11 mammalian FVIII genes. Line J lists the XTEN insertion points by amino acid number that were obtained based by combining the multiple measurements above.
FIG. 8 depicts the sites in a FVIII B-domain deleted sequence (SEQ ID NO: 1594) identified as active insertion points for XTEN using the information depicted in FIG. 8 and as confirmed in the assays of Example 34.
FIG. 9 depicts the range of sites in a FVIII B-domain deleted sequence (SEQ ID NO: 1595) identified for insertion of XTEN using the information depicted in FIG. 8 and or Example 34 plus a span of amino acids around each insertion point that are considered suitable for insertion of XTEN.
FIG. 10 is a schematic of the assembly of a CFXTEN library created by identifying insertion points as described for FIG. 7 followed by insertion of single XTEN (black bars) at the various insertion points using molecular biology techniques. The constructs are expressed and recovered, then evaluated for FVIII activity and pharmacokinetic properties to identify those CFXTEN configurations that result in enhanced properties.
FIG. 11 is a schematic of the assembly of a CFXTEN component library in which segments of FVIII BDD domains, either singly or linked to various lengths of XTEN (black bars) are assembled in a combinatorial fashion into libraries of genes encoding the CFXTEN, which can then be evaluated for FVIII activity and pharmacokinetic properties to identify those CFXTEN configurations that result in enhanced properties.
FIG. 12 illustrates several examples of CFXTEN configurations with XTEN (shown as thick, wavy lines), with certain XTEN releasable by inserting cleavage sequences (indicated by black triangles) that are cleavable by procoagulant proteases. FIG. 12A illustrates a scFVIII with two terminal releasable XTENS. FIG. 12B illustrates the same configuration as FIG. 12A but with an additional non-releasable XTEN linking the A3 and C1 domains. FIG. 12C illustrates a mature heterodimer FVIII with two terminal releasable XTEN. FIG. 12D illustrates the same configuration as 10C but with an additional non-releasable XTEN linking the A3 and C1 domains.
FIG. 13 is a schematic flowchart of representative steps in the assembly, production and the evaluation of an XTEN.
FIG. 14 is a schematic flowchart of representative steps in the assembly of a CFXTEN polynucleotide construct encoding a fusion protein. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a library to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The resulting pool of ligation products is gel-purified and the band with the desired length of XTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene is cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is then performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, resulting in the gene 500 encoding an FVIII-XTEN fusion protein.
FIG. 15 is a schematic flowchart of representative steps in the assembly of a gene encoding fusion protein comprising a CF and XTEN, its expression and recovery as a fusion protein, and its evaluation as a candidate CFXTEN product.
FIG. 16 illustrates the use of donor XTEN sequences to produce truncated XTENs. FIG. 16A provides the sequence of AG864 (SEQ ID NO: 1596), with the underlined sequence used to generate a sequence length of 576 (SEQ ID NO: 1597). FIG. 16B provides the sequence of AG864 (SEQ ID NO: 1598), with the underlined sequence used to generate a sequence length of 288 (SEQ ID NO: 1599). FIG. 16C provides the sequence of AG864 (SEQ ID NO: 1600), with the underlined sequence used to generate a sequence length of 144 (SEQ ID NO: 1601). FIG. 16D provides the sequence of AE864 (SEQ ID NO: 1602), with the underlined sequence used to generate a sequence length of 576 (SEQ ID NO: 1603). FIG. 16E provides the sequence of AE864 (SEQ ID NO: 1604), with the underlined sequence used to generate a sequence length of 288 (SEQ ID NO: 1605). FIG. 16F provides the sequence of AE864 (SEQ ID NO: 1606) used to generate four sequences of 144 length (SEQ ID NOS1607-1610, respectively, in order of appearance) (the double underline indicates the first amino acid in the 144 sequence with the single underline representing the balance of that sequence).
FIG. 17 is a schematic representation of the design of Factor VIII-XTEN expression vectors with different strategies introducing XTEN elements into the FVIII coding sequence. FIG. 17A shows an expression vector encoding XTEN fused to the 3′ end of the sequence encoding FVIII. FIG. 17B depicts an expression vector encoding an XTEN element inserted into the middle of the coding sequence encoding a single FVIII. FIG. 17C depicts an expression vector encoding two XTEN elements: one inserted internal to the FVIII coding sequence, and the other fused to the 3′ end of the FVIII coding sequence.
FIG. 18 illustrates the process of combinatorial gene assembly of genes encoding XTEN. In this case, the genes are assembled from 6 base fragments and each fragment is available in 4 different codon versions (A, B, C and D). This allows for a theoretical diversity of 4096 in the assembly of a 12 amino acid motif.
FIG. 19 shows the pharmacokinetic profile (plasma concentrations) in cynomolgus monkeys after single doses of different compositions of GFP linked to unstructured polypeptides of varying length, administered either subcutaneously or intravenously, as described in Example 41. The compositions were GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576 and XTEN_AD836-GFP. Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are presented as the plasma concentration versus time (h) after dosing and show, in particular, a considerable increase in half-life for the XTEN_AD836-GFP, the composition with the longest sequence length of XTEN. The construct with the shortest sequence length, the GFP-L288 had the shortest half-life.
FIG. 20 shows an SDS-PAGE gel of samples from a stability study of the fusion protein of XTEN_AE864 fused to the N-terminus of GFP (see Example 42). The GFP-XTEN was incubated in cynomolgus plasma and rat kidney lysate for up to 7 days at 37° C. In addition, GFP-XTEN administered to cynomolgus monkeys was also assessed. Samples were withdrawn at 0, 1 and 7 days and analyzed by SDS PAGE followed by detection using Western analysis with antibodies against GFP.
FIG. 21 shows results of a size exclusion chromatography analysis of glucagon-XTEN construct samples measured against protein standards of known molecular weight, with the graph output as absorbance versus retention volume, as described in Example 40. The glucagon-XTEN constructs are 1) glucagon-Y288; 2) glucagonY-144; 3) glucagon-Y72; and 4) glucagon-Y36. The results indicate an increase in apparent molecular weight with increasing length of XTEN moiety (see Example 40 for data).
FIG. 22 shows results of a Western blot of proteins expressed by cell culture of cells transformed with constructs as designated (Example 25). The samples in lanes 1-12 were: MW Standards, FVIII (42.5 ng), pBC0100B, pBC0114A, pBC0100, pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0149, and pBC0146, respectively. Lanes 8, 9 and 12 show bands consistent with a FVIII with a C-terminal XTEN288, with an estimated MW of 95 kDa. Lanes 7 and 11 show bands consistent with a FVIII with a C-terminal XTEN42, with an estimated MW of 175 kDa. Lanes 2-6 show bands consistent with FVIII and heavy chain. Lanes 10 and 23 show bands consistent with heavy chain. Lane 7 shows a band consistent with heavy chain and an attached XTEN42.
FIG. 23 shows the results of FVIII assay on samples obtained from FVIII and von Willebrand factor double knock-out mice with hydrodynamic plasmid DNA injection, as detailed in Example 36.
FIG. 24 is a graphic and tabular portrayal of the pharmacokinetic properties of rBDD-FVIII and the purified CFXTEN fusion proteins pBC0145 and pBC0146 (with C-terminal XTEN) administered to either HemA or FVIII/VWF double knock-out mice as described in Example 30, showing the enhanced half-life of the CFXTEN in both strains of mice.
FIG. 25 is a graphic and tabular portrayal of the pharmacokinetic properties of rBDD-FVIII and the CFXTEN fusion proteins pSD0050 and pSD0062 (with internal inserted XTEN) administered to either HemA ( FIG. 25A ) or FVIII/VWF double knock-out mice ( FIG. 25B ) using a cell culture PK assay in HemA mice. Dose, 5-minute recovery, and half-life (T1/2) are shown, as described in Example 32, underscoring the enhanced recovery and half-life of the CFXTEN compared to the positive control FVIII in both strains of mice.
FIG. 26 is a graphic depiction of a titration of GMA8021 FVIII inhibitor using the pBC0114 BDD-FVIII AND CFXTEN construct LSD0049.002 with three 144 amino acid XTEN insertions at residues 18, 745 and 2332. The data indicate a right-shift of approximately 0.7 order of magnitude in the amount of antibody in μg/ml required to inhibit the CFXTEN to the 50% level, compared to FVIII positive control.
FIG. 27 is a schematic of the logic flow chart of the algorithm SegScore. In the figure the following legend applies: i, j—counters used in the control loops that run through the entire sequence; HitCount—this variable is a counter that keeps track of how many times a subsequence encounters an identical subsequence in a block; SubSeqX—this variable holds the subsequence that is being checked for redundancy; SubSeqY—this variable holds the subsequence that the SubSeqX is checked against; BlockLen—this variable holds the user determined length of the block; SegLen—this variable holds the length of a segment. The program is hardcoded to generate scores for subsequences of lengths 3, 4, 5, 6, 7, 8, 9, and 10; Block—this variable holds a string of length BlockLen. The string is composed of letters from an input XTEN sequence and is determined by the position of the i counter; SubSeqList—this is a list that holds all of the generated subsequence scores.
FIG. 28 depicts the application of the algorithm SegScore to a hypothetical XTEN of 11 amino acids (SEQ ID NO: 1591) in order to determine the repetitiveness. An XTEN sequence consisting of N amino acids is divided into N-S+1 subsequences of length S(S=3 in this case). A pair-wise comparison of all subsequences is performed and the average number of identical subsequences is calculated to result in the subsequence score of 1.89.
FIG. 29 is a graph of the individual construct values of the ratio of FVIII activity in the assayed CFXTEN to that of the pBC114 FVIII positive control after exposure to the GMA8021 antibody to FVIII, grouped according to the number of XTEN in the construct fusion protein (see Example 28). The results show an essentially linear relationship in the ability of the CFXTEN to retain FVIII activity with increasing number of incorporated XTEN.
FIG. 30 depicts the primary sequence and domain structure of mature B-domain deleted (BDD) human FVIII construct (Example 46). The location of the introduced NheI and ClaI restriction sites is shown. Note that the amino acid numbering corresponds to the amino acid positions in the primary sequence of mature FVIII ( FIG. 30 ). Individual domains are bounded by gray lines/boxes with domain identification in gray text. Acidic regions (a1, a2, a3) are indicated with dashed boxes. Solid wedges/triangles indicate sites of thrombin cleavage in the activation of FVIII to FVIIIa. Unfilled wedges/triangle indicates the site of intracellular proteolytic processing to the two-chained form of FVIII. Hexagons indicate sites of N-linked glycosylation. Circles indicate sites of Tyr sulfation. Unique non-native restriction sites (NheI, GCTAG; ClaI, ATCGAT) introduced into cDNA to facilitate XTEN insertion/recombination are highlighted in gray with double underline.
FIG. 31 provides graphical representation of the FVIII construct described in FIG. 30 , indicating the domain organization and the location of native and non-native restriction sites.
FIG. 32 shows the graphical ASAView outputs for structural datasets 2R7E, 3CDZ, and PM0076106. Accessible Solvent Areas (ASA) for the amino acids in domains A1, A2, A3, C1 and C2 are shown. Analyses were performed on X-ray crystallographic coordinates 3CDZ (Ngo et al., Structure 16: 597-606 (2008)) and 2R7E (Shen et al., Blood 111:1240-1247 (2008)) deposited in the Protein Data Bank maintained by the Research Collaboratory for Structural Bioinformatics (RCSB; http://www.rcsb.org/pdb), as well as on atomic coordinates PM0076106 for the predicted refined FVIII structure derived from a molecular dynamics simulation study (Venkateswarlu, BMC Struct. Biol. 10:7 (2010)) deposited in the Protein Model Database (http://ml.caspur.it/PMDB/main.php) maintained by Consorzio Interuniversitario per le Applicazioni di Supercalcolo per Università e Riserca (CASPUR) and the Department of Biochemical Sciences of the University of Rome.
FIG. 33 shows a structural representation of the location of XTEN insertion sites. The central drawing corresponding to the crystal structure of FVIII (PDB: 2R7E) is surrounded by detailed view of domains A1, A2, A3, C1 and C2. Beta strands and alpha helices are shown as ribbon representation. Loops are shown as alpha carbon pipes. The amino acids at XTEN insertion sites are shown as CPK sphere representation. The number in each graph indicate the location of the XTEN insertion sites according to the numbering in FIG. 30 .
FIG. 34 shows a structural representation of the location of XTEN insertion sites shown in FIG. 33 wherein the resulting recombinant FVIII protein displays FVIII activity.
FIG. 35 shows a structural representation of the location of XTEN insertion sites shown in FIG. 34 wherein the resulting recombinant FVIII protein displays FVIII activity.
FIG. 36 shows a structural representation of the location of XTEN insertion sites shown in FIG. 35 wherein the resulting recombinant FVIII protein displays FVIII activity.
FIG. 37 shows a ClustalW multiple sequence alignment of domains A1, A2, A3, C1 and C2 of FVIII showing the location of XTEN insertions resulting in recombinant FVIII proteins displaying FVIII activity (black box, white text) or displaying no FVIII activity (grey box, bold text).
FIG. 38 shows a DSSP graphical representation of the secondary structure of the two polypeptide chains in a native active human FVIII crystal structure deposited under the identifier 2R7E at the Protein Data Bank (see Example 47). Amino acid sequence numbering is the same as in the protein sequence in FIG. 30 . The beta sheet regions are shown as filled arrows and are designated 31 to P66. The location of the XTEN permissive loops is denoted by crosshatched boxes. Domain A1 XTEN permissive loops are designated Loop A1-1 and Loop A1-2. Domain A2 XTEN permissive loops are designated Loop A2-1 and Loop A2-2. Domain A3 XTEN permissive loops are designated Loop A3-1 and Loop A3-2.
FIG. 39 shows a DSSP graphical representation of the secondary structure of the two polypeptide chains in a native active human FVIII crystal structure deposited under the identifier 2R7E at the Protein Data Bank (see Example 47). Amino acid sequence numbering is the same as in the protein sequence in FIG. 30 . The beta sheet regions are shown as filled arrows and are designated 31 to P66. The location of the XTEN permissive loops is denoted by crosshatched boxes. Domain A1 XTEN permissive loops are designated Loop A1-1 and Loop A1-2. Domain A2 XTEN permissive loops are designated Loop A2-1 and Loop A2-2. Domain A3 XTEN permissive loops are designated Loop A3-1 and Loop A3-2.
FIG. 40 shows a ClustalW multiple sequence alignment of domains A1, A2, A3, C1 and C2 of FVIII showing the location of XTEN insertions resulting in recombinant FVIII proteins displaying FVIII activity (black box, white text) or displaying no FVIII activity (grey box, bold text). The locations of the XTEN permissive loops are indicated by dashed rectangles (see Example 47).
FIG. 41 . FIG. 41A presents a front view structural representation of human FVIII (PDB:2R7E) showing the location of domains A1, A2, A3, C1 and C2 (circled in dashed lined) and the locations of XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1 and A3-2 highlighted as CPK sphere representations. FIG. 41B presents a side view structural representation of human FVIII (PDB:2R7E) showing the location of domains A1, A2, A3, C1 and C2 (circled in dashed lined) and the locations of XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1 and A3-2 highlighted as CPK sphere representations.
FIG. 42 shows the top view structural representations of isolated human FVIII (PDB:2R7E) A domains showing the location of XTEN permissive loops highlighted as CPK sphere representations. FIGS. 42B , 42 D and 42 F show side view structural representations of isolated human FVIII (PDB:2R7E) A domains showing the location of XTEN permissive loops highlighted as CPK sphere representations.
FIG. 43 shows sequences of various factor VIII B-domain deletions and individual mutations. Lines 4-10 show various B-domain deletions with indicated XTEN linking the flanking B-domain residual or A3 domain residues. The R1648A mutation is indicated by arrow in line 5 and 8, while the Y1680F mutation is indicated by arrow in lines 8-10.
FIG. 44 is a bar graph of chromogenic and aPTT assay activity of various CFXTEN with single XTEN insertions (Example 49).
FIG. 45 is a bar graph of chromogenic and aPTT assay activity of various CFXTEN with 2 XTEN insertions (Example 49).
FIG. 46 is a bar graph of chromogenic and aPTT assay activity of various CFXTEN with 3 XTEN insertions (Example 49).
FIG. 47 is a graph of plasma levels in DKO mice of various administered CFXTEN with single XTEN insertions compared to a BDD-FVIII control, demonstrating the 10- to 20-fold longer half-life achieved by the XTEN insertions at various locations (Example 50).
FIG. 48 is a graph of plasma levels in DKO mice of various administered CFXTEN with one, two, and three XTEN insertions compared to a BDD-FVIII control, demonstrating the increases in half-life achieved by the inclusion of additional XTEN insertions compared to single or two insertions (Example 51).
FIG. 49 are graphs of the plotted inhbition curves for remaining factor VIII procoagulant activity in samples assayed in the Bethesda assay with three hemophilia patient sera ( FIGS. 49A-C ) or sheep anti-FVII ( FIG. 49D ) described in Example 52, demonstrating a clear left-shift of the inhibition curve for the two CFXTEN molecules compared to the FVIII not linked to XTEN.



DETAILED DESCRIPTION OF THE INVENTION
Before the embodiments of the invention are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.
DEFINITIONS
In the context of the present application, the following terms have the meanings ascribed to them unless specified otherwise:
As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
The term “domain,” when used in reference to a factor VIII polypeptide refers to either a full length domain or a functional fragment thereof, for example, full length or functional fragments of the A1 domain, A2 domain, A3 domain, B domain, C1 domain, and/or C2 domain of factor VIII.
The term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).
The term “non-naturally occurring,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
The terms “hydrophilic” and “hydrophobic” refer to the degree of affinity that a substance has with water. A hydrophilic substance has a strong affinity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water. Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is a scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, which is listed in Hopp, T P, et al., Proc Natl Acad Sci USA (1981) 78:3824. Examples of “hydrophilic amino acids” are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of “hydrophobic amino acids” are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
A “fragment” when applied to a protein, is a truncated form of a native biologically active protein that retains at least a portion of the therapeutic and/or biological activity. A “variant”. when applied to a protein is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared with the reference biologically active protein. As used herein, the term “biologically active protein moiety” includes proteins modified deliberately, as for example, by site directed mutagenesis, synthesis of the encoding gene, insertions, or accidentally through mutations.
The term “sequence variant” means polypeptides that have been modified compared to their native or original sequence by one or more amino acid insertions, deletions, or substitutions. Insertions may be located at either or both termini of the protein, and/or may be positioned within internal regions of the amino acid sequence. A non-limiting example is insertion of an XTEN sequence within the sequence of the biologically-active payload protein. In deletion variants, one or more amino acid residues in a polypeptide as described herein are removed. Deletion variants, therefore, include all fragments of a payload polypeptide sequence. In substitution variants, one or more amino acid residues of a polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art.
As used herein, “internal XTEN” refers to XTEN sequences that have been inserted into the sequence of the coagulation factor. Internal XTENs can be constructed by insertion of an XTEN sequence into the sequence of a coagulation factor such as FVIII, either by insertion between two adjacent amino acids within a domain (“intradomain”) or between two domains (“interdomain”) of the coagulation factor or wherein XTEN replaces a partial, internal sequence of the coagulation factor.
As used herein, “terminal XTEN” refers to XTEN sequences that have been fused to or in the N- or C-terminus of the coagulation factor or to a proteolytic cleavage sequence or linker at the N- or C-terminus of the coagulation factor. Terminal XTENs can be fused to the native termini of the coagulation factor. Alternatively, terminal XTENs can replace a portion of a terminal sequence of the coagulation factor.
The term “XTEN release site” refers to a cleavage sequence in CFXTEN fusion proteins that can be recognized and cleaved by a mammalian protease, effecting release of an XTEN or a portion of an XTEN from the CFXTEN fusion protein. As used herein, “mammalian protease” means a protease that normally exists in the body fluids, cells or tissues of a mammal. XTEN release sites can be engineered to be cleaved by various mammalian proteases (a.k.a. “XTEN release proteases”) such as FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17, MMP-20, or any protease that is present during a clotting event. Other equivalent proteases (endogenous or exogenous) that are capable of recognizing a defined cleavage site can be utilized. The cleavage sites can be adjusted and tailored to the protease utilized.
The term “within”, when referring to a first polypeptide being linked to a second polypeptide, encompasses linking that connects the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, as well as insertion of the first polypeptide into the sequence of the second polypeptide. For example, when an XTEN is linked “within” a domain of a factor VIII polypeptide, the XTEN may be linked to the N-terminus, the C-terminus, or may be inserted in said domain.
As used herein, the term “site,” when used to refer to an insertion site of an XTEN within or to a biological polypeptide such as a factor VIII, represents the amino acid position at which the XTEN is linked. When numbered sites are described, such as a first, second, third, fourth, fifth, or sixth site for the insertion of an XTEN within or to the factor VIII, each site will be understood to represent a distinct site in the factor VIII; e.g., the second site is a different factor VIII location from the first site, the third site is different from the second and the first, etc.
“Activity” or “procoagulant activity” as applied to form(s) of a CFXTEN polypeptide provided herein, refers to the ability to bind to a target coagulation protein substrate or cofactor and promote a clotting event, whether measured by an in vitro, ex vivo or in vivo assay. Such assays include, but are not limited to, one-stage clotting assays, two-stage clotting assays, chromogenic assays, and ELISA assays.
“Biological activity” refers to an in vitro or in vivo biological function or effect, including but not limited to either receptor or ligand binding, or an effect on coagulation generally known in the art for the FVIII coagulation factor, or a cellular, physiologic, or clinical response, including arrest of a bleeding episode.
As used herein, the term “ELISA” refers to an enzyme-linked immunosorbent assay as described herein or as otherwise known in the art.
A “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of this invention.
“Isolated” when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.”
An “isolated” polynucleotide or polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.
A “chimeric” protein contains at least one fusion polypeptide comprising at least one region in a different position in the sequence than that which occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion polypeptide; or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
“Conjugated”, “linked,” “fused,” and “fusion” are used interchangeably herein. These terms refer to the joining together of two or more chemical elements, sequences or components, by whatever means including chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and in reading phase or in-frame. An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).
In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
“Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
The terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
The term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.
“Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed as a recombinant protein in a host cell.
The terms “gene” and “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.
“Homology” or “homologous” or “sequence identity” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences. Polypeptides that are homologous preferably have sequence identities that are at least 70%, preferably at least 80%, even more preferably at least 90%, even more preferably at least 95-99%, and most preferably 100% identical.
“Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, linking them together. To ligate the DNA fragments or genes together, the ends of the DNA must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.
The terms “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Generally, stringency of hybridization is expressed, in part, with reference to the temperature and salt concentration under which the wash step is carried out. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60° C. for long polynucleotides (e.g., greater than 50 nucleotides)—for example, “stringent conditions” can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and three washes for 15 min each in 0.1×SSC/1% SDS at 60° C. to 65° C. Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSC concentration may be varied from about 0.1 to 2×SSC, with SDS being present at about 0.1%. Such wash temperatures are typically selected to be about 5° C. to 20° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3 rd edition, Cold Spring Harbor Laboratory Press, 2001. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
The terms “percent identity,” percentage of sequence identity,” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of matched positions (at which identical residues occur in both polypeptide sequences), dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. When sequences of different length are to be compared, the shortest sequence defines the length of the window of comparison. Conservative substitutions are not considered when calculating sequence identity.
“Percent (%) sequence identity,” with respect to the polypeptide sequences identified herein, is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
The term “non-repetitiveness” as used herein in the context of a polypeptide refers to a lack or limited degree of internal homology in a peptide or polypeptide sequence. The term “substantially non-repetitive” can mean, for example, that there are few or no instances of four contiguous amino acids in the sequence that are identical amino acid types or that the polypeptide has a subsequence score (defined infra) of 10 or less or that there is no a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence. The term “repetitiveness” as used herein in the context of a polypeptide refers to the degree of internal homology in a peptide or polypeptide sequence. In contrast, a “repetitive” sequence may contain multiple identical copies of short amino acid sequences. For instance, a polypeptide sequence of interest may be divided into n-mer sequences and the number of identical sequences can be counted. Highly repetitive sequences contain a large fraction of identical sequences while non-repetitive sequences contain few identical sequences. In the context of a polypeptide, a sequence can contain multiple copies of shorter sequences of defined or variable length, or motifs, in which the motifs themselves have non-repetitive sequences, rendering the full-length polypeptide substantially non-repetitive. The length of polypeptide within which the non-repetitiveness is measured can vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids, or from about 9 to about 14 amino acids. “Repetitiveness” used in the context of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.
A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
“Serum degradation resistance,” as applied to a polypeptide, refers to the ability of the polypeptides to withstand degradation in blood or components thereof, which typically involves proteases in the serum or plasma. The serum degradation resistance can be measured by combining the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about 37° C. The samples for these time points can be run on a Western blot assay and the protein is detected with an antibody. The antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein's size is identical to that of the injected protein, then no degradation has occurred. In this exemplary method, the time point where 50% of the protein is degraded, as judged by Western blots or equivalent techniques, is the serum degradation half-life or “serum half-life” of the protein.
The term “t 1/2 ” as used herein means the terminal half-life calculated as ln(2)/K el . K el is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. Half-life typically refers to the time required for half the quantity of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes. The terms “t 1/2 ”, “terminal half-life”, “elimination half-life” and “circulating half-life” are used interchangeably herein.
“Active clearance” means the mechanisms by which a protein is removed from the circulation other than by filtration or coagulation, and which includes removal from the circulation mediated by cells, receptors, metabolism, or degradation of the protein.
“Apparent molecular weight factor” and “apparent molecular weight” are related terms referring to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid sequence. The apparent molecular weight is determined using size exclusion chromatography (SEC) or similar methods by comparing to globular protein standards, and is measured in “apparent kD” units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the actual molecular weight; the latter predicted by adding, based on amino acid composition, the calculated molecular weight of each type of amino acid in the composition or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel.
The terms “hydrodynamic radius” or “Stokes radius” is the effective radius (Rh in nm) of a molecule in a solution measured by assuming that it is a body moving through the solution and resisted by the solution's viscosity. In the embodiments of the invention, the hydrodynamic radius measurements of the XTEN fusion proteins correlate with the ‘apparent molecular weight factor’, which is a more intuitive measure. The “hydrodynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its ability to migrate in gels of macromolecules. The hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. Most proteins have globular structure, which is the most compact three-dimensional structure a protein can have with the smallest hydrodynamic radius. Some proteins adopt a random and open, unstructured, or ‘linear’ conformation and as a result have a much larger hydrodynamic radius compared to typical globular proteins of similar molecular weight.
“Physiological conditions” refers to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject. A host of physiologically relevant conditions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers are listed in Sambrook et al. (2001). Physiologically relevant temperature ranges from about 25° C. to about 38° C., and preferably from about 35° C. to about 37° C.
A “reactive group” is a chemical structure that can be coupled to a second reactive group. Examples for reactive groups are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups, azide groups. Some reactive groups can be activated to facilitate coupling with a second reactive group. Non-limiting examples for activation are the reaction of a carboxyl group with carbodiimide, the conversion of a carboxyl group into an activated ester, or the conversion of a carboxyl group into an azide function.
“Controlled release agent”, “slow release agent”, “depot formulation” and “sustained release agent” are used interchangeably to refer to an agent capable of extending the duration of release of a polypeptide of the invention relative to the duration of release when the polypeptide is administered in the absence of agent. Different embodiments of the present invention may have different release rates, resulting in different therapeutic amounts.
The terms “antigen”, “target antigen” and “immunogen” are used interchangeably herein to refer to the structure or binding determinant that an antibody fragment or an antibody fragment-based therapeutic binds to or has specificity against.
The term “payload” as used herein refers to a protein or peptide sequence that has biological or therapeutic activity; the counterpart to the pharmacophore of small molecules. Examples of payloads include, but are not limited to, coagulation factors, cytokines, enzymes, hormones, and blood and growth factors.
The term “antagonist”, as used herein, includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. Methods for identifying antagonists of a polypeptide may comprise contacting a native polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide. In the context of the present invention, antagonists may include proteins, nucleic acids, carbohydrates, antibodies or any other molecules that decrease the effect of a biologically active protein.
The term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native polypeptide may comprise contacting a native polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.
As used herein, “treat” or “treating,” or “palliating” or “ameliorating” are used interchangeably and mean administering a drug or a biologic to achieve a therapeutic benefit, to cure or reduce the severity of an existing condition, or to achieve a prophylactic benefit, prevent or reduce the likelihood of onset or severity the occurrence of a condition. By therapeutic benefit is meant eradication or amelioration of the underlying condition being treated or one or more of the physiological symptoms associated with the underlying condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying condition.
A “therapeutic effect” or “therapeutic benefit,” as used herein, refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a fusion protein of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition or symptom of the disease (e.g., a bleed in a diagnosed hemophilia A subject), or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologically active protein, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
The term “therapeutically effective dose regimen”, as used herein, refers to a schedule for consecutively administered multiple doses (i.e., at least two or more) of a biologically active protein, either alone or as a part of a fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.
I). General Techniques
The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3 rd edition, Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology”, F. M. Ausubel, et al. eds., 1987; the series “Methods in Enzymology,” Academic Press, San Diego, Calif.; “PCR 2: a practical approach”, M. J. MacPherson, B. D. Hames and G. R. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman's The Pharmacological Basis of Therapeutics,” 11 th Edition, McGraw-Hill, 2005; and Freshney, R. I., “Culture of Animal Cells: A Manual of Basic Technique,” 4 th edition, John Wiley & Sons, Somerset, N.J., 2000, the contents of which are incorporated in their entirety herein by reference.
II). Coagulation Factor VIII
The present invention relates, in part, to compositions comprising factor VIII coagulation factor (CF) linked to one or more extended recombinant proteins (XTEN), resulting in a CFXTEN fusion protein composition. As used herein, “CF” refers to factor V111 (FVIII) or mimetics, sequence variants and truncated versions of FVIII, as described below.
“Factor VIII” or “FVIII” or “FVIII protein” means a blood coagulation factor protein and species (including human, porcine, canine, rat or murine FVIII proteins) and sequence variants thereof that includes, but is not limited to the 2351 amino acid single-chain precursor protein (with a 19-amino acid hydrophobic signal peptide), the mature 2332 amino acid factor VIII cofactor protein of approximately 270-330 kDa with the domain structure A1-A2-B-A3-C1-C2, as well as the nonenzymatic “active” or cofactor form of FVIII (FVIIIa) that is a circulating heterodimer of two chains that form as a result of proteolytic cleavage after R1648 of a heavy chain form composed of A1-A2-B (in the range of 90-220 kD) of amino acids 1-1648 (numbered relative to the mature FVIII form) and a light chain A3-C1-C2 of 80 kDa of amino acids 1649-2232, each of which is depicted schematically in FIG. 1 . Further, and as used herein, each of A1, A2 and the A3 domain encompasses acidic spacer regions; a1, a2, and a3 acidic regions, respectively. Thus, it will be understood that CFXTEN constructs described as having A1, A2, A3, B, C1 and C2 domains include the a1, a2 and a3 acidic regions. As used herein, “Factor VIII” or “FVIII” or “FVIII polypeptide” also includes variant forms, including proteins with substitutions, additions and/or deletions so long as the variant retains a desired biological activity such as procoagulant activity. Myriad functional FVIII variants have been constructed and can be used as recombinant FVIII proteins as described herein. See PCT Publication Nos. WO 2011/069164 A2, WO 2012/006623 A2, WO 2012/006635 A2, or WO 2012/006633 A2, all of which are incorporated herein by reference in their entireties. A great many functional FVIII variants are known. In addition, hundreds of nonfunctional mutations in FVIII have been identified in hemophilia patients. See, e.g., Cutler et al., Hum. Mutat. 19:274-8 (2002), incorporated herein by reference in its entirety. In addition, comparisons between FVIII from humans and other species have identified conserved residues that are likely to be required for function. See, e.g., Cameron et al., Thromb. Haemost. 79:317-22 (1998) and U.S. Pat. No. 6,251,632, incorporated herein by reference in their entireties.
In one embodiment, the human factor VIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; B, residues Ser741-Arg1648; A3, residues Ser1649-Asn2019; C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1649-Tyr2332. In another embodiment, residues Arg336-Arg372 is usually referred to as the a1 region, and the Arg372 is cleaved by thrombin. In certain embodiments, the a2 region is part of the A1 domain. In another embodiment, residues Glu1649-Arg1689, is referred to as the a3 acidic region. In certain embodiments, the a3 acidic region is a part of the A3 domain. In another embodiment, a native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are the structurally-related “A domains,” B is the “B domain,” C1 and C2 are the structurally-related “C domains,” and a1, a2 and a3 are acidic spacer regions. In the foregoing formula and referring to the primary amino acid sequence position in FIG. 30 , the A1 domain of human FVIII extends from Ala1 to about Arg336, the a1 spacer region extends from about Met337 to about Arg372, the A2 domain extends from about Ser373 to about Tyr719, the a2 spacer region extends from about Glu720 to about Arg740, the B domain extends from about Ser741 to about Arg 1648, the a3 spacer region extends from about Glu1649 to about Arg1689, the A3 domain extends from about Ser1690 to about Asn2019, the C1 domain extends from about Lys2020 to about Asn2172, and the C2 domain extends from about Ser2173 to Tyr2332 (Saenko et al., 2005, J Thromb Hemostasis, 1, 922-930). Other than specific proteolytic cleavage sites, designation of the locations of the boundaries between the domains and regions of FVIII can vary in different literature references. The boundaries noted herein are therefore designated as approximate by use of the term “about.”
Such factor VIII include truncated sequences such as B-domain deleted “BDD” sequences in which a portion or the majority of the B domain sequence is deleted (such as BDD sequences disclosed or referenced in U.S. Pat. Nos. 6,818,439 and 7,632,921). An example of a BDD FVIII is REFACTO® or XYNTHA® (recombinant BDD FVIII), which comprises a first polypeptide corresponding to amino acids 1 to 743 of FIG. 30 , fused to a second polypeptide corresponding to amino acids 1638 to 2332 of FIG. 30 . Exemplary BDD FVIII constructs which can be used to produce recombinant proteins of the invention include, but are not limited to FVIII with a deletion of amino acids corresponding to amino acids 747-1638 of mature human FVIII ( FIG. 30 ) (Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety), and FVIII with a deletion of amino acids corresponding to amino acids 771-1666 or amino acids 868-1562 of mature human FVIII ( FIG. 30 ) (Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety).
In addition, sequences that include heterologous amino acid insertions or substitutions (such as aspartic acid substituted for valine at position 75), or single chain FVIII (scFVIII) in which the heavy and light chains are covalently connected by a linker. As used herein, “FVIII” shall be any functional form of factor VIII molecule with the typical characteristics of blood coagulation factor VIII capable of correcting human factor VIII deficiencies when administered to such a subject, e.g., a subject with hemophilia A. FVIII or sequence variants have been isolated, characterized, and cloned, as described in U.S. Pat. Nos. 4,757,006; 4,965,199; 5,004,804; 5,198,349, 5,250,421; 5,919,766; 6,228,620; 6,818,439; 7,138,505; 7,632,921; and 20100081615.
Human factor VIII is encoded by a single-copy gene residing at the tip of the long arm of the X chromosome (q28). It comprises nearly 186,000 base pairs (bp) and constitutes approximately 0.1% of the X-chromosome (White, G. C. and Shoemaker, C. B., Blood (1989) 73:1-12). The human FVIII amino acid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199, which is incorporated herein by reference in its entirety. Native mature human FVIII derived from the cDNA sequence (i.e., without the secretory signal peptide but prior to other post-translational processing) is presented as FIG. 3 .
The DNA encoding the mature factor VIII mRNA is found in 26 separate exons ranging in size from 69 to 3,106 bp. The 25 intervening intron regions that separate the exons range in size from 207 to 32,400 bp. The complete gene consists of approximately 9 kb of exon and 177 kb of intron. The three repeat A domains have approximately 30% sequence homology. The B domain contains 19 of the approximately 25 predicted glycosylation sites, and the A3 domain is believed to contain a binding site for the von Willebrand factor. The tandem C domains follow the A3 domain and have approximately 37% homology to each other (White, G. C. and Shoemaker, C. B., Blood (1989) 73:1-12).
The B domain separates the A2 and A3 domains of native factor FVIII in the newly synthesized precursor single-chain molecule. The precise boundaries of the B domain have been variously reported as extending from amino acids 712 to 1648 of the precursor sequence (Wood et al., Nature (1984) 312:330-337) or amino acids 741-1648 (Pipe, S W, Haemophilia (2009) 15:1187-1196 and U.S. Pat. No. 7,560,107) or amino acids 740-1689 (Toole, J J. Proc. Natl. Acad. Sci. USA (1986) 83:5939-5942). As used herein, “B domain” means amino acids 741-1648 of mature factor VIII. As used herein, “FVIII B domain deletion” or “FVIII BDD” means a FVIII sequence with any, a fragment of, or all of amino acids 741 to 1648 deleted. In one embodiment, FVIII BDD variants retain remnant amino acids of the B domain from the N-terminal end (“B1” as used herein) and C-terminal end (“B2” as used herein). In one FVIII BDD variant, the B domain remnant amino acids are SFSQNPPVLKRHQR (SEQ ID NO: 1614). In one FVIII BDD variant, the B1 remnant is SFS and the B2 remnant is QNPPVLKRHQR (SEQ ID NO: 1615). In another FVIII BDD variant, the B1 remnant is SFSQN (SEQ ID NO: 1616) and the B2 remnant is PPVLKRHQR (SEQ ID NO: 1617). A “B-domain-deleted factor VIII,” “FVIII BDD,” or “BDD FVIII” may have the full or partial deletions disclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of which is incorporated herein by reference in its entirety. In some embodiments, a B-domain-deleted factor VIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No. 6,346,513). In another embodiment, a B-domain deleted factor VIII is the 5743/Q1638 B-domain deleted factor V111 (SQ version factor VIII) (e.g., factor VIII having a deletion from amino acid 744 to amino acid 1637, e.g., factor VIII having amino acids 1-743 and amino acids 1638-2332 of full-length factor VIII). In some embodiments, a B-domain-deleted factor VIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620). In some embodiments, a B-domain-deleted factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2 , and example 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments, a B-domain-deleted factor VIII has a deletion of most of the B domain, but still contains amino-terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain, as disclosed in WO 91/09122, which is incorporated herein by reference in its entirety. In some embodiments, a B-domain-deleted factor VIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety. A B-domain-deleted factor VIII may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of factor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety. Additional B domain deletions that are part of the invention include: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 though 1648 (Pasek (PCT application No. 88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the foregoing deletions may be made in any factor VIII sequence utilized in the embodiments of the present invention.
Proteins involved in clotting include factor I, factor II, factor III, factor IV, factor V, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, Protein C, and tissue factor (collectively or individually “clotting protein(s)”). The interaction of the major clotting proteins in the intrinsic and extrinsic clotting pathways is showed in FIG. 2 . The majority of the clotting proteins are present in zymogen form, but when activated, exhibit a procoagulant protease activity in which they activate another of the clotting proteins, contributing to the intrinsic or extrinsic coagulation pathway and clot formation. In the intrinsic pathway of the coagulation cascade, FVIII associates with a complex of activated factor IX, factor X, calcium, and phospholipid. The factor VIII heterodimer has no enzymatic activity, but the heterodimer becomes active as a cofactor of the enzyme factor IXa after proteolytic activation by thrombin or factor Xa, with the activity of factor VIIIa characterized by its ability to form a membrane binding site for factors IXa and X in a conformation suitable for activation of the factor X by factor IXa. Upon cleavage by thrombin, activated FVIII (FVIIIa) dissociates from von Willebrand factor and binds to negatively charged phospholipid PL, and the resulting complex participates as a cofactor to factor IXa in the factor X activating (tenase) complex. Within the C2 domain and amino acid residues 1649 through 1689 in the A3 domain are von Willebrand factor (vWF) binding sites that act to complex with von Willebrand factor, the resulting circulating complex protects FVIII from rapid degradation in the blood (Weiss H J, et al. Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand's disease. J Clin Invest (1977) 60:390).
Activated factor VIII is a heterotrimer comprised of the A1 domain and the A2 domain and the light chain including domains A3-C1-C2. The activation of factor IX is achieved by a two-step removal of the activation peptide (Ala 146-Arg 180) from the molecule (Bajaj et al., Human factor 1× and factor IXa, in METHODS IN ENZYMOLOGY. 1993). The first cleavage is made at the Arg 145-Ala 146 site by either factor XIa or factor VIIa/tissue factor. The second, and rate limiting cleavage is made at Arg 180-Val 181. The activation removes 35 residues. Activated human factor IX exists as a heterodimer of the C-terminal heavy chain (28 kDa) and an N-terminal light chain (18 kDa), which are held together by one disulfide bridge attaching the enzyme to the Gla domain. Factor IXa in turn activates factor X in concert with activated factor VIII. Alternatively, factors 1× and X can both be activated by factor VIIa complexed with lipidated tissue factor, generated via the extrinsic pathway. Factor Xa then participates in the final common pathway whereby prothrombin is converted to thrombin, and thrombin, in turn converts fibrinogen to fibrin to form the clot.
Defects in the coagulation process can lead to bleeding disorders (coagulopathies) in which the time taken for clot formation is prolonged. Such defects can be congenital or acquired. For example, hemophilia A and B are inherited diseases characterized by deficiencies in FVIII and FIX, respectively. Stated differently, biologically active factor VIII corrects the coagulation defect in plasma derived from individuals afflicted with hemophilia A. Recombinant FVIII has been shown to be effective and has been approved for the treatment of hemophilia A in adult and pediatric patients, and also is used to stop bleeding episodes or prevent bleeding associated with trauma and/or surgery. Current therapeutic uses of factor VIII can be problematic in the treatment of individuals exhibiting a deficiency in factor VIII, as well as those individuals with Von Willebrand's disease. In addition, individuals receiving factor VIII in replacement therapy frequently develop antibodies to these proteins that often reduce or eliminate the procoagulant activity of the bound FVIII. Continuing treatment is exceedingly difficult because of the presence of these antibodies that reduce or negate the efficacy of the treatment.
In one aspect, the invention contemplates inclusion of FVIII sequences in the CFXTEN fusion protein compositions that are identical to human FVIII, sequences that have homology to FVIII sequences, sequences that are natural, such as from humans, non-human primates, mammals (including domestic animals), or truncated version of FVIII; all of which retain at least a portion of the procoagulant activity of native FVIII and that are useful for preventing, treating, mediating, or ameliorating hemophilia
A or bleeding episodes related to trauma, surgery, or deficiency of coagulation factor VIII. Sequences with homology to FVIII may be found by standard homology searching techniques, such as NCBI BLAST, or in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, The Universal Protein Resource (UniProt) and subscription provided databases such as GenSeq (e.g., Derwent).
In one embodiment, the FVIII incorporated into the subject CFXTEN compositions is a recombinant polypeptide with a sequence corresponding to a FVIII protein found in nature. In another embodiment, the FVIII is a non-natural FVIII sequence variant, fragment, homolog, or a mimetic of a natural sequence that retains at least a portion of the procoagulant activity of the corresponding native FVIII. In another embodiment, the FVIII is a truncated variant with all or a portion of the B domain deleted (“FVIII BDD”), which can be in either heterodimeric form or can remain as a single chain (“scFVIII”), the latter described in Meulien et al., Protein Eng. (1988) 2(4):301-306. Non-limiting examples of FVIII BDD are factor VIII sequences in which the amino acids are deleted between residue number 741 and residue number 1640 (numbered relative to native, mature FVIII), or between residue number 745 and residue number 1640, or between residue number 745 and residue number 1640, or between residue number 741 and residue number 1690, or between residue number 745 and residue number 1667, or between residue number 745 and residue number 1657, or between residue number 747 and residue number 1642, or between residue number 751 and residue number 1667.
In another embodiment, heterologous sequences are incorporated into the FVIII, which may include XTEN, as described more fully below. Table 1 provides a non-limiting list of amino acid sequences of FVIII that are encompassed by the CFXTEN fusion proteins of the invention. In some embodiments, FVIII incorporated into CFXTEN fusion proteins include proteins that have at least about 70% sequence identity, or alternatively 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an amino acid sequence of comparable length selected from Table 1.





TABLE 1



FVIII amino acid sequences







SEQ

Name

ID

(source)
Amino Acid Sequence
NO:







FVIII
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK
1

precursor
SFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM


polypeptide
ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN


(human)
GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF



AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS



VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL



FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD



DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG



PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR



PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR



CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR



SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY



ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN



SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPS



TRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLS



DLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNE



KLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQL



DTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLF



KGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVW



QNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKE



GPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSV



EGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEK



KIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVL



QDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQ



QNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNE



KEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPA



ASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTY



KKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQ



GTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWK



SQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCS



QNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQK



KTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPL



YRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPR



KNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLL



VCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPT



FKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR



KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNK



CQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDL



LAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD



SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDA



QITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVT



GVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVN



SLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
2

mature
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS


(human)
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD



PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAID



SNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST



SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSL



SEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFK



VSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR



MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFL



PESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEF



TKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLP



QIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTA



HFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPL



EETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHS



IPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQG



AKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKV



ELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFL



RVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLN



ACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSD



QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS



SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE



DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH



MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEF



ALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGL



VMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETV



EMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASG



QYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL



YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHP



THYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKA



RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF



LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH



QIALRMEVLGCEAQDLY




FVIII
MQVELYTCCFLCLLPFSLSATRKYYLGAVELSWDYMQSDLLSALHADTSFSSRVP
3

(Canine)
GSLPLTTSVTYRKTVFVEFTDDLFNIAKPRPPWMGLLGPTIQAEVYDTVVIVLKN



MASHPVSLHAVGVSYWKASEGAEYEDQTSQKEKEDDNVIPGESHTYVWQVLKE



NGPMASDPPCLTYSYFSHVDLVKDLNSGLIGALLVCKEGSLAKERTQTLQEFVLL



FAVFDEGKSWHSETNASLTQAEAQHELHTINGYVNRSLPGLTVCHKRSVYWHVI



GMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTFLMDLGQFLLFCHIPSH



QHDGMEAYVKVDSCPEEPQLRMKNNEDKDYDDGLYDSDMDVVSFDDDSSSPFI



QIRSVAKKHPKTWVHYIAAEEEDWDYAPSGPTPNDRSHKNLYLNNGPQRIGKKY



KKVRFVAYTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI



NYVTPLHTGRLPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSS



FINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENRSWYLTEN



MQRFLPNADVVQPHDPEFQLSNIMHSINGYVFDNLQLSVCLHEVAYWYILSVGA



QTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFR



NRGMTALLKVSSCNRNIDDYYEDTYEDIPTPLLNENNVIKPRSFSQNSRHPSTKEK



QLKATTTPENDIEKIDLQSGERTQLIKAQSVSSSDLLMLLGQNPTPRGLFLSDLREA



TDRADDHSRGAIERNKGPPEVASLRPELRHSEDREFTPEPELQLRLNENLGTNTTV



ELKKLDLKISSSSDSLMTSPTIPSDKLAAATEKTGSLGPPNMSVHFNSHLGTIVFGN



NSSHLIQSGVPLELSEEDNDSKLLEAPLMNIQESSLRENVLSMESNRLFKEERIRGP



ASLIKDNALFKVNISSVKTNRAPVNLTTNRKTRVAIPTLLIENSTSVWQDIMLERN



TEFKEVTSLIHNETFMDRNTTALGLNHVSNKTTLSKNVEMAHQKKEDPVPLRAE



NPDLSSSKIPFLPDWIKTHGKNSLSSEQRPSPKQLTSLGSEKSVKDQNFLSEEKVVV



GEDEFTKDTELQEIFPNNKSIFFANLANVQENDTYNQEKKSPEEIERKEKLTQENV



ALPQAHTMIGTKNFLKNLFLLSTKQNVAGLEEQPYTPILQDTRSLNDSPHSEGIHM



ANFSKIREEANLEGLGNQTNQMVERFPSTTRMSSNASQHVITQRGKRSLKQPRLS



QGEIKFERKVIANDTSTQWSKNMNYLAQGTLTQIEYNEKEKRAITQSPLSDCSMR



NHVTIQMNDSALPVAKESASPSVRHTDLTKIPSQHNSSHLPASACNYTFRERTSGV



QEGSHFLQEAKRNNLSLAFVTLGITEGQGKFSSLGKSATNQPMYKKLENTVLLQP



GLSETSDKVELLSQVHVDQEDSFPTKTSNDSPGHLDLMGKIFLQKTQGPVKMNK



TNSPGKVPFLKWATESSEKIPSKLLGVLAWDNHYDTQIPSEEWKSQKKSQTNTAF



KRKDTILPLGPCENNDSTAAINEGQDKPQREAMWAKQGEPGRLCSQNPPVSKHH



QREITVTTLQPEEDKFEYDDTFSIEMKREDFDIYGDYENQGLRSFQKKTRHYFIAA



VERLWDYGMSRSPHILRNRAQSGDVQQFKKVVFQEFTDGSFTQPLYRGELNEHL



GLLGPYIRAEVEDNIVVTFKNQASRPYSFYSSLISYDEDEGQGAEPRRKFVNPNET



KIYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRSNTLNPA



HGRQVTVQEFALVFTIFDETKSWYFTENLERNCRAPCNVQKEDPTLKENFRFHAI



NGYVKDTLPGLVMAQDQKVRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMA



VYNLYPGVFETVEMLPSQVGIWRIECLIGEHLQAGMSTLFLVYSKKCQTPLGMAS



GHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSWIKVDLLAPMIIHGI



MTQGARQKFSSLYVSQFIIMYSLDGNKWHSYRGNSTGTLMVFFGNVDSSGIKHNI



FNPPIIAQYIRLHPTHYSIRSTLRMELLGCDFNSCSMPLGMESKAISDAQITASSYLS



SMLATWSPSQARLHLQGRTNAWRPQANNPKEWLQVDFRKTMKVTGITTQGVKS



LLISMYVKEFLISSSQDGHNWTLFLQNGKVKVFQGNRDSSTPVRNRLEPPLVARY



VR LHPQSWAHHIALRLEVLGCDTQQPA




FVIII (Pig)
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
4


DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD



PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAID



SNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST



SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSL



SEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFK



VSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR



MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFL



PESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEF



TKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLP



QIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTA



HFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPL



EETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHS



IPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQG



AKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKV



ELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFL



RVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLN



ACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSD



QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS



SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE



DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH



MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEF



ALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGL



VMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETV



EMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASG



QYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL



YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHP



THYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKA



RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF



LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH



QIALRMEVLGCEAQDLY




FVIII
AIRRYYLGAVELSWNYIQSDLLSVLHTDSRFLPRMSTSFPFNTSIMYKKTVFVEYK
5

(Mouse)
DQLFNIAKPRPPWMGLLGPTIWTEVHDTVVITLKNMASHPVSLHAVGVSYWKAS



EGDEYEDQTSQMEKEDDKVFPGESHTYVWQVLKENGPMASDPPCLTYSYMSHV



DLVKDLNSGLIGALLVCKEGSLSKERTQMLYQFVLLFAVFDEGKSWHSETNDSY



TQSMDSASARDWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEIHSIF



LEGHTFFVRNHRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKV



DSCPEESQWQKKNNNEEMEDYDDDLYSEMDMFTLDYDSSPFIQIRSVAKKYPKT



WIHYISAEEEDWDYAPSVPTSDNGSYKSQYLSNGPHRIGRKYKKVRFIAYTDETF



KTRETIQHESGLLGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVSPLHARRLPR



GIKHVKDLPIHPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINPERDLASGLIGP



LLICYKESVDQRGNQMMSDKRNVILFSIFDENQSWYITENMQRFLPNAAKTQPQD



PGFQASNIMHSINGYVFDSLELTVCLHEVAYWHILSVGAQTDFLSIFFSGYTFKHK



MVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFRKRGMTALLKVSSCDKST



SDYYEEIYEDIPTQLVNENNVIDPRSFFQNTNHPNTRKKKFKDSTIPKNDMEKIEPQ



FEEIAEMLKVQSVSVSDMLMLLGQSHPTPHGLFLSDGQEAIYEAIHDDHSPNAIDS



NEGPSKVTQLRPESHHSEKIVFTPQPGLQLRSNKSLETTIEVKWKKLGLQVSSLPS



NLMTTTILSDNLKATFEKTDSSGFPDMPVHSSSKLSTTAFGKKAYSLVGSHVPLN



ASEENSDSNILDSTLMYSQESLPRDNILSIENDRLLREKRFHGIALLTKDNTLFKDN



VSLMKTNKTYNHSTTNEKLHTESPTSIENSTTDLQDAILKVNSEIQEVTALIHDGT



LLGKNSTYLRLNHMLNRTTSTKNKDIFHRKDEDPIPQDEENTIMPFSKMLFLSESS



NWFKKTNGNNSLNSEQEHSPKQLVYLMFKKYVKNQSFLSEKNKVTVEQDGFTK



NIGLKDMAFPHNMSIFLTTLSNVHENGRHNQEKNIQEEIEKEALIEEKVVLPQVHE



ATGSKNFLKDILILGTRQNISLYEVHVPVLQNITSINNSTNTVQIHMEHFFKRRKDK



ETNSEGLVNKTREMVKNYPSQKNITTQRSKRALGQFRLSTQWLKTINCSTQCIIKQ



IDHSKEMKKFITKSSLSDSSVIKSTTQTNSSDSHIVKTSAFPPIDLKRSPFQNKFHV



QASSYIYDFKTKSSRIQESNNFLKETKINNPSLAILPWNMFIDQGKFTSPGKSNTNS



VTYKKRENIIFLKPTLPEESGKIELLPQVSIQEEEILPTETSHGSPGHLNLMKEVFLQ



KIQGPTKWNKAKRHGESIKGKTESSKNTRSKLLNHHAWDYHYAAQIPKDMWKS



KEKSPEIISIKQEDTILSLRPHGNSHSIGANEKQNWPQRETTWVKQGQTQRTCSQIP



PVLKRHQRELSAFQSEQEATDYDDAITIETIEDFDIYSEDIKQGPRSFQQKTRHYFI



AAVERLWDYGMSTSHVLRNRYQSDNVPQFKKVVFQEFTDGSFSQPLYRGELNEH



LGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYKEDQRGEEPRRNFVKPNET



KIYFWKVQHHMAPTEDEFDCKAWAYFSDVDLERDMHSGLIGPLLICHANTLNPA



HGRQVSVQEFALLFTIFDETKSWYFTENVKRNCKTPCNFQMEDPTLKENYRFHAI



NGYVMDTLPGLVMAQDQRIRWYLLSMGNNENIQSIHFSGHVFTVRKKEEYKMA



VYNLYPGVFETLEMIPSRAGIWRVECLIGEHLQAGMSTLFLVYSKQCQIPLGMAS



GSIRDFQITASGHYGQWAPNLARLHYSGSINAWSTKEPFSWIKVDLLAPMIVHGIK



TQGARQKFSSLYISQFIIMYSLDGKKWLSYQGNSTGTLMVFFGNVDSSGIKHNSF



NPPIIARYIRLHPTHSSIRSTLRMELMGCDLNSCSIPLGMESKVISDTQITASSYFTN



MFATWSPSQARLHLQGRTNAWRPQVNDPKQWLQVDLQKTMKVTGIITQGVKSL



FTSMFVKEFLISSSQDGHHWTQILYNGKVKVFQGNQDSSTPMMNSLDPPLLTRYL



RIHPQIWEHQIALRLEILGCEAQQQY




FVIII BDD
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK
6

variant
SFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM


(U.S. Pat.
ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN


No.
GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF


7632921,
AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS


SEQ ID
VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL


NO: 3)
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD



DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG



PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR



PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR



CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR



SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY



ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN



SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL



KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF



IAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE



HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN



ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTL



NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF



HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK



MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG



MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII



HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIK



HNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS



YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ



GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPL



LTRYLRIHPQSWVHQIALRMEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
7

BDD-2
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY



DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR



NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT



FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD



EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF



DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD



QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK



AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW



APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII



MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR



STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ



GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ



DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR



MEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
8

BDD-3
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS


(G1648)
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDY



DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR



NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT



FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD



EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF



DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD



QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK



AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW



APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII



MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR



STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ



GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ



DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR



MEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
9

BDD-4
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQQSPRSFQKKTRHYFIAAVERLWDY



GMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIR



AEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV



QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVT



VQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD



TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG



VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ



ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQ



KFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI



RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWS



PSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMY



VKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS



WVHQIALRMEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
10

BDD-5
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT



DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEED



QRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV



HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAP



CNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIH



FSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS



TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKE



PFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT



LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG



MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD



FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGN



QDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
11

BDD-6
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD



TISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR



AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFR



NQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF



DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDE



TKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR



IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAG



IWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAP



KLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMY



SLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTL



RMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRS



NAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH



QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEV



LGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
12

BDD-7
VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQSPRSFQKKTRHYFIAAVERLWDYG



MSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRA



EVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQ



HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTV



QEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTL



PGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVF



ETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQIT



ASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF



SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL



HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS



KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVK



EFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSW



VHQIALRMEVLGCEAQDLY




FVIII
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK
13

BDD-8
SFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM


precursor
ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN


(U.S. Pat.
GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF


No.
AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS


6818439
VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL


SEQ ID
FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD


NO: 47)
DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG



PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR



PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR



CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR



SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY



ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN



SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL



KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF



IAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE



HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN



ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTL



NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF



HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK



MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG



MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII



HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIK



HNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS



YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ



GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPL



LTRYLRIHPQSWVHQIALRMEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
14

BDD-9
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS


mature
EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV


(U.S. Pat.
DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM


No.
QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL


6818439)
EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY



DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR



NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT



FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD



EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF



DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD



QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK



AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW



APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII



MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR



STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ



GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ



DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR



MEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
15

BDD-10
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDY



DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR



NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT



FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD



EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF



DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD



QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK



AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW



APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII



MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR



STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ



GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ



DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR



MEVLGCEAQDLY




FVIII
ATRATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF
16

BDD-11
VEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSY



WKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSY



LSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETK



NSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEV



HSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAY



VKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK



HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMA



YTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYS



RRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDL



ASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPA



GVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSG



YTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS



SCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQ



EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSS



PHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVED



NIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM



APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFA



LFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLV



MAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVE



MLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ



YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI



SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTH



YSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL



HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS



SSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA



LRMEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
17

BDD-12
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDY



DDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR



NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT



FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD



EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF



DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD



QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK



AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW



APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII



MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR



STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ



GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ



DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR



MEVLGCEAQDLY




FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT
18

BDD-13
DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS



EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV



DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM



QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL



EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV



DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT



WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE



TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP



KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL



IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL



EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK



HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK



NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY



DDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR



NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT



FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD



EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF



DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD



QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK



AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW



APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII



MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR



STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ



GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ



DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR



MEVLGCEAQDLY



























































































































































































































































































































































































































































































































































































































The present invention also contemplates CFXTEN comprising FVIII with various amino acid deletions, insertions and substitutions made in the FVIII sequences of Table 1 that retain procoagulant activity. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 2. In embodiments of the CFXTEN in which the sequence identity of the FVIII is less than 100% compared to a specific sequence disclosed herein, the invention contemplates substitution of any of the other 19 natural L-amino acids for a given amino acid residue of the given FVIII, which may be at any position within the sequence of the FVIII, including adjacent amino acid residues. If any one substitution results in an undesirable change in procoagulant activity, then one of the alternative amino acids can be employed and the construct protein evaluated by the methods described herein (e.g., the assays of Table 49), or using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934, the content of which is incorporated by reference in its entirety, or using methods generally known in the art. In a preferred substitution, the FVIII component of the CFXTEN embodiments is modified by replacing the R1648 residue (numbered relative to the native mature form of FVIII) with glycine or alanine to prevent proteolytic processing to the heterodimer form. In another substitution, the FVIII component of the CFXTEN embodiments is modified by replacing the Y1680 residue (numbered relative to the native mature form of FVIII) with phenylalanine. In another embodiment, the FVIII component of the CFXTEN embodiments is modified by replacing the Y1680 residue (numbered relative to the native mature form of FVIII) with phenylalanine and the R1648 residue (numbered relative to the native mature form of FVIII) with glycine or alanine.
In one embodiment, the FVIII of the fusion protein composition has one or more amino acid substitutions designed to reduce the binding of FVIII inhibitors at epitopes recognized by the antibodies of Table 9, including but not limited to substitutions at Lys(377), Lys(466), Lys(380), Ser(488), Arg(489), Arg(490), Leu(491), Lys(493), Lys(496), His(497), Lys(499), Lys(512), Lys(523), Lys(556), Met (2199), Phe(2200), Leu(2252), Val(2223), and Lys(2227). In addition, variants can include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at or near the N- or C-terminus of the full-length native amino acid sequence or of a domain of a FVIII so long as the variant retains some if not all of the procoagulant activity of the native peptide. The resulting FVIII sequences that retain at least a portion (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% or more) of the procoagulant activity in comparison to native circulating FVIII are considered useful for the fusion protein compositions of this invention. Examples of FVIII variants are known in the art, including those described in U.S. Pat. Nos. 6,316,226; 6,818,439; 7,632,921; 20080227691, which are incorporated herein by reference. In one embodiment, a FVIII sequence variant has an aspartic acid substituted for valine at amino acid position 75 (numbered relative to the native mature form of FVIII).





TABLE 2



Exemplary conservative amino acid substitutions






Original Residue
Exemplary Substitutions





Ala (A)
val; leu; ile


Arg (R)
lys; gln; asn


Asn (N)
gin; his; lys; arg


Asp (D)
Glu


Cys (C)
Ser


Gln (Q)
Asn


Glu (E)
Asp


Gly (G)
Pro


His (H)
asn: gin: lys: arg


Ile (I)
leu; val; met; ala; phe: norleucine


Leu (L)
norleucine: ile: val; met; ala: phe


Lys (K)
arg: gin: asn


Met (M)
leu; phe; ile


Phe (F)
leu: val: ile; ala


Pro (P)
Gly


Ser (S)
Thr


Thr (T)
Ser


Trp (W)
Tyr


Tyr (Y)
Trp: phe: thr: ser


Val (V)
Ile; leu; met; phe; ala; norleucine









































III). Extended Recombinant Polypeptides
In one aspect, the invention provides XTEN polypeptide compositions that are useful as fusion protein partner(s) to link to and/or incorporate within a FVIII polypeptide, resulting in a CFXTEN fusion protein. XTEN are generally polypeptides with non-naturally occurring, substantially non-repetitive sequences having a low degree of or no secondary or tertiary structure under physiologic conditions. XTEN typically have from about 36 to about 3000 amino acids of which the majority or the entirety are small hydrophilic amino acids. As used herein, “XTEN” specifically excludes whole antibodies or antibody fragments (e.g. single-chain antibodies and Fc fragments). XTEN polypeptides have utility as a fusion protein partners in that they serve various roles, conferring certain desirable pharmacokinetic, physicochemical, pharmacologic, and pharmaceutical properties when linked to a FVIII protein to a create a CFXTEN fusion protein. Such CFXTEN fusion protein compositions have enhanced properties compared to the corresponding FVIII not linked to XTEN, making them useful in the treatment of certain conditions related to FVIII deficiencies or bleeding disorders, as more fully described below.
The selection criteria for the XTEN to be fused to the FVIII proteins used to create the inventive fusion proteins compositions generally relate to attributes of physical/chemical properties and conformational structure of the XTEN that is, in turn, used to confer enhanced pharmaceutical, pharmacologic, and pharmacokinetic properties to the FVIII fusion proteins compositions. The unstructured characteristic and physical/chemical properties of the XTEN result, in part, from the overall amino acid composition disproportionately limited to 4-6 hydrophilic amino acids, the linking of the amino acids in a quantifiable non-repetitive design, and the length of the XTEN polypeptide. In an advantageous feature common to XTEN but uncommon to polypeptides, the properties of XTEN disclosed herein are not tied to absolute primary amino acid sequences, as evidenced by the diversity of the exemplary sequences of Table 4 that, within varying ranges of length, possess similar properties, many of which are documented in the Examples. The XTEN of the present invention may exhibit one or more, or all of the following advantageous properties: unstructured conformation, conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, a defined degree of charge, and increased hydrodynamic (or Stokes) radii; properties that can make them particularly useful as fusion protein partners. Non-limiting examples of the enhanced properties that XTEN confer on the fusion proteins comprising FVIII fused to XTEN, compared to FVIII not linked to XTEN, include increases in the overall solubility and/or metabolic stability, reduced susceptibility to proteolysis, reduced immunogenicity, reduced rate of absorption when administered subcutaneously or intramuscularly, reduced binding to FVIII clearance receptors, reduced reactivity to anti-payload antibodies, enhanced interactions with substrate, and/or enhanced pharmacokinetic properties when administered to a subject. The enhanced pharmacokinetic properties of the CFXTEN compositions compared to FVIII not linked to XTEN include longer terminal half-life (e.g., two-fold, three-fold, four-fold or more), increased area under the curve (AUC) (e.g., 25%, 50%, 100% or more), lower volume of distribution, and enhanced absorption after subcutaneous or intramuscular injection (an advantage compared to commercially-available forms of FVIII that must be administered intravenously). In addition, it is believed that the CFXTEN compositions comprising cleavage sequences (described more fully, below) permit sustained release of biologically active FVIII, such that the administered CFXTEN acts as a depot. It is specifically contemplated that the inventive CFXTEN fusion proteins can exhibit one or more or any combination of the improved properties disclosed herein. As a result of these enhanced properties, it is believed that CFXTEN compositions permit less frequent dosing compared to FVIII not linked to XTEN when administered at comparable dosages. Such CFXTEN fusion protein compositions have utility to treat certain factor VIII-related conditions, as described herein.
A variety of methods and assays are known in the art for determining the physical/chemical properties of proteins such as the CFXTEN compositions comprising XTEN. Such properties include but are not limited to secondary or tertiary structure, solubility, protein aggregation, stability, absolute and apparent molecular weight, purity and uniformity, melting properties, contamination and water content. Methods to assay these properties include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.
The XTEN component(s) of the CFXTEN are designed to behave like denatured peptide sequences under physiological conditions, despite the extended length of the polymer. “Denatured” describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperature. Peptides in denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by a lack of long-range interactions as determined by NMR. “Denatured conformation” and “unstructured conformation” are used synonymously herein. In some embodiments, the invention provides XTEN sequences that, under physiologic conditions, are largely devoid of secondary structure. In other cases, the XTEN sequences are substantially devoid of secondary structure under physiologic conditions such that the XTEN can adopt random coil conformation. “Largely devoid,” as used in this context, means that at least 50% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure as measured or determined by the means described herein. “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure, as measured or determined by the methods described herein.
A variety of methods have been established in the art to discern the presence or absence of secondary and tertiary structures in a given polypeptide. In particular, secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra, as does the lack of these structure elements. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe-Robson (“GOR”) algorithm (Garnier J, Gibrat J F, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553), as described in US Patent Application Publication No. 20030228309A1. For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as the total and/or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure).
In one embodiment, the XTEN sequences used in the subject fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In another embodiment, the XTEN sequences of the fusion protein compositions have a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. The XTEN sequences of the fusion protein compositions have a high degree of random coil percentage, as determined by the GOR algorithm. In some embodiments, an XTEN sequence have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and most preferably at least about 99% random coil, as determined by the GOR algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm and at least about 90% random coil, as determined by the GOR algorithm. In other embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2% at least about 90% random coil, as determined by the GOR algorithm.
1. Non-Repetitive Sequences
It is contemplated that the XTEN sequences of the CFXTEN embodiments are substantially non-repetitive. In general, repetitive amino acid sequences have a tendency to aggregate or form higher order structures, as exemplified by natural repetitive sequences such as collagens and leucine zippers. These repetitive amino acids may also tend to form contacts resulting in crystalline or pseudocrystalline structures. In contrast, the low tendency of non-repetitive sequences to aggregate enables the design of long-sequence XTENs with a relatively low frequency of charged amino acids that would otherwise be likely to aggregate if the sequences were repetitive. The non-repetitiveness of a subject XTEN can be observed by assessing one or more of the following features. In one embodiment, a “substantially non-repetitive” XTEN sequence has about 36, or at least 72, or at least 96, or at least 144, or at least 288, or at least 400, or at least 500, or at least 600, or at least 700, or at least 800, or at least 864, or at least 900, or at least 1000, or at least 2000, to about 3000 or more amino acid residues, or has a length ranging from about 36 to about 3000, about 100 to about 500, about 500 to about 1000, about 1000 to about 3000 amino acids and residues, in which no three contiguous amino acids in the sequence are identical amino acid types unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues. In another embodiment, as described more fully below, a “substantially non-repetitive” XTEN sequence comprises motifs of 9 to 14 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.
The degree of repetitiveness of a polypeptide or a gene can be measured by computer programs or algorithms or by other means known in the art. According to the current invention, algorithms to be used in calculating the degree of repetitiveness of a particular polypeptide, such as an XTEN, are disclosed herein, and examples of sequences analyzed by algorithms are provided (see Examples, below). In one aspect, the repetitiveness of a polypeptide of a predetermined length can be calculated (hereinafter “subsequence score”) according to the formula given by Equation 1:







Subsequence





score








∑

i
=
1

m







Count
i


m




I








wherein: m=(amino acid length of polypeptide)-(amino acid length of subsequence)+1; and Count i =cumulative number of occurrences of each unique subsequence within sequence i




An algorithm termed “SegScore” was developed to apply the foregoing equation to quantitate repetitiveness of polypeptides, such as an XTEN, providing the subsequence score wherein sequences of a predetermined amino acid length “n” are analyzed for repetitiveness by determining the number of times (a “count”) a unique subsequence of length “s” appears in the set length, divided by the absolute number of subsequences within the predetermined length of the sequence. FIG. 27 depicts a logic flowchart of the SegScore algorithm, while FIG. 28 portrays a schematic of how a subsequence score is derived for a fictitious XTEN with 11 amino acids and a subsequence length of 3 amino acid residues. For example, a predetermined polypeptide length of 200 amino acid residues has 192 overlapping 9-amino acid subsequences and 198 3-mer subsequences, but the subsequence score of any given polypeptide will depend on the absolute number of unique subsequences and how frequently each unique subsequence (meaning a different amino acid sequence) appears in the predetermined length of the sequence.
In the context of the present invention, “subsequence score” means the sum of occurrences of each unique 3-mer frame across 200 consecutive amino acids of the cumulative XTEN polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from 200 consecutive amino acids of repetitive and non-repetitive polypeptides are presented in Example 45. In one embodiment, the invention provides a CFXTEN comprising one XTEN in which the XTEN has a subsequence score less than 12, more preferably less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5. In another embodiment, the invention provides CFXTEN comprising at least two to about six XTEN in which 200 amino acids of the XTEN have a subsequence score of less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5. In the embodiments of the CFXTEN fusion protein compositions described herein, an XTEN component of a fusion protein with a subsequence score of 10 or less (i.e., 9, 8, 7, etc.) is also substantially non-repetitive.
It is believed that the non-repetitive characteristic of XTEN of the present invention together with the particular types of amino acids that predominate in the XTEN, rather than the absolute primary sequence, confers many of the enhanced physicochemical and biological properties of the CFXTEN fusion proteins. These enhanced properties include a higher degree of expression of the fusion protein in the host cell, greater genetic stability of the gene encoding XTEN, a greater degree of solubility, less tendency to aggregate, and enhanced pharmacokinetics of the resulting CFXTEN compared to fusion proteins comprising polypeptides having repetitive sequences. These enhanced properties permit more efficient manufacturing, lower cost of goods, and facilitate the formulation of XTEN-comprising pharmaceutical preparations containing extremely high protein concentrations, in some cases exceeding 100 mg/ml. Furthermore, the XTEN polypeptide sequences of the embodiments are designed to have a low degree of internal repetitiveness in order to reduce or substantially eliminate immunogenicity when administered to a mammal. Polypeptide sequences composed of short, repeated motifs largely limited to only three amino acids, such as glycine, serine and glutamate, may result in relatively high antibody titers when administered to a mammal despite the absence of predicted T-cell epitopes in these sequences. This may be caused by the repetitive nature of polypeptides, as it has been shown that immunogens with repeated epitopes, including protein aggregates, cross-linked immunogens, and repetitive carbohydrates are highly immunogenic and can, for example, result in the cross-linking of B-cell receptors causing B-cell activation. (Johansson, J., et al. (2007) Vaccine, 25:1676-82; Yankai, Z., et al. (2006) Biochem Biophys Res Commun, 345:1365-71; Hsu, C. T., et al. (2000) Cancer Res, 60:3701-5); Bachmann M F, et al. Eur J. Immunol. (1995) 25(12):3445-3451).
2. Exemplary Sequence Motifs
The present invention encompasses XTEN used as fusion partners that comprise multiple units of shorter sequences, or motifs, in which the amino acid sequences of the motifs are non-repetitive. The non-repetitive property is met despite the use of a “building block” approach using a library of sequence motifs that are multimerized to create the XTEN sequences. Thus, while an XTEN sequence may consist of multiple units of as few as four different types of sequence motifs, because the motifs themselves generally consist of non-repetitive amino acid sequences, the overall XTEN sequence is designed to render the sequence substantially non-repetitive.
In one embodiment, an XTEN has a substantially non-repetitive sequence of greater than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues, or even longer wherein at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs, and wherein each of the motifs has about 9 to 36 amino acid residues. In other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 14 amino acid residues. In still other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues. In these embodiments, it is preferred that the sequence motifs are composed of substantially (e.g., 90% or more) or exclusively small hydrophilic amino acids, such that the overall sequence has an unstructured, flexible characteristic. Examples of amino acids that are included in XTEN are, e.g., arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine. As a result of testing variables such as codon optimization, assembly polynucleotides encoding sequence motifs, expression of protein, charge distribution and solubility of expressed protein, and secondary and tertiary structure, it was discovered that XTEN compositions with the enhanced characteristics disclosed herein mainly or exclusively include glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues wherein the sequences are designed to be substantially non-repetitive. In one embodiment, XTEN sequences have predominately four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P) that are arranged in a substantially non-repetitive sequence that is greater than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues in length. In some embodiment, an XTEN sequence is made of 4, 5, or 6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, XTEN have sequences of greater than about 36 to about 1000, or about 100 to about 2000, or about 400 to about 3000 amino acid residues wherein at least about 80% of the sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues and wherein at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or 100% of each of the motifs consists of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 40%, or about 30%, or 25%, or about 17%. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues consisting of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 40%, or 30%, or about 25%. In yet other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).
In still other embodiments, XTENs comprise substantially non-repetitive sequences of greater than about 36 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the sequence consists of non-overlapping sequence motifs of 9 to 14 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif. In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif. In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs consist of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif. In yet other embodiments, XTENs consist of 12 amino acid sequence motifs wherein the amino acids are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif, and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. The foregoing embodiments are examples of substantially non-repetitive XTEN sequences. Additional examples are detailed below.
In some embodiments, the invention provides CFXTEN compositions comprising one, or two, or three, or four, five, six or more non-repetitive XTEN sequence(s) of about 36 to about 1000 amino acid residues, or cumulatively about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multiple units of four or more non-overlapping sequence motifs selected from the amino acid sequences of Table 3, wherein the overall sequence remains substantially non-repetitive. In some embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units of non-overlapping sequences selected from a single motif family selected from Table 3, resulting in a family sequence. As used herein, “family” means that the XTEN has motifs selected only from a single motif category from Table 3; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in the XTEN not from a family motif are selected to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, or to achieve a better linkage to a FVIII coagulation factor component of the CFXTEN. In some embodiments of XTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or of the AE motif family, or of the AF motif family, or of the AG motif family, or of the AM motif family, or of the AQ motif family, or of the BC family, or of the BD family, with the resulting XTEN exhibiting the range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 3. These sequences can be selected to achieve desired physical/chemical characteristics, including such properties as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred by the amino acid composition of the motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues.





TABLE 3



XTEN Sequence Motifs of 12 Amino Acids and Motif

Families







SEQ



ID

Motif Family
MOTIF SEQUENCE
NO:



AD
GESPGGSSGSES
19



AD
GSEGSSGPGESS
20



AD
GSSESGSSEGGP
21



AD
GSGGEPSESGSS
22



AE, AM
GSPAGSPTSTEE
23



AE, AM, AQ
GSEPATSGSETP
24



AE, AM, AQ
GTSESATPESGP
25



AE, AM, AQ
GTSTEPSEGSAP
26



AF, AM
GSTSESPSGTAP
27



AF, AM
GTSTPESGSASP
28



AF, AM
GTSPSGESSTAP
29



AF, AM
GSTSSTAESPGP
30



AG, AM
GTPGSGTASSSP
31



AG, AM
GSSTPSGATGSP
32



AG, AM
GSSPSASTGTGP
33



AG, AM
GASPGTSSTGSP
34



AQ
GEPAGSPTSTSE
35



AQ
GTGEPSSTPASE
36



AQ
GSGPSTESAPTE
37



AQ
GSETPSGPSETA
38



AQ
GPSETSTSEPGA
39



AQ
GSPSEPTEGTSA
40



BC
GSGASEPTSTEP
41



BC
GSEPATSGTEPS
42



BC
GTSEPSTSEPGA
43



BC
GTSTEPSEPGSA
44



BD
GSTAGSETSTEA
45



BD
GSETATSGSETA
46



BD
GTSESATSESGA
47



BD
GTSTEASEGSAS
48



* Denotes individual motif sequences that, when used together in various permutations, results in a “family sequence”

















































































In some embodiments of XTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, with the resulting XTEN exhibiting the range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 3, selected to achieve desired physicochemical characteristics, including such properties as net charge, lack of secondary structure, or lack of repetitiveness that may be conferred by the amino acid composition of the motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs or portions of the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN of about 36, about 42, about 72, about 144, about 288, about 576, about 864, about 1000, about 2000 to about 3000 amino acid residues, or any intermediate length. Non-limiting examples of XTEN family sequences useful for incorporation into the subject CFXTEN are presented in Table 4. It is intended that a specified sequence mentioned relative to Table 4 has that sequence set forth in Table 4, while a generalized reference to an AE144 sequence, for example, is intended to encompass any AE sequence having 144 amino acid residues; e.g., AE144 — 1A, AE144 — 2A, etc., or a generalized reference to an AG144 sequence, for example, is intended to encompass any AG sequence having 144 amino acid residues, e.g., AG144 — 1, AG144 — 2, AG144_A, AG144_B, AG144_C, etc.





TABLE 4



XTEN Polypeptides







SEQ

XTEN

ID

Name
Amino Acid Sequence
NO:







AE42
GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS
49



AE42_1
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS
50



AE42_2
PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG
51



AE42_3
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP
52



AG42_1
GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGP
53



AG42_2
GPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP
54



AG42_3
SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA
55



AG42_4
SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
56



AE48
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS
57



AM48
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS
58



AE144
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS
59


EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP



ATSGSETPGTSTEPSEGSAP




AE144_
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
60

1A
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES



ATPESGPGTSTEPSEGSAPG




AE144_
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
61

2A
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES



ATPESGPGTSESATPESGPG




AE144_
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
62

2B
TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES



ATPESGPGTSESATPESGPG




AE144_
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
63

3A
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG



SPTSTEEGTSTEPSEGSAPG




AE144_
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
64

3B
TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG



SPTSTEEGTSTEPSEGSAPG




AE144_
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
65

4A
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES



ATPESGPGTSTEPSEGSAPG




AE144_
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
66

4B
TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES



ATPESGPGTSTEPSEGSAPG




AE144_
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
67

5A
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG



SPTSTEEGSPAGSPTSTEEG




AE144_
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE
68

6B
PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPG




AF144
GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGS
69


TSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPS



GESSTAPGTSPSGESSTAP




AG144_
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA
70

1
STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST



GTGPGSSPSASTGTGPGASP




AG144_
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
71

2
GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPG



ASPGTSSTGSPGTPGSGTASSS




AG144_
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
72

A
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA



SPGTSSTGSPGASPGTSSTGSP




AG144_
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG
73

B
SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGA



SPGTSSTGSPGASPGTSSTGSP




AG144_
GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPG
74

C
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS



TPSGATGSPGASPGTSSTGSP




AG144_
GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG
75

F
SSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS



TPSGATGSPGASPGTSSTGSP




AG144_
GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG
76

3
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA



SPGTSSTGSPGASPGTSSTGSP




AG144_
GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG
77

4
ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP



GSGTASSSPGSSTPSGATGSP




AE288_
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT
78

1
STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA



GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESA



TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE



GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




AE288_
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT
79

2
STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA



GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT



SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT



STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP




AG288_
PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP
80

1
GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG



SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS



PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP



GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS




AG288_
GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG
81

2
ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGA



SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST



PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG



TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP




AF504
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
82


SXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA



SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG



SGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS



GATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTS



STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST



GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG



SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP



GASPGTSSTGSP




AF540
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT
83


STPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPS



GESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS



GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESP



GPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP



GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGT



STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSE



SPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGE



SSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP




AD576
GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPG
84


SSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSS



ESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGG



EPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGG



SSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSS



GSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEG



GPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGG



PGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSS



GSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSG



SSESGSSEGGPGSEGSSGPGESS




AE576
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
85


STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE



SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP



SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE



GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES



GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP



GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST



EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA



TPESGPGTSTEPSEGSAP




AF576
GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT
86


STPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPS



GESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS



GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESP



GPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP



GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGT



STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSE



SPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGE



SSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGS



ASPGTSTPESGSASP




AG576
PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSP
87


GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG



ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGA



SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP



GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA



STGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS



STGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA



SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT



GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTG



PGSSPSASTGTGPGASPGTSSTGS




AE624
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE
88


GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT



SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST



EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA



TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE



GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE



TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP



GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE



SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP



SEGSAP




AD836
GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESG
89


ESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGES



PGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSES



GSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGG



SSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGP



GESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSES



GSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGS



ESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGP



GESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSES



GSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEP



SESGSSGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGP



GESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSES



GSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGS



ESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS




AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT
90


STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE



SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP



SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE



GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES



GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP



GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST



EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA



TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG



SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE



TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP



GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT



STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




AF864
GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGT
91


STPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPS



GESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTA



ESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGS



ASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPG



PGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPG



STSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGAS



ASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSES



PSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAE



SPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST



APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP



GSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGT



SPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSS



TAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG



ATGSP




AG864_
GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG
92

2
SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA



SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG



SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS



GATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTS



STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST



GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG



SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP



GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG



ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS



TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS



ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA



STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS



STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST



GSP




AM875
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS
93


TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSE



SATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEP



SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP



ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE



TPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAP



GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGA



SASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSE



SPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATS



GSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESS



TAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSAS



PGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPG



SSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSS



TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTE



PSEGSAP




AE912
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE
94


GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT



SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST



EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA



TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE



GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE



TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP



GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS



EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE



SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP



SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP



ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES



GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP



GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT



STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP




AM923
MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAP
95


GSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGS



TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPA



GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS



PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSE



GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST



EEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAP



GSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGT



SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSP



SGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESA



TPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSG



SETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGT



APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGP



GASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGS



SPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP




AM1318
GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS
96


TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSE



SATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEP



SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP



ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE



TPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAP



GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGP



EPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPA



GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSST



AESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGES



STAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPES



GPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAP



GTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGS



SPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGAS



PGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESA



TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSS



TGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSE



TPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEE



GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGS



STPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTS



STAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAG



SPTSTEEGTSTEPSEGSAP




BC 864
GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGS
97


EPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTST



EPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPAT



SGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSG



TEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTE



PSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPS



GSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGS



GASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTST



EPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEP



SEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSE



PGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPG



SAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPS



GSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGS



GASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA




BD864
GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETA
98


GSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETA



GTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETA



GTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSAS



GSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEA



GSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA



GTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSAS



GSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEA



GSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSAS



GSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETA



GTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETA



GTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETA



GTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETA



GTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGA



GTSESATSESGAGSETATSGSETA




AE948
GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS
99


PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEP



ATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT



SGSETPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSG



SETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES



GPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP



GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGT



SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE



SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEP



SEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPT



STEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSE



TPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE



GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT



STEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE



SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA



TPESGPGTSESATPESGP




AE1044
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGT
100


STEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSE



SATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEP



SEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP



ESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGS



APGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAP



GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGT



SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST



EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGS



PTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP



ESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPES



GPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEE



GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT



STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEP



ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP



SEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT



STEEGTSESATPESGPGTSESATPESGPGTST




AE1140
GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGS
101


EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSE



SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGS



PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP



ESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES



GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE



GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGS



PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA



GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEP



SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPT



STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSE



TPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAP



GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS



PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEP



ATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEP



SEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT



STEEGTSTEPSEGSAPGSPAGSPTSTEEGSPA




AE1236
GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT
102


STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEP



ATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGS



PTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT



STEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE



TPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP



GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGT



SESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTST



EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA



TPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATP



ESGPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE



TPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP



GSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGT



SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTST



EPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESA



TPESGPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSG



SETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP




AE1332
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGT
103


STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPA



GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPAT



SGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG



SETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES



GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAP



GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS



EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST



EPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGS



PTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATP



ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE



TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP



GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGS



EPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTST



EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT



SGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATP



ESGPGTSESATPESGPGTSTEPSEGSAPGTST




AE1428
GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGT
104


STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPA



GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT



SGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE



GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGS



APGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP



GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSE



SATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPAT



SGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPT



STEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES



GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP



GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT



SESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSE



SATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP



SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSE



GSAPGSPAGSPTSTEEGTSESATPESGPGSPA




AE1524
GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGS
105


PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTST



EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT



SGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT



STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPES



GPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEE



GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGS



EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEP



ATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGS



PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP



ESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTST



EEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGP



GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGT



SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPA



GSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA



TPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE



GSAPGTSTEPSEGSAPGTSESATPESGPGSPA




AE1620
GSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGT
106


SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST



EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESA



TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATP



ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES



GPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE



GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT



STEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEP



ATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP



SEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSE



GSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTST



EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGP



GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS



EPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSE



SATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESA



TPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE



GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST




AE1716
GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS
107


PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSE



SATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESA



TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE



GSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGS



APGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE



GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS



PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTST



EPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT



SGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT



STEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPES



GPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE



GSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGS



PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPA



GSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP



SEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSG



SETPGSPAGSPTSTEEGTSESATPESGPGTSE




AE1812
GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT
108


STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE



SATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA



TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE



GSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS



APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETP



GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS



PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTST



EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA



TPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSE



GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES



GPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP



GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT



SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP



ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESA



TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP



ESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEP




AE1908
GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGS
109


PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTST



EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSTEP



SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP



ESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS



APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGP



GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT



STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSE



SATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEP



SEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSE



GSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGS



APGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETP



GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGS



PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST



EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP



SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP



ESGPGSPAGSPTSTEEGTSESATPESGPGSEP




AE2004A
GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGS
110


PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST



EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEP



SEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSG



SETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES



GPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE



GTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGS



PAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSE



SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP



SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP



ESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES



GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEE



GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS



PAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEP



ATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP



SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPT



STEEGTSTEPSEGSAPGTSESATPESGPGTSE




AG948
GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG
111


TPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSS



TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASP



GTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPG



TSSTGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSG



ATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSS



TGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS



SSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG



PGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGP



GSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPG



SSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS



PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTP



SGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG



TASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGA



TGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTAS



SSPGSSTPSGATGSPGSSTPSGATGSP




AG1044
GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG
112


TPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSS



PSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPG



SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPG



TSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTS



STGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGAT



GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG



SPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP



GASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPG



TPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSS



TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASP



GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS



GATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS



TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSS



TGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAT



GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGT



GPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSST




AG1140
GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG
113


SSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS



TPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS



ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPS



GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSG



ATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGA



TGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSST



GSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATG



SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGP



GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG



TPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSS



PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPG



SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS



GATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG



ATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSAST



GTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGAT



GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSST




AG1236
GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG
114


ASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTP



GSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPS



ASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSPSA



STGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSG



ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGA



TGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST



GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATG



SPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP



GSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG



SSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGA



SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSP



SASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPG



TSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSG



TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGT



ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA



SSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP




AG1332
GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPG
115


SSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSS



TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP



SGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA



STGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS



STGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT



GSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATG



SPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSP



GSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG



TPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS



TPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTP



SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPS



GATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS



STGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST



GSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG



SPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGP



GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG




AG1428
GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPG
116


TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSS



TPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP



GTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS



GTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS



TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTA



SSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGT



GPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG



PGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP



GASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPG



SSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA



SPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASP



GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGS



GTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG



ATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST



GTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT



GSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASP




AG1524
GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG
117


TPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTP



GSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASP



GTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS



GTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG



ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA



SSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS



SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP



GSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG



TPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSS



TPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPS



ASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG



TASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGA



TGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT



GSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG



SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSP



GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPG




AG1620
GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPG
118


ASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSS



PSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTP



SGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGT



SSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST



GTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST



GSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT



GPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGS



PGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSP



GASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG



TPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS



TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPS



ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSA



STGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSG



ATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSAST



GTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS



SSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST




AG1716
GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPG
119


SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSS



PSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPG



SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPS



GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT



ASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSST



GSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS



SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGP



GTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG



TPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTP



GSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASP



GTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS



GTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG



ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSAST



GTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST



GSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG



SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG




AG1812
GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG
120


SSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSS



PSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP



SGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSG



TASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGT



ASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT



GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG



SPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSP



GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG



ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSS



TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPG



SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPG



TSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSG



TASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGA



TGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT



GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG



SPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASP




AG1908
GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPG
121


SSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGA



SPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSP



SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS



GTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSAS



TGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST



GTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST



GSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS



SPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGP



GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPG



SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS



TPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPG



SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS



GATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS



TGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSAST



GTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT



GSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSP




AG2004A
GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG
122


SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGA



SPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST



PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPG



TSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTS



STGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA



SSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS



SPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP



GASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPG



SSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSS



PSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP



GTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGS



GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSG



ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSS



TGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGAT



GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG



SPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASP




AE72B
SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE
123


PATSGSETPG




AE72C
TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS
124


TEPSEGSAPG




AE108A
TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA
125


PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS




AE108B
GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS
126


EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP




AE144A
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE
127


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS



PTSTEEGSPAGSPTSTEEGS




AE144B
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP
128


AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG



SPTSTEEGTSTEPSEGSAPG




AE180A
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS
129


TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET



PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS




AE216A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE
130


SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG



PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG



TSTEPSEGSAPGSEPATSGSETPGTSESAT




AE252A
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES
131


GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP



GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS



PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST



EPSE




AE288A
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
132


SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE



TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP



GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT



STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA




AE324A
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG
133


SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG



PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG



SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS



ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE



PSEGSAPGSEPATS




AE360A
PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS
134


TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG



PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG



SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS



ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG



SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT




AE396A
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS
135


TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG



PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG



SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP



AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA



TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS




AE432A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE
136


SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE



EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG



SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP



AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG



SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS



GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS




AE468A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE
137


SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA



PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG



TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT



PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG



SAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT




AE504A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS
138


TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET



PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG



SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES



ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS



EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS



ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG



PGTSTEPS




AE540A
TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE
139


GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS



APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP



GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS



PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP



ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS



PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT



STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE



TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP




AE576A
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
140


ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS



APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP



GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS



PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSE



SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP



SEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT



STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES



GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP



GSEPATSGSETPGTSESA




AE612A
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS
141


TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA



PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG



TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG



SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT



PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE



SGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA



PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG



SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT




AE648A
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG
142


SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET



PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG



TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE



PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES



ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS



EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE



SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG



PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG



TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS



TEPSEGSAPGSEPATSGSETPGTSESAT




AE684A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
143


SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG



PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG



TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS



TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP



TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE



SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA



PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG



SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE



PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA



TS




AE720A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS
144


EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG



SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG



PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG



TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS



TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP



TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE



SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA



PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG



SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE



PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE




AE756A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS
145


EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG



SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG



PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG



TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS



TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP



TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE



SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA



PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG



SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE



PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA



TSGSETPGTSES




AE792A
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE
146


SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA



PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG



TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE



PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE



PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT



PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG



SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG



PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG



TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS



ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES



ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS



EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS




AE828A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE
147


SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA



PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG



TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTS



ESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES



ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS



EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS



ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG



PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG



TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS



TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG



SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT



PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG



SAPGSEPATSGSETPGTSESAT




AG72A
GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS
148


PGTPGSGTASS




AG72B
GSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG
149


TPGSGTASSSP




AG72C
SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSST
150


PSGATGSPGA




AG108A
SASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPG
151


TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASP




AG108B
PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP
152


GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS




AG144A
PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP
153


GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPG



ASPGTSSTGSPGTPGSGTASSS




AG144B
PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS
154


ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA



STGTGPGASPGTSSTGSPGASP




AG180A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
155


TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS



TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS




AG216A
TGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS
156


TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST



GSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG



SPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG




AG252A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
157


TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS



TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS



SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS



PGASPG




AG288A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
158


TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS



TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS



SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS



PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS




AG324A
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
159


STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA



SSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG



SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP



GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG



TPGSGTASSSPGSSTP




AG360A
TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
160


STGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT



GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT



GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG



PGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP



GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG




AG396A
GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT
161


ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA



SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG



SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP



GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG



SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSS



TPSGATGSPGSSTPSGATGSPGASPGT




AG432A
GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
162


ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA



SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS



SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP



GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPG



SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS



TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP



S




AG468A
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
163


STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT



GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG



SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP



GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG



SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP



GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP



GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG




AG504A
TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS
164


STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT



GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG



SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP



GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG



SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP



GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP



GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS



GTASSSPGSSTP




AG540A
TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS
165


STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTA



SSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG



SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP



GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG



TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA



SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP



GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSA



STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG




AG576A
TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS
166


TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA



TGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT



GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS



SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP



GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG



SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSS



TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTP



SGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS



GATGSPGSSTPSGATGSPGASPG




AG612A
STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT
167


GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG



SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP



GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG



ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS



TPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASP



GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPG



TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG



TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS



TGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS




AG648A
GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG
168


ATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS



TGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST



GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG



SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP



GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG



ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS



TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS



ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA



STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS



STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP




AG684A
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG
169


ATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA



SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS



SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSP



GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPG



SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGA



SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG



SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA



STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST



GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTG



TGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG



SPGASPG




AG720A
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG
170


ATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS



TGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST



GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG



SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSP



GSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG



ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGA



SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG



SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG



TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS



STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTG



TGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG




AG756A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
171


TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS



TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS



SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS



PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP



GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG



ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP



GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP



GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG



TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG



ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST



GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG



TGPGASPGTSSTGSPGASPG




AG792A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
172


TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS



TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS



SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS



PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP



GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG



ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP



GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP



GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG



TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG



ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST



GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG



TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG




AG828A
TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS
173


TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS



TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS



SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS



PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP



GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG



ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP



GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP



GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG



TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG



ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST



GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG



TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG



SPGSSPSASTGTGPGTPGSGTASSSPGSSTP




AG288_
GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG
1699

DE
ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA



SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST



PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS



ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP













































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































In other embodiments, the CFXTEN composition comprises one or more non-repetitive XTEN sequences of lengths ranging from about 36 to about 3000 amino acid residues, wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of non-overlapping 36 amino acid sequence motifs selected from one or more of the polypeptide sequences of Tables 13-17, either as a family sequence, or where motifs are selected from two or more families of motifs.
In those embodiments wherein the XTEN component of the CFXTEN fusion protein has less than 100% of its amino acids consisting of 4, 5, or 6 types of amino acid selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequence consisting of the sequence motifs from Table 3 or the XTEN sequences of Tables 4, and 13-17, the other amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. The XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are either interspersed throughout the XTEN sequence, are located within or between the sequence motifs, or are concentrated in one or more short stretches of the XTEN sequence, e.g., to create a linker between the XTEN and the FVIII components. In such cases where the XTEN component of the CFXTEN comprises amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids be hydrophobic residues such that the resulting sequences generally lack secondary structure, e.g., not having more than 2% alpha helices or 2% beta-sheets, as determined by the methods disclosed herein. Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequences to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid desamidation). Thus, in some embodiments, the XTEN component of the CFXTEN fusion protein comprising other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) have a sequence with less than 5% of the residues contributing to alpha-helices and beta-sheets as measured by the Chou-Fasman algorithm and have at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm.
3. Length of Sequence
In another aspect, the invention provides XTEN of varying lengths for incorporation into CFXTEN compositions wherein the length of the XTEN sequence(s) are chosen based on the property or function to be achieved in the fusion protein. Depending on the intended property or function, the CFXTEN compositions comprise short or intermediate length XTEN located internal to the FVIII sequence or between FVIII domains and/or longer XTEN sequences that can serve as carriers, located in the fusion proteins as described herein. While not intended to be limiting, the XTEN or fragments of XTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer lengths of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, the XTEN for incorporation into the subject CFXTEN encompass XTEN or fragments of XTEN with lengths of about 6, or about 12, or about 36, or about 40, or about 42, or about 72 or about 96, or about 144, or about 288, or about 400, or about 500, or about 576, or about 600, or about 700, or about 800, or about 864, or about 900, or about 1000, or about 1500, or about 2000, or about 2500, or up to about 3000 amino acid residues in length. Alternatively, the XTEN sequences can be about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. The precise length of an XTEN incorporated into the subject CFXTEN can vary without adversely affecting the activity of a CFXTEN composition. In one embodiment, one or more of the XTEN used in the CFXTEN disclosed herein has 36 amino acids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from one of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD. In another embodiment, two or more of the XTEN used in the CFXTEN disclosed herein has 36 amino acids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from two of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD, with combinations of AE and AG family sequences preferred. In some embodiments, CFXTEN comprising one or more of the XTEN used herein contain XTEN selected from any one of the sequences in Table 4, which may be linked to the FVIII component directly or via spacer sequences disclosed herein.
In particular CFXTEN configuration designs, where the XTEN serve as a flexible linker, or are inserted in external loops or unordered regions of the FVIII sequence to increase the bulk, flexibility, or hydrophilicity of the region, or are designed to interfere with clearance receptors for FVIII to enhance pharmacokinetic properties, or to interfere with binding of FVIII inhibitors or other anti-FVIII antibodies, or where a short or intermediate length of XTEN is used to facilitate tissue penetration or to vary the strength of interactions of the CFXTEN fusion protein with its target, or where it is desirable to distribute the cumulative length of XTEN in segments of short or intermediate length at multiple locations within the FVIII sequence, the invention contemplates CFXTEN compositions with one, two, three, four, five or more short or intermediate XTEN sequences inserted between or within one or more FVIII domains or within external loops, or at other sites in the FVIII sequence such as, but not limited to, locations at or proximal to the insertion sites identified in Table 5, Table 6, Table 7, Table 8, and Table 9 or as illustrated in FIGS. 8-9 . In one embodiment of the foregoing, the CFXTEN fusion protein contains multiple XTEN segments, e.g., at least two, or at least three, or at least four, or at least five or more XTEN segments in which the XTEN segments can be identical or they can be different and wherein the CFXTEN retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the procoagulant activity of native FVIII when assayed by one of the assays disclosed herein. In other particular CFXTEN configuration designs, where the XTEN serves as a carrier to increase the bulk of the fusion protein, or to vary the strength of interactions of the CFXTEN fusion protein with its target, or to enhance the pharmacokinetic properties of the fusion protein, the invention contemplates CFXTEN compositions with one or more intermediate or longer length XTEN sequences inserted at the C-terminus, within the B domain (or the residual of the BDD sequence) between or within one or more FVIII domains, within external loops, or at other sites in the FVIII sequence such as, but not limited to, insertion sites identified in Table 5, Table 6, Table 7, Table 8, and Table 9 or as illustrated in FIGS. 8-9 . However, it is believed that the incorporation of multiple XTEN of short to intermediate lengths into CFXTEN compositions confers enhanced properties on the fusion proteins compared to CFXTEN fusion proteins with the same number of amino acids in fewer but longer length XTEN, yet still results in compositions with procoagulant activity and extended half-life; the rationale of which is detailed herein regarding the derived radii of multiple XTEN.
In the embodiments wherein the CFXTEN fusion proteins comprise multiple XTEN sequences, the cumulative length of the total residues in the XTEN sequences is greater than about 100 to about 3000, or about 200 to about 2000, or about 400 to about 1000 amino acid residues and the XTEN can be identical or they can be different in sequence, net charge, or in length. In one embodiment of CFXTEN comprising multiple XTEN, the individual XTEN sequences each exhibit at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a motif or an XTEN selected from Tables 3, 4, and 13-17 or a fragment thereof, when optimally aligned with a sequence of comparable length.
As described more fully below, methods are disclosed in which the CFXTEN are designed by selecting the length of the XTEN and its site of incorporation within the CFXTEN to confer a target half-life, retention of procoagulant activity, reduced binding to FVIII inhibitors or an enhanced physicochemical property (e.g., stability or solubility) of a CFXTEN fusion protein, encoding constructs are created and expressed and the recombinant CFXTEN fusion proteins are isolated and recovered. In general, XTEN cumulative lengths longer that about 400 residues incorporated into the CFXTEN compositions result in longer half-life compared to shorter cumulative lengths, e.g., shorter than about 280 residues. In one embodiment, CFXTEN fusion proteins designs are contemplated that comprise at least a single XTEN as a carrier, with a long sequence length of at least about 400, or at least about 600, or at least about 800, or at least about 900, or at least about 1000 or more amino acids. In another embodiment, multiple XTEN are incorporated into the fusion protein to achieve cumulative lengths of at least about 400, or at least about 600, or at least about 800, or at least about 900, or at least about 1000 or more amino acids, wherein the XTEN can be identical or they can be different in sequence or length. As used herein, “cumulative length” is intended to encompass the total length, in amino acid residues, when more than one XTEN is incorporated into the CFXTEN fusion protein. Both of the foregoing embodiments are designed to confer increased bioavailability and/or increased terminal half-life after administration to a subject compared to CFXTEN comprising shorter cumulative XTEN lengths, yet still result in a procoagulant activity and hemostasis effect. When administered subcutaneously or intramuscularly, the C max is reduced but the area under the curve (AUC) is increased in comparison to a comparable dose of a CFXTEN with shorter cumulative length XTEN or FVIII not linked to XTEN, thereby contributing to the ability to maintain effective levels of the CFXTEN composition for a longer period of time and permitting increased periods of 2, 4, 7, 10, 14 or 21 days between dosing, as described more fully below. Thus, the XTEN confers the property of a depot to the administered CFXTEN, in addition to the other physicochemical properties described herein.
When XTEN are used as a carrier, the invention takes advantage of the discovery that increasing the length of the non-repetitive, unstructured polypeptides enhances the unstructured nature of the XTENs and correspondingly enhances the physical/chemical and pharmacokinetic properties of fusion proteins comprising the XTEN carrier. As described more fully in the Examples, proportional increases in the length of the XTEN, even if created by a repeated order of single family sequence motifs (e.g., the four AE motifs of Table 3), result in a sequence with a higher percentage (e.g., 90% or more) of random coil formation, as determined by GOR algorithm, or reduced content of alpha-helices or beta-sheets (e.g., less than 2%), as determined by Chou-Fasman algorithm, compared to shorter XTEN lengths. In addition, increasing the length of the unstructured polypeptide fusion partner, as described in the Examples, results in a fusion protein with a disproportionate increase in terminal half-life (e.g., as much as 50, 100, 200 or more hours) compared to fusion proteins with unstructured polypeptide partners with shorter sequence lengths. The enhanced pharmacokinetic properties of the CFXTEN in comparison to FVIII not linked to XTEN are described more fully, below.
In another aspect, the invention provides methods to create XTEN of short or intermediate lengths from longer “donor” XTEN sequences, wherein the longer donor XTEN sequence is truncated at the N-terminus, or the C-terminus, or a fragment is created from the interior of a donor sequence, thereby resulting in a short or intermediate length XTEN. In non-limiting examples, as schematically depicted in FIG. 16A-C , an AG sequence of 864 amino acid residues can be truncated to yield an AG sequence with 144 residues, an AG sequence with 288 residues, an AG sequence with 576 residues, or other intermediate lengths, while the AE sequence of 864 residues (as depicted in FIG. 16D , E) can be truncated to yield multiple AE sequences of 144 residues, an AE sequence with 288 or 576 residues or other shorter or intermediate lengths. It is specifically contemplated that such an approach can be utilized with any of the XTEN embodiments described herein or with any of the sequences listed in Tables 4 or 13-17 to result in XTEN of a desired length. In preferred embodiments, the CFXTEN comprising multiple XTEN have XTEN exhibiting at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to sequences selected from AE42 — 1, AE42 — 2, AE42 — 3, AG42 — 1, AG42 — 2, AG42 — 3, AG42 — 4, AE1441 — 1A, AE144 — 2A, AE144 — 2B, AE144 — 3A, AE144 — 3B, AE144 — 4A, AE144 — 4B, AE144 — 5A, AE144 — 6B, AG144 — 1, AG144 — 2, AG144_A, AG144_B, AG144_C, AG144_F, AG144 — 3, AG144 — 4, AE288 — 1, AE288 — 2, AG288 — 1, AG288 — 2, and AG288 DE.
4. Net Charge
In other embodiments, the unstructured characteristic of an XTEN polypeptide can be enhanced by incorporation of amino acid residues with a net charge and/or reduction of the overall percentage (e.g. less than 5%, or 4%, or 3%, or 2%, or 1%) of hydrophobic amino acids in the XTEN sequence. The overall net charge and net charge density is controlled by modifying the content of charged amino acids in the XTEN sequences, either positive or negative, with the net charge typically represented as the percentage of amino acids in the polypeptide contributing to a charged state beyond those residues that are cancelled by a residue with an opposite charge. In some embodiments, the net charge density of the XTEN of the compositions may be above +0.1 or below −0.1 charges/residue. By “net charge density” of a protein or peptide herein is meant the net charge divided by the total number of amino acids in the protein or propeptide. In other embodiments, the net charge of an XTEN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more. Based on the net charge, some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, the XTEN will have an isoelectric point between 1.5 and 4.5 and carry a net negative charge under physiologic conditions.
Since most tissues and surfaces in a human or animal have a net negative charge, in some embodiments the XTEN sequences are designed to have a net negative charge to minimize non-specific interactions between the XTEN containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. Not to be bound by a particular theory, an XTEN can adopt open conformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptide that individually carry a net negative charge and that are distributed across the sequence of the XTEN polypeptide. In some embodiments, the XTEN sequence is designed with at least 90% or 95% of the charged residues separated by other residues such as serine, alanine, threonine, proline or glycine, which leads to a more uniform distribution of charge, better expression or purification behavior. Such a distribution of net negative charge in the extended sequence lengths of XTEN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. In preferred embodiments, the negative charge of the subject XTEN is conferred by incorporation of glutamic acid residues. Generally, the glutamic residues are spaced uniformly across the XTEN sequence. In some cases, the XTEN can contain about 10-80, or about 15-60, or about 20-50 glutamic residues per 20 kDa of XTEN that can result in an XTEN with charged residues that would have very similar pKa, which can increase the charge homogeneity of the product and sharpen its isoelectric point, enhance the physicochemical properties of the resulting CFXTEN fusion protein for, and hence, simplifying purification procedures. For example, where an XTEN with a negative charge is desired, the XTEN can be selected solely from an AE family sequence, which has approximately a 17% net charge due to incorporated glutamic acid, or can include varying proportions of glutamic acid-containing motifs of Table 3 to provide the desired degree of net charge. Non-limiting examples of AE XTEN include, but are not limited to the 36, 42, 144, 288, 576, 624, 864, and 912 AE family sequences of Tables 4 and 14 or fragments thereof. In one embodiment, an XTEN sequence of Tables 4, or 13-17 can be modified to include additional glutamic acid residues to achieve the desired net negative charge. Accordingly, in one embodiment the invention provides XTEN in which the XTEN sequences contain about 1%, 2%, 4%, 8%, 10%, 15%, 17%, 20%, 25%, or even about 30% glutamic acid. In one embodiment, the invention contemplates incorporation of up to 5% aspartic acid residues into XTEN in addition to glutamic acid in order to achieve a net negative charge.
In other embodiments, where no net charge is desired, the XTEN can be selected from, for example, AG XTEN components, such as the AG motifs of Table 3, or those AM motifs of Table 3 that have no net charge. Non-limiting examples of AG XTEN include, but are not limited to 36, 42, 144, 288, 576, and 864 AG family sequences of Tables 4 and 16, or fragments thereof. In another embodiment, the XTEN can comprise varying proportions of AE and AG motifs (in order to have a net charge that is deemed optimal for a given use or to maintain a given physicochemical property.
Not to be bound by a particular theory, the XTEN of the CFXTEN compositions with the higher net charge are expected to have less non-specific interactions with various negatively-charged surfaces such as blood vessels, tissues, or various receptors, which would further contribute to reduced active clearance. Conversely, it is believed that the XTEN of the CFXTEN compositions with a low (or no) net charge would have a higher degree of interaction with surfaces that can potentiate the activity of the associated coagulation factor, given the known contribution of cell (e.g., platelets) and vascular surfaces to the coagulation process and the intensity of activation of coagulation factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry (2000) 39(32):9850-9858).
The XTEN of the compositions of the present invention generally have no or a low content of positively charged amino acids. In some embodiments, the XTEN may have less than about 10% amino acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2%, or less than about 1% amino acid residues with a positive charge. However, the invention contemplates constructs where a limited number of amino acids with a positive charge, such as lysine, are incorporated into XTEN to permit conjugation between the epsilon amine of the lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule to be conjugated to the XTEN backbone. In one embodiment of the foregoing, the XTEN of the subject CFXTEN has between about 1 to about 100 lysine residues, or about 1 to about 70 lysine residues, or about 1 to about 50 lysine residues, or about 1 to about 30 lysine residues, or about 1 to about 20 lysine residues, or about 1 to about 10 lysine residues, or about 1 to about 5 lysine residues, or alternatively only a single lysine residue. Using the foregoing lysine-containing XTEN, fusion proteins can be constructed that comprise XTEN, a FVIII coagulation factor, plus a chemotherapeutic agent or other coagulation factor or cofactor useful in the treatment of coagulopathy conditions, wherein the maximum number of molecules of the agent incorporated into the XTEN component is determined by the numbers of lysines or other amino acids with reactive side chains (e.g., cysteine) incorporated into the XTEN.
As hydrophobic amino acids impart structure to a polypeptide, the invention provides that the content of hydrophobic amino acids in the XTEN will typically be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. In one embodiment, the amino acid content of methionine and tryptophan in the XTEN component of a CFXTEN fusion protein is typically less than 5%, or less than 2%, and most preferably less than 1%. In another embodiment, the XTEN of the subject CFXTEN compositions will have a sequence that has less than 10% amino acid residues with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 5% of the total XTEN sequence.
5. Low Immunogenicity
In another aspect, the XTEN sequences provided herein have a low degree of immunogenicity or are substantially non-immunogenic. Several factors can contribute to the low immunogenicity of XTEN, e.g., the non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the XTEN sequence.
Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid sequences of the protein antigen. The precise folding of the protein brings these sequences into a well-defined, stable spatial configurations, or epitopes, that can be recognized as “foreign” by the host humoral immune system, resulting in the production of antibodies to the protein or the activation of a cell-mediated immune response. In the latter case, the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition that is a function of the peptide binding specificity of that individual's HLA-DR allotype. Engagement of a MHC Class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
The ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) is dependent on a number of factors; most notably its primary sequence. In one embodiment, a lower degree of immunogenicity is achieved by designing XTEN sequences that resist antigen processing in antigen presenting cells, and/or choosing sequences that do not bind MHC receptors well. The invention provides CFXTEN fusion proteins with substantially non-repetitive XTEN polypeptides designed to reduce binding with MHC II receptors, as well as avoiding formation of epitopes for T-cell receptor or antibody binding, resulting in a low degree of immunogenicity. Avoidance of immunogenicity can attribute to, at least in part, a result of the conformational flexibility of XTEN sequences; i.e., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular interest are sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result in conformational epitopes. The administration of fusion proteins comprising XTEN, using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the XTEN sequence, and also reduce the immunogenicity of the FVIII fusion partner in the CFXTEN compositions.
In one embodiment, the XTEN sequences utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating less immunogenic proteins has been disclosed previously; see for example WO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This is achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and, in particular, 9-mer sequences that are not human, and then altering the design of the XTEN sequence to eliminate or disrupt the epitope sequence. In some embodiments, the XTEN sequences are substantially non-immunogenic by the restriction of the numbers of epitopes of the XTEN predicted to bind MHC receptors. With a reduction in the numbers of epitopes capable of binding to MHC receptors, there is a concomitant reduction in the potential for T cell activation as well as T cell helper function, reduced B cell activation or upregulation and reduced antibody production. The low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as, e.g., TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown in Example 46. The TEPITOPE score of a given peptide frame within a protein is the log of the K d (dissociation constant, affinity, off-rate) of the binding of that peptide frame to multiple of the most common human MHC alleles, as disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555). The score ranges over at least 20 logs, from about 10 to about −10 (corresponding to binding constraints of 10 e 10 K d to 10 e −10 K d ), and can be reduced by avoiding hydrophobic amino acids that serve as anchor residues during peptide display on MHC, such as M, I, L, V, F. In some embodiments, an XTEN component incorporated into a CFXTEN does not have a predicted T-cell epitope at a TEPITOPE threshold score of about −5, or −6, or −7, or −8, or −9, or at a TEPITOPE score of −10. As used herein, a score of “−9” is a more stringent TEPITOPE threshold than a score of −5.
In another embodiment, the inventive XTEN sequences, including those incorporated into the subject CFXTEN fusion proteins, are rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the XTEN, reducing the processing of XTEN into small peptides that can bind to MHC II receptors. In another embodiment, the XTEN sequence is rendered substantially non-immunogenic by the use a sequence that is substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Accordingly, the reduced TEPITOPE score and elimination of known proteolytic sites from the XTEN render the XTEN compositions, including the XTEN of the CFXTEN fusion protein compositions, substantially unable to be bound by mammalian receptors, including those of the immune system or active clearance receptors that target FVIII. In one embodiment, an XTEN of a CFXTEN fusion protein can have >100 nM K d binding to a mammalian receptor, or greater than 500 nM K d , or greater than 1 μM K d towards a mammalian cell surface receptor or circulating polypeptide receptor.
Additionally, the non-repetitive sequence and corresponding lack of epitopes of XTEN limit the ability of B cells to bind to or be activated by XTEN. A repetitive sequence is recognized and can form multivalent contacts with even a few B cells and, as a consequence of the cross-linking of multiple T-cell independent receptors, can stimulate B cell proliferation and antibody production. In contrast, while an XTEN can make contacts with many different B cells over its extended sequence, each individual B cell may only make one or a small number of contacts with an individual XTEN due to the lack of repetitiveness of the sequence. Not being to be bound by any theory, XTENs typically have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In one embodiment, the CFXTEN have reduced immunogenicity as compared to the corresponding FVIII that is not fused to an XTEN. In one embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-CFXTEN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In another embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-FVIII IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In another embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-XTEN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In the foregoing embodiments, the mammal can be a mouse, a rat, a rabbit, or a cynomolgus monkey.
An additional feature of XTENs with non-repetitive sequences relative to sequences with a high degree of repetitiveness is non-repetitive XTENs form weaker contacts with antibodies. Antibodies are multivalent molecules. For instance, IgGs have two identical binding sites and IgMs contain 10 identical binding sites. Thus antibodies against repetitive sequences can form multivalent contacts with such repetitive sequences with high avidity, which can affect the potency and/or elimination of such repetitive sequences. In contrast, antibodies against non-repetitive XTENs may yield monovalent interactions, resulting in less likelihood of immune clearance such that the CFXTEN compositions can remain in circulation for an increased period of time. In addition, it is believed, as schematically portrayed in FIG. 6 , the flexible unstructured nature of XTEN provides steric shielding of FVIII regions proximal to the XTEN site of insertion and providing steric hindrance to binding by FVIII inhibitors.
In another aspect, a subject XTEN useful as a fusion partner has a high hydrodynamic radius; a property that in some embodiments confers a corresponding increased apparent molecular weight to the CFXTEN fusion protein incorporating the XTEN, while in other embodiments enhances steric hindrance to FVIII inhibitors and to anti-FVIII antibodies, reducing their ability to bind to CFXTEN. As detailed in Example 26, the linking of XTEN to therapeutic protein sequences results in CFXTEN compositions that can have increased hydrodynamic radii, increased apparent molecular weight, and increased apparent molecular weight factor compared to a therapeutic protein not linked to an XTEN. For example, in therapeutic applications in which prolonged half-life is desired, compositions in which an XTEN with a high hydrodynamic radius is incorporated into a fusion protein comprising a therapeutic protein can effectively enlarge the hydrodynamic radius of the composition beyond the glomerular pore size of approximately 3-5 nm (corresponding to an apparent molecular weight of about 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55:1261-1277), resulting in reduced renal clearance of circulating proteins with a corresponding increase in terminal half-life and other enhanced pharmacokinetic properties. The hydrodynamic radius of a protein is conferred by its molecular weight as well as by its structure, including shape or compactness. Not to be bound by a particular theory, the XTEN can adopt open conformations due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure. The open, extended and unstructured conformation of the XTEN polypeptide can have a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and/or molecular weight that have secondary and/or tertiary structure, such as typical globular proteins. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. Example 26 demonstrates that increases in XTEN length result in proportional increase in the hydrodynamic radius, apparent molecular weight, and/or apparent molecular weight factor, and thus permit the tailoring of CFXTEN to desired cut-off values of apparent molecular weights or hydrodynamic radii. Accordingly, in certain embodiments, the CFXTEN fusion protein can be configured with an XTEN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or about 12 nm, or about 15 nm, or about 20 nm, or about 30 nm or more. In the foregoing embodiments, the large hydrodynamic radius conferred by the XTEN in a CFXTEN fusion protein can lead to reduced clearance of the resulting fusion protein, an increase in terminal half-life, and an increase in mean residence time.
Generally, the actual molecular weight of the mature form of FVIII component is about 265 kDa, while in the case of a FVIII BDD, it is about 165 kDa. The actual molecular weight of a CFXTEN fusion protein for comprising a FVIII BDD plus one or more XTEN ranges from about 200 to about 270 kDa, depending on the length of the XTEN components. As described in the Examples, when the molecular weights of the CFXTEN fusion proteins are derived from size exclusion chromatography analyses, the open conformation of the XTEN due to the low degree of secondary structure results in an increase in the apparent molecular weight of the fusion proteins into which they are incorporated. In some embodiments, the CFXTEN comprising a FVIII and at least one or more XTEN exhibits an apparent molecular weight of at least about 400 kD, or at least about 500 kD, or at least about 700 kD, or at least about 1000 kD, or at least about 1400 kD, or at least about 1600 kD, or at least about 1800 kD, or at least about 2000 kD. Accordingly, the CFXTEN fusion proteins comprising one or more XTEN exhibit an apparent molecular weight that is about 1.3-fold greater, or about 2-fold greater, or about 3-fold greater or about 4-fold greater, or about 8-fold greater, or about 10-fold greater, or about 12-fold greater, or about 15-fold greater than the actual molecular weight of the fusion protein. In one embodiment, the isolated CFXTEN fusion protein of any of the embodiments disclosed herein exhibit an apparent molecular weight factor under physiologic conditions that is greater than about 1.3, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 10, or greater than about 15. In another embodiment, the CFXTEN fusion protein has, under physiologic conditions, an apparent molecular weight factor that is about 3 to about 20, or is about 5 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual molecular weight of the fusion protein. It is believed that the increased apparent molecular weight of the subject CFXTEN compositions enhances the pharmacokinetic properties of the fusion proteins by a combination of factors, which include reduced active clearance, reduced binding by FVIII inhibitors, and reduced loss in capillary and venous bleeding.
IV). CFXTEN Compositions
The present invention provides compositions comprising fusion proteins having factor VIII linked to one or more XTEN sequences, wherein the fusion protein acts to replace or augment the amount of existing FVIII in the intrinsic or contact activated coagulation pathway when administered into a subject. The invention addresses a long-felt need in increasing the terminal half-life of exogenously administered factor VIII to a subject in need thereof. One way to increase the circulation half-life of a therapeutic protein is to ensure that renal clearance or metabolism of the protein is reduced. Another way to increase the terminal half-life is to reduce the active clearance of the therapeutic protein, whether mediated by receptors, active metabolism of the protein, or other endogenous mechanisms. Both may be achieved by conjugating the protein to a polymer, which, on one hand, is capable of conferring an increased molecular size (or hydrodynamic radius) to the protein and, hence, reduced renal clearance, and, on the other hand, interferes with binding of the protein to clearance receptors or other proteins that contribute to metabolism or clearance. Thus, certain objects of the present invention include, but are not limited to, providing improved FVIII molecules with a longer circulation or terminal half-life, decreasing the number or frequency of necessary administrations of FVIII compositions, retaining at least a portion of the activity compared to native coagulation factor VIII, and/or enhancing the ability to treat coagulation deficiencies and uncontrolled bleedings more efficiently, more effectively, more economically, and/or with greater safety compared to presently available factor VIII preparations.
Accordingly, the present invention provides recombinant factor VIII fusion protein compositions comprising an FVIII covalently linked to one or more extended recombinant polypeptides (“XTEN”), resulting in a CFXTEN fusion protein composition. The term “CFXTEN”, as used herein, is meant to encompass fusion polypeptides that comprise at least one payload region comprising a FVIII or a portion of a FVIII that is capable of procoagulant activity associated with a FVIII coagulation factor and at least one other region comprising one or more XTEN polypeptides that may be interspersed within the payload region and/or attached to the terminus. In one embodiment, the FVIII is native FVIII. In another embodiment, the FVIII is a sequence variant, fragment, homolog, or mimetic of a natural sequence that retains at least a portion of the procoagulant activity of native FVIII, as disclosed herein. Non-limiting examples of FVIII suitable for inclusion in the compositions include the sequences of Table 1 or sequences having at least 80%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to a sequence of Table 1. In a preferred embodiment, the FVIII is a B-domain deleted (BDD) FVIII sequence variant, such as those BDD sequences from Table 1 or other such sequences known in the art. In another preferred embodiment, the CFXTEN comprises a B-domain deleted (BDD) FVIII sequence variant expressed with the native 19 amino acid signal sequence, which is cleaved during the maturation of the protein.
The compositions of the invention include fusion proteins that are useful, when administered to a subject in need thereof, for mediating or preventing or ameliorating a condition associated with factor VIII deficiencies or defects in endogenously produced FVIII, or bleeding disorders associated with trauma, surgery, factor VIII deficiencies or defects. Of particular interest are CFXTEN fusion protein compositions for which an increase in a pharmacokinetic parameter, increased solubility, increased stability, or some other enhanced pharmaceutical property compared to native FVIII is sought, or for which increasing the terminal half-life would improve efficacy, safety, or result in reduced dosing frequency and/or improve patient management. The CFXTEN fusion proteins of the embodiments disclosed herein exhibit one or more or any combination of the improved properties and/or the embodiments as detailed herein. In some embodiments, the CFXTEN fusion composition remains at a level above a threshold value of at least 0.01-0.05, or 0.05 to 0.1, or 0.1 to 0.4 IU/ml when administered to a subject, for a longer period of time when compared to a FVIII not linked to XTEN and administered at a comparable dose to a subject in need thereof (e.g., a subject such as a human or mouse or monkey with hemophilia A).
The FVIII of the subject compositions, particularly those disclosed in Table 1, together with their corresponding nucleic acid and amino acid sequences, are available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, The Universal Protein Resource (UniProt), subscription provided databases such as GenSeq (e.g., Derwent), as well as in the patent and primary literature. Polynucleotide sequences applicable for expressing the subject CFXTEN sequences may be a wild type polynucleotide sequence encoding a given FVIII (e.g., either full length or mature), or in some instances the sequence may be a variant of the wild type polynucleotide sequence (e.g., a polynucleotide which encodes the wild type biologically active protein, wherein the DNA sequence of the polynucleotide has been optimized, for example, for expression in a particular species, or a polynucleotide encoding a variant of the wild type protein, such as a site directed mutant or an allelic variant. It is well within the ability of the skilled artisan to use a wild-type or consensus cDNA sequence or a codon-optimized variant of a FVIII to create CFXTEN constructs contemplated by the invention using methods known in the art and/or in conjunction with the guidance and methods provided herein, and described more fully in the Examples.
In one embodiment, a CFXTEN fusion protein comprises a single FVIII molecule exhibiting at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity to a sequence of Table 1 linked to a single XTEN (e.g., an XTEN as described above) including, but not limited to sequences of the AE or AG family with 42, 144, 288, 576, or 864 amino acids, as set forth in Table 4. In another embodiment, the CFXTEN comprises a single FVIII linked to two XTEN, wherein the XTEN may be identical or they may be different. In another embodiment, the CFXTEN fusion protein comprises a single FVIII molecule linked to one, two, three, four, five, six or more XTEN sequences, in which the FVIII is a sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table 1, when optimally aligned, and the one or more XTEN are each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to one or more sequences selected from any one of Tables 3, 4, and 13-17, when optimally aligned. In the foregoing embodiment, where the CFXTEN has two or more XTEN, the XTEN may be identical or they may be different sequences. In yet another embodiment, the CFXTEN fusion protein comprises a single FVIII exhibiting at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to sequences of comparable length selected from Table 1, when optimally aligned, with the portions interspersed with and linked by three, four, five, six or more XTEN sequences that may be identical or may be different and wherein each has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to sequences selected from any one of Tables 3, 4, and 13-17, or fragments thereof, when optimally aligned. In yet another embodiment, the invention provides a CFXTEN fusion protein comprising a sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity to a sequence from Table 21, when optimally aligned.
1. CFXTEN Fusion Protein Configurations
The invention provides CFXTEN fusion protein compositions with the CF and XTEN components linked in specific N- to C-terminus configurations.
In one embodiment of the CFXTEN composition, the invention provides a fusion protein of formula I:
(XTEN) x -CF-(XTEN) y I

wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with sequenced from Table 1; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. Accordingly, the CFXTEN fusion composition can have XTEN-CF, XTEN-CF-XTEN, or CF-XTEN configurations.
In another embodiment of the CFXTEN composition, the invention provides a fusion protein of formula II:
(XTEN) x -(S) x -(CF)-(XTEN) y II

wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein, wherein the fusion protein is of formula III:
(XTEN) x -(S) x -(CF)-(S) y -(XTEN) y III

wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequence set for in Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula IV:
(A1)-(XTEN) u -(A2)-(XTEN) v -(B)-(XTEN) w -(A3)-(XTEN) x -(C1)-(XTEN) y -(C2)-(XTEN) z IV

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; v is either 0 or 1; w is either 0 or 1; x is either 0 or 1; y is either 0 or 1; y is either 0 or 1 with the proviso that u+v+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula V:
(XTEN) t -(S) a -(A1)-(S) b -(XTEN) u -(S) b -(A2)-(S) c -(XTEN) v -(S) c -(B)-(S) d -(XTEN) w -(S) d -(A3)-(S) e -(XTEN) x -(S) e -(C1)-(S) f -(XTEN) y -(S) f -(C2)-(S) g -(XTEN) z V

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t+u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VI:
(XTEN) u -(S) a -(A1)-(S) b -(XTEN) v -(S) b -(A2)-(S) c -(XTEN) w -(S) c -(A3)-(S) d -(XTEN) x -(S) d -(C1)-(S) e -(XTEN) y -(S) e -(C2)-(S) f -(XTEN) z VI

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VII:
(SP)-(XTEN) x -(CS) x -(S) x -(FVIII — 1-745)-(S)-(XTEN)-(S)-(FVIII — 1635-2332)-(S) z -(CS) z -(XTEN) z VII

wherein independently for each occurrence, SP is a signal peptide, preferably with sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is a cleavage sequence listed in Table 12, S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include amino acids compatible with restrictions sites, “FVIII — 1-745” is residues 1-745 of Factor FVIII and “FVIII — 1635-2332” is residues 1635-2332 of FVIII, x is either 0 or 1, y is either 0 or 1, and z is either 0 or 1, wherein x+y+z>2; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity sequences set forth in Table 4. In one embodiment of formula VII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VIII:
(A1)-(S) a -(XTEN) v -(S) a -(A2)-(B1)-(S) b -(XTEN) w -(S) b -(B2)-(A3)-(S)-(XTEN) x -(S)-(C1)-(S) d -(XTEN) y -(S) d -(C2)-(S) e -(XTEN) z VIII

wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; B1 is a fragment of the B domain that can have from residue 741 to 743-750 of FVIII or alternatively from about residue 741 to about residues 745 of FVIII; B2 is a fragment of the B domain that can have from residues 1635-1686 to 1689 of FVIII or alternatively from about residue 1640 to about residues 1689 of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In one embodiment of formula VIII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula IX:
(A1 N )-(S) a -(XTEN) t -(S) b -(A1 C )-(A2 N )-(S) c -(XTEN) u -(S) d -(A2c)-(B N )-(S) e -(XTEN) v -(S) f (Bc)-(A3 N )-(S) g -(XTEN) w -(S) h -(A3 C )-(C1 N )-(S) i -(XTEN) x -(S) j -(C1 C )-(C2 N )-(S) k -(XTEN) y -(S)-(C2 C )-(S) m -(XTEN) z IX

wherein independently for each occurrence, A1 N is a fragment of the A1 domain from at least residue number 1 (numbered relative to native, mature FVIII) to no more than residue number 371, A1 is a fragment of the A1 domain from at least residue number 2 to no more than residue number 372; A2 N is a fragment of the A2 domain from at least residue number 373 to no more than residue number 739, A2 c is a fragment of the A2 domain from at least residue number 374 to no more than residue number 740; B N is a fragment of the B domain from at least residue number 741 to no more than residue number 1647, Be is a fragment of the B domain from at least residue number 742 to no more than residue number 1648; A3 N is a fragment of the A3 domain from at least residue number 1649 to no more than residue number 2019, A3, is a fragment of the A3 domain from at least residue number 1650 to no more than residue number 2019; C1 N is a fragment of the C1 domain from at least residue number 2020 to no more than residue number 2171, C1, is a fragment of the C1 domain from at least residue number 2021 to no more than residue number 2172; C2 N is a fragment of the C2 domain from at least residue number 2173 to no more than residue number 2331, C2, is a fragment of the C2 domain from at least residue number 2174 to no more than residue number 2332; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0 or 1; k is either 0 or 1; 1 is either 0 or 1; m is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; w is 0 or 1, x is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t+u+v+w+x+y+z≧1; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least 90% identity to sequences set forth in Table 4. In one embodiment of formula IX, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula IX, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.
The embodiments of formulae IV-VIII encompass CFXTEN configurations wherein one or more XTEN of lengths ranging from about 6 amino acids to ≧1000 amino acids (e.g., sequences selected from any one of Tables 3, 4, and 13-17 or fragments thereof, or sequences exhibiting at least about 90-99% or more sequence identity thereto) are inserted and linked between adjoining domains of the factor VIII or are linked to the N- or C-terminus of the FVIII. In other embodiments of formulae V-VIII, the invention further provides configurations wherein the XTEN are linked to FVIII domains via spacer sequences which can optionally comprise amino acids compatible with restrictions sites or can include cleavage sequences (e.g., the sequences of Tables 11 and 12, described more fully below) such that the XTEN encoding sequence can be, in the case of a restriction site, integrated into a CFXTEN construct and, in the case of a cleavage sequence, the XTEN can be released from the fusion protein by the action of a protease appropriate for the cleavage sequence.
The embodiments of formulae VI-VIII differ from those of formula V in that the FVIII component of formulae VI-VIII are only the B-domain deleted forms (“FVIII BDD”) of factor VIII that retain short residual sequences of the B-domain, non-limiting examples of sequences of which are provided in Table 1, wherein one or more XTEN or fragments of XTEN of lengths ranging from about 6 amino acids to ≧1000 amino acids (e.g., sequences selected from any one of Tables 3, 4, and 13-17) are inserted and linked between adjoining domains of the factor VIII and/or between the remnants of the B domain residues, such as those of Table 8. The embodiment of formula IX generally differs from those of the other formulae in that the one or more XTEN are each inserted within domains of FVIII rather than between domains, and/or has an XTEN linked to the C-terminus of the FVIII (or is linked via a spacer sequence to the C-terminus of the FVIII).
In some embodiments of a CFXTEN, the fusion protein comprises a B-domain deleted form of FVIII wherein the B-domain deletion starts from a first position at about amino acid residue number 745 and ends at a second position at amino acid residue number 1635 to about 1690 with reference to the full-length human factor VIII sequence and an XTEN links the first position and the second position of the B-domain deletion. In one embodiment of the foregoing, the first position and the second position of the B-domain deletion are selected from the positions of Table 8. In another embodiment of the foregoing, at least one XTEN links the first and second position wherein the at least one XTEN links factor VIII amino acid residue 745 and amino acid residue 1640, or amino acid residue 741 and amino acid residue 1640, or amino acid residue 741 and amino acid residue 1690, or amino acid residue 745 and amino acid residue 1667, or amino acid residue 745 and amino acid residue 1657, or amino acid residue 745 and amino acid residue 1657, or amino acid residue 747 and amino acid residue 1642, or amino acid residue 751 and amino acid residue 1667. In one embodiment of the CFXTEN, wherein the factor VIII comprises an XTEN linking a first position and a second position of a B-domain deletion described in the embodiments of this paragraph, the XTEN is a sequence having at least 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity compared to a sequence of comparable length selected from any one of Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned, wherein the CFXTEN retains at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII.
The invention contemplates all possible permutations of insertions of XTEN between or within the domains of FVIII or at or proximal to the insertion points of Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9 , with optional linking of an additional XTEN to the N- or C-terminus of the FVIII, optionally linked via an additional cleavage sequence selected from Table 12, resulting in a CFXTEN composition; non-limiting examples of which are portrayed in FIGS. 5 and 12 .
In one embodiment, the CFXTEN comprises a FVIII BDD sequence of Table 1 in which one or more XTEN that each has at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or more sequence identity compared to a sequence from any one of Tables 3, 4, and 13-17 or fragments thereof are inserted between any two of the residual B domain amino acids of the FVIII BDD sequence, resulting in a single chain FVIII fusion protein, wherein the CFXTEN retains at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII. In the foregoing embodiment, the CFXTEN can have an additional XTEN sequence of any one of Tables 4, and 13-17 linked to the N- or C-terminus of the fusion protein. In another embodiment, a CFXTEN comprises at least a first XTEN inserted at a site set forth in Table 8, wherein the CFXTEN retains at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII. In one embodiment of a fusion protein of formula VII, the CFXTEN comprises a FVIII BDD sequence of Table 1 in which two or more XTEN that each has at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity compared to a sequence from any one of Tables 3, 4, and 13-17 or fragments thereof are linked to a FVIII-BDD sequence in which at least one XTEN is inserted from about 3 to about 20 amino acid residues to the C-terminus side of the FVIII cleavage site amino acid R740 and from about 3 to about 20 amino acid residues to the N-terminus side of the FVIII cleavage site amino acid R1689 of the residual B domain amino acids of the FVIII BDD sequence, resulting in a single chain FVIII fusion protein, and one or two XTEN are linked by a cleavage sequence to the N- and/or C-terminus of the FVIII-BDD sequence, wherein the CFXTEN exhibits at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII after release of the XTEN by cleavage of the cleavage sequences.
In one embodiment, the A3 domain comprises an a3 acidic region or a portion thereof. In another embodiment, at least one XTEN is inserted within the a3 acidic region or the portion thereof, N-terminus of the a3 acidic region or the portion thereof, C-terminus of the a3 acidic region or the portion thereof, or a combination thereof. In certain embodiments, at least one XTEN is inserted within the C2 domain, N-terminus of C2 domain, C-terminus of C2 domain, or a combination thereof. In still other embodiments, the Factor VIII comprises all or portion of B domain. In yet other embodiments, at least one XTEN is inserted within all or a portion of B domain, N-terminus of B domain, C-terminus of B domain, or a combination thereof.
2. CFXTEN Fusion Protein Configurations with Internal XTEN
In another aspect, the invention provides CFXTEN configured with one or more XTEN sequences located internal to the FVIII sequence. In one embodiment, invention provides CFXTEN configured with one or more XTEN sequences located internal to the FVIII sequence to confer properties such as, but not limited to, increased stability, increased resistance to proteases, increased resistance to clearance mechanisms including but not limiting to interaction with clearance receptors or FVIII inhibitors, and increased hydrophilicity, compared to FVIII without the incorporated XTEN.
The invention contemplates that different configurations or sequence variants of FVIII can be utilized as the platform into which one or more XTEN are inserted. These configurations include, but are not limited to, native FVIII, FVIII BDD, and single chain FVIII (scFVIII), and variants of those configurations. In the case of scFVIII, the invention provides CFXTEN that can be constructed by replacing one or multiple amino acids of the processing site of FVIII. In one embodiment, the scFVIII utilized in the CFXTEN is created by replacing the R1648 in the FVIII sequence RHQREITR (SEQ ID NO: 1698) with glycine or alanine to prevent proteolytic processing to the heterodimer form. It is specifically contemplated that any of the CFXTEN embodiments disclosed herein with a 1648 FVIII residue can have a glycine or alanine substitution for the arginine at position 1648. In some embodiments, the invention provides CFXTEN comprising scFVIII wherein parts of the sequence surrounding the R1648 processing site are replaced with XTEN, as illustrated in FIGS. 10A and 10B . In one embodiment, at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% or more of the B-domain is replaced with an XTEN sequence disclosed herein, including one or more of the R740, R1648, or R1689 cleavage sites. In another embodiment, the CFXTEN has the FVIII sequence of the B-domain between the FXIa cleavage sites at R740 and R1689 (with at least 1-5 adjacent B-domain amino acids also retained between the cut site and the start of the XTEN to permit the protease to access the cut site) replaced with XTEN. In another embodiment, the CFXTEN has the FVIII sequence of the B-domain between the FXIa cleavage site at N745 and P1640 replaced with XTEN. In other embodiments, the invention provides CFXTEN FVIII BDD sequence variants in which portions of the B-domain are deleted but only one of the FXI R740 or R1689 activation sites (and 1-5 adjacent amino acids of the B-domain) are left within the construct, wherein the XTEN remains attached at one end to either the light or heavy chain after cleavage by FXIa, as illustrated in FIGS. 5B and 5D . In one embodiment of the foregoing, the CFXTEN comprises a FVIII BDD sequence in which the amino acids between N745 to P1640 or between S743 to Q1638 or between P747 to V1642 or between N745 and Q1656 or between N745 and S1657 or between N745 and T1667 or between N745 and Q1686 or between R747 and V1642 or between T751 and T1667 are deleted and an XTEN sequence is linked between these amino acids, connecting the heavy and light chains, and can further comprise additional XTEN inserted either in external surface loops, between FVIII domains, or at the N- or C-termini of the FVIII BDD sequence, such as one or more insertion sites from Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9 . In another embodiment of the foregoing, the CFXTEN comprises a FVIII BDD sequence in which the amino acids between K713 to Q1686 or between residues 741 and 1648 are deleted and an XTEN linked between the two amino acids, and additional XTEN can be inserted either in surface loops, between FVIII domains, or at the N- or C-termini of the FVIII BDD sequence, including but not limited to one or more insertion sites from Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9 . In some embodiments such CFXTEN sequences can have one or more XTEN exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to an XTEN sequence from any one of Tables 4 and 13-17.
The invention contemplates other CFXTEN with internal XTEN in various configurations; schematics of exemplary configurations are illustrated in FIGS. 5 and 10 . The regions suitable for XTEN insertion sites include the known domain boundaries of FVIII, exon boundaries, known surface (external) loops and solvent accessible surface area sites identified by X-ray crystallography analysis, and structure models derived from molecular dynamic simulations of FVIII, regions with a low degree of order (assessed by programs described in FIG. 7 legend), regions of low homology/lack of conservation across different species, and hydrophilic regions. In another embodiment, XTEN insertion sites were selected based on FVIII putative clearance receptor binding sites. In another embodiment, CFXTEN comprises XTEN inserted at locations not within close proximity to mutations implicated in hemophilia A listed in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database were eliminated (Kemball-Cook G, et al. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4. Nucleic Acids Res. (1998) 26(1):216-219). In another embodiment, potential sites for XTEN insertion include residues within FVIII epitopes that are capable of being bound by anti-FVIII antibodies occurring in sensitized hemophiliacs and that do not otherwise serve as protein interactive sites. Regions and/or sites that are considered for exclusion as XTEN insertion sites include residues/regions of factor VIII that are important in various interactions including other clotting proteins, residues surrounding each arginine activating/inactivating cleavage site acted on by the proteases thrombin, factor Xa, activated protein C, residues surrounding the signal peptide processing site (residue 1) if the construct contains the signal peptide, regions known to interact with other proteins such as FIXa, FX/FXa, thrombin, activated protein C, protein S cofactor to Protein C, von Willebrand factor, sites known to interact with phospholipid cofactors in coagulation, residues involved in domain interactions, residues coordinating Ca ++ or Cu ++ ions, cysteine residues involved in S—S intramolecular bonds, documented amino acid insertion and point mutation sites in FVIII produced in hemophilia A subjects affecting procoagulant activity, and mutation sites in FVIII made in a research lab that affect procoagulant activity. Sites considered for either insertion (to prolong half-life) or for exclusion (needed to remove spent FVIIIa or FXa) include regions known to interact with heparin sulfate proteoglycan (HSPG) or low-density lipoprotein receptor-related protein (LPR).
By analysis of the foregoing criteria, as described in Example 34, different insertion sites or ranges of insertions sites across the FVIII BDD sequence have been identified and/or confirmed as candidates for insertion of XTEN, non-limiting examples of which are listed in Table 5, Table 6, Table 7, Table 8, and Table 9 and are shown schematically in FIGS. 8 and 9 . In one embodiment, CFXTEN comprise XTEN insertions between the individual domains of FVIII, i.e., between the A1 and A2, or between the A2 and the B, or between the B and the A3, or between the A3 and the C1, or between the C1 and the C2 domains. In another embodiment, CFXTEN comprises XTEN inserted within the B domain or between remnant residues of the BDD sequence. In another embodiment, CFXTEN comprises XTEN inserted at known exon boundaries of the encoding FVIII gene as exons represent evolutionary conserved sequence modules that have a high probability of functioning in the context of other protein sequences. In another embodiment, CFXTEN comprise XTEN inserted within surface loops identified by the x-ray structure of FVIII. In another embodiment, CFXTEN comprise XTEN inserted within regions of low order identified as having low or no detected electron density by X-ray structure analysis. In another embodiment, CFXTEN comprise XTEN inserted within regions of low order, predicted by structure prediction algorithms such as, but not limited to FoldIndex, RONN, and Kyte & Doolitlle algorithms. In another embodiment, CFXTEN comprise XTEN inserted within sequence areas of high frequency of hydrophilic amino acids. In another embodiment, CFXTEN comprise XTEN inserted within epitopes capable of being bound by naturally-occurring anti-FVIII antibodies in sensitized hemophiliacs. In another embodiment, CFXTEN comprise XTEN inserted within sequence areas of low sequence conservation and/or differences in sequence segment length across FVIII sequences from different species. In another embodiment, CFXTEN comprise XTEN linked to the N-terminus and/or C-terminus. In another embodiment, the invention provides CFXTEN configurations with inserted XTEN selected from two or more of the criteria from the embodiments listed above. In another embodiment, the invention provides CFXTEN configurations with at least one, alternatively at least two, alternatively at least three, alternatively at least four, alternatively at least five or more XTEN inserted into a factor VIII sequence wherein the points of insertion are at or proximal to the N- or C-terminus side of the at least one, two, three, four, or five, or six or more amino acids selected from the insertion residue amino acids of Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9 , or alternatively within one, or within two, or within three, or within four, or within five, or within six amino acids of the insertion residue amino acids from Table 5, Table 6, Table 7, Table 8, and Table 9, or within the various spans of the insertion residue amino acids schematically portrayed for an exemplary FVIII BDD sequence in FIG. 9 .
As described above, the one or more internally-located XTEN or a fragment of XTEN can have a sequence length of 6 to 1000 or more amino acid residues. In some embodiments, wherein the CFXTEN have one or two or three or four or five or more XTEN sequences internal to the FVIII, the XTEN sequences can be identical or can be different. In one embodiment, each internally-located XTEN has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to comparable lengths or fragments of XTEN or motifs selected from any one of Tables 3, 4, and 13-17, when optimally aligned. In another embodiment, the invention provides a CFXTEN configured with one or more XTEN inserted internal to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, wherein the insertions are located at the insertion points or range of insertion points indicated in Table 5, Table 6, Table 7, Table 8, and Table 9, FIG. 8 or within the range of insertions as illustrated in FIG. 9 . It will be understood by those of skill in the art that an XTEN inserted within the FVIII sequence at an insertion point of Table 5, Table 6, Table 7, Table 8, and Table 9 is linked by its N- and C-termini to flanking FVIII amino acids (or via a linking spacer or cleavage sequences, as described above), while an XTEN linked to the N- or C-terminus of FVIII would only be linked to a single FVIII amino acid (or to a linking spacer or cleavage sequence amino acid, as described above). By way of example only, variations of CFXTEN with three internal XTEN could have: XTEN (as described herein) incorporated between FVIII BDD residues 741 and 1640, residues 18 and 19, and residues 1656 and 1657; or XTEN incorporated between FVIII BDD residues 741 and 1640, residues 1900 and 1901, and at the C-terminus at residue 2332; or XTEN incorporated between FVIII BDD residues 26 and 27, residues 1656 and 1657, and residues 1900 and 1901; or XTEN incorporated between FVIII BDD residues 741 and 1640, residues 1900 and 1901, and at the C-terminus at residue 2332.
In evaluating the CFXTEN fusion proteins with XTEN inserted in the locations from Table 5, it was discovered that insertions in certain regions of the FVIII sequence resulted in CFXTEN with good expression and retention of procoagulant activity. Accordingly, in preferred embodiments, the invention provides CFXTEN fusion proteins configured with one, or two, or three, or four, or five, or six or more XTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an XTEN selected from any one of Tables 4, and 13-17 inserted internal or linked to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, wherein the insertions are located at an insertion point within one, or two, or three, or four, or five, or six or more ranges set forth in Table 7. In the foregoing embodiments, the CFXTEN fusion proteins with the XTEN insertions retain at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the procoagulant activity compared to the corresponding FVIII not linked to XTEN.
In evaluating the CFXTEN fusion proteins with XTEN inserted in one or more locations from Table 5, it was surprisingly discovered that a high percentage of fusion proteins with the XTEN insertions retained procoagulant activity, as described in Example 25. Accordingly, the invention provides CFXTEN fusion proteins configured with one, two, three, four, five, six or more XTEN wherein the resulting fusion protein exhibits at least about 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% or more of the procoagulant activity compared to the corresponding FVIII not linked to XTEN when assayed by a coagulation assay described herein. In a preferred embodiment, the invention provides CFXTEN fusion proteins comprising one, or two, or three, or four, or five, or six or more XTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an XTEN selected from any one of Tables 4, and 13-17 linked to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, wherein the insertions are located at one or more insertion points selected from Table 5, Table 6, Table 7, Table 8, and Table 9, and wherein the resulting fusion protein exhibits at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% or more procoagulant activity compared to the corresponding FVIII not linked to XTEN, when assayed in vitro by an assay described herein (e.g., a chromogenic assay). As the subject CFXTEN fusion proteins typically exhibit increased terminal half-life compared to native FVIII, it will be appreciated by one of skill in the art that a CFXTEN with lower procoagulant activity relative to an equimolar amount of native FVIII would nevertheless be acceptable when administered as a therapeutic composition to a subject in need thereof. In another embodiment, the CFXTEN fusion proteins comprising one, or two, or three, or four, or five or more XTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an XTEN selected from any one of Tables 4, and 13-17 linked to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, wherein the insertions are located at one or more insertion points or the range of insertion points selected from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein the resulting fusion protein exhibits at least about 0.5 IU/ml, or at least about 0.75 IU/ml, or at least about 1.0 IU/ml, or at least about 1.5 IU/ml, or at least about 2.0 IU/ml, or at least about 2.5 IU/ml, or at least about 3 IU/ml, or at least about 4 IU/ml, or at least about 5 IU/ml, or at least about 7 IU/ml, or at least about 10 IU/ml, or at least about 20 IU/ml, or at least about 30 IU/ml FVIII activity when expressed in cell culture medium and assayed in a chromogenic assay, wherein the culture and expression are according to methods described herein; e.g., the methods of Example 25.
It is believed that the discovery of the insertions sites wherein the FVIII retains at least a portion of its procoagulant activity would also permit the insertion of other peptides and polypeptides with either unstructured or structured characteristics that are associated with the prolongation of half-life when fused to a FVIII protein in one or more of those same sites. Non-limiting examples include albumin, albumin fragments, Fc fragments of immunoglobulins, the 3 subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, a HAP sequence, a transferrin, the PAS polypeptides of U.S. Pat Application No. 20100292130, polyglycine linkers, polyserine linkers, peptides and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) with varying degrees of secondary structure from less than 50% to greater than 50%, amongst others, would be suitable for insertion in the identified active insertions sites of FVIII.
In the fusion protein embodiments described herein, the CFXTEN fusion protein can further comprise one or more cleavage sequence from Table 12 or other sequences known in the art, the cleavage sequence being located between or within 6 amino acid residues of the intersection of the FVIII and the XTEN sequences, which may include two cleavage sequences in a given internal XTEN sequence. In one embodiment, the CFXTEN comprising cleavage sequences has two identical cleavage sequences, each located at or near the respective ends of one or more internal XTEN such that the XTEN is released from the fusion protein when cleaved by the protease that binds to and cleaves that sequence. The sequences that can be cleaved are described more fully below and exemplary sequences are provided
TABLE 5 Insertion locations for XTEN linked to the FVIII BDD sequence XTEN FVIII BDD Insertion Insertion Downstream FVIII No. Point Residue Sequence Domain 1 0 (N-terminus) ATR A1 2 3 R RYY A1 3 17 M QSD A1 4 18 Q SDL A1 5 22 G ELP A1 6 24 L PVD A1 7 26 V DAR A1 8 28 A RFP A1 9 32 P RVP A1 10 38 F PFN A1 11 40 F NTS A1 12 41 N TSV A1 13 60 N IAK A1 14 61 I AKP A1 15 65 R PPW A1 16 81 Y DTV A1 17 111 G AEY A1 18 116 D QTS A1 19 119 S QRE A1 20 120 Q REK A1 21 128 V FPG A1 22 129 F PGG A1 23 130 P GGS A1 24 182 G SLA A1 25 185 A KEK A1 26 188 K TQT A1 27 205 G KSW A1 28 210 S ETK A1 29 211 E TKN A1 30 216 L MQD A1 31 220 R DAA A1 32 222 A ASA A1 33 223 A SAR A1 34 224 S ARA A1 35 230 K MHT A1 36 243 P GLI A1 37 244 G LIG A1 38 250 R KSV A1 39 318 D GME A1 40 333 P QLR A1 42 334 Q LRM A1 43 336 R MKN a1 44 339 N NEE a1 45 345 D YDD a1 46 357 V VRF a1 47 367 S FIQ a1 48 370 S RPY a1 49 375 A KKH A2 50 376 K KHP A2 51 378 H PKT A2 52 399 V LAP A2 53 403 D DRS A2 54 405 R SYK A2 55 409 S QYL A2 56 416 P QRI A2 57 434 E TFK A2 58 438 T REA A2 59 441 A IQH A2 60 442 I QHE A2 61 463 I IFK A2 62 487 Y SRR A2 63 490 R LPK A2 64 492 P KGV A2 65 493 K GVK A2 66 494 G VKH A2 67 500 D FPI A2 68 506 G EIF A2 69 518 E DGP A2 70 556 K ESV A2 71 565 Q IMS A2 72 566 I MSD A2 73 598 P AGV A2 74 599 A GVQ A2 75 603 L EDP A2 76 616 S ING A2 77 686 G LWI A2 78 713 K NTG A2 79 719 Y EDS A2 80 730 L LSK A2 81 733 K NNA A2 82 745 N PPV B 83 1640 P PVL B 84 1652 R TTL B 85 1656 Q SDQ A3 86 1685 N QSP A3 87 1711 M SSS A3 88 1713 S SPH A3 89 1720 N RAQ A3 90 1724 S GSV A3 91 1725 G SVP A3 92 1726 S VPQ A3 93 1741 G SFT A3 94 1744 T QPL A3 95 1749 R GEL A3 96 1773 V TFR A3 97 1792 Y EED A3 98 1793 E EDQ A3 99 1796 Q RQG A3 100 1798 Q GAE A3 101 1799 G AEP A3 102 1802 P RKN A3 103 1803 R KNF A3 104 1807 V KPN A3 105 1808 K PNE A3 106 1827 K DEF A3 107 1844 E KDV A3 108 1861 N TLN A3 109 1863 L NPA A3 110 1896 E RNC A3 111 1900 R APC A3 112 1904 N IQM A3 113 1905 I QME A3 114 1910 P TFK A3 115 1920 A ING A3 116 1937 D QRI A3 117 1981 G VFE A3 118 2019 N KCQ A3 119 2020 K CQT C1 120 2044 G QWA C1 121 2068 F SWI C1 122 2073 V DLL C1 123 2090 R QKF C1 124 2092 K FSS C1 125 2093 F SSL C1 126 2111 K WQT C1 127 2115 Y RGN C1 128 2120 T GTL C1 129 2125 V FFG C1 130 2171 L NSC C1 131 2173 S CSM C2 132 2188 A QIT C2 133 2223 V NNP C2 134 2224 N NPK C2 135 2227 K EWL C2 136 2268 G HQW C2 137 2277 N GKV C2 138 2278 G KVK C2 139 2290 F TPV C2 140 2332 Y C terminus CT of FVIII
Indicates an insertion point for XTEN based on the amino acid number of mature full-length human FVIII, wherein the insertion could be either on the N- or C-terminal side of the indicated amino acid Downstream sequence in FVIII BDD with 746-1639 deletion






TABLE 6



Exemplary insertion locations for XTEN linked to

a FVIII polypeptide













Distance


XTEN

FVIII BDD

from


Insertion
Insertion
Downstream
FVIII
insertion

No.
Point
Residue
Sequence
Domain
residue










9
32
P
RVP
A1
−3, +6



31
220
R
DAA
A1
—



34
224
S
ARA
A1
+5



43
336
R
MKN
a1
−1, +6



44
339
N
NEE
a1
−4, +5



52
399
V
LAP
A2
−6, +3



56
416
P
QRI
A2
+6



75
603
L
EDP
A2
_6, +6



85
1656
Q
SDQ
B
−3, +6



87
1711
M
SSS
A3
−6, +1



91
1725
G
SVP
A3
+6



113
1905
I
QME
A3
+6



114
1910
P
TFK
A3
−5, +6




















































Distance from insertion residue refers to the relative number of amino acids away from the N-terminus (negative numbers) or C-terminus (positive numbers) of the designated insertion residue (residue “0”) where an insertion may be made. The designation “−x” refers to an insertion site which is x amino acids away on the N-terminal side of the designated insertion residue. Similarly, the designation “+x” refers to an insertion site which is x amino acids away on the C-terminal side of the designated insertion residue.
For example, “−1, +2” indicates that the insertion is made at the N-terminus or C-terminus, if amino acid residues denoted −1, 0, +1 or +2.
TABLE 7 Further exemplary insertion locations for XTEN linked to a FVIII polypeptide XTEN Insertion First Insertion No. Point Range Residue FVIII Domain 3 18-32 Q A1 8 40 F A1 18 211-224 E A1 27 336-403 R A1, A2 43 599 A A2 47 745-1640 N B 50 1656-1728 Q B, A3 57 1796-1804 R A3 65 1900-1912 R A3 81 2171-2332 L C1, C2
indicates range of insertion sites numbered relative to the amino acid number of mature human FVIII

TABLE 8 Exemplary XTEN insertion locations within B-domain deleted variants of a FVIII polypeptide XTEN Insertion First Insertion Second Insertion Point Range Residue Residue 740-1640 R P 740-1690 R S 741-1648 S R 743-1638 S Q 745-1638 N Q 745-1640 N P 745-1656 N Q 745-1657 N S 745-1667 N T 745-1686 N Q 747-1642 R V 751-1667 T T
indicates the amino acids linked within the B-domain deleted variant and adjacent A3 domain, with the amino acids numbered relative to the amino acid number of mature human FVIII indicates the amino acids linked by an XTEN inserted in the BDD-FVIII






TABLE 9



Exemplary insertion locations for XTEN linked to

a FVIII polypeptide resulting in procoagulant

activity








XTEN

FVIII BDD



Insertion
Insertion
Downstream
FVIII

No.
Point
Residue
Sequence
Domain









2
3
R
RYY
A1



4
18
Q
SDL
A1



5
22
G
ELP
A1



7
26
V
DAR
A1



9
32
P
RVP
A1



11
40
F
NTS
A1



18
116
D
QTS
A1



19
119
S
QRE
A1



26
188
K
TQT
A1



29
211
E
TKN
A1



30
216
L
MQD
A1



31
220
R
DAA
A1



34
224
S
ARA
A1



35
230
K
MHT
A1



40
333
P
QLR
A1



43
336
R
MKN
a1



44
339
N
NEE
a1



52
399
V
LAP
A2



53
403
D
DRS
A2



55
409
S
QYL
A2



56
416
P
QRI
A2



60
442
I
QHE
A2



62
487
Y
SRR
A2



63
490
R
LPK
A2



66
494
G
VKH
A2



69
518
E
DGP
A2



74
599
A
GVQ
A2



75
603
L
EDP
A2



78
713
K
NTG
A2



82
745
N
PPV
B



85
1656
Q
SDQ
A3



87
1711
M
SSS
A3



89
1720
N
RAQ
A3



91
1725
G
SVP
A3



99
1796
Q
RQG
A3



102
1802
P
RKN
A3



110
1896
E
RNC
A3



111
1900
R
APC
A3



112
1904
N
IQM
A3



113
1905
I
QME
A3



114
1910
P
TFK
A3



121
2068
F
SWI
C1



130
2171
L
NSC
C1



135
2227
K
EWL
C2



137
2277
N
GKV
C2



140
2332
Y
C terminus of
C2




FVIII



Downstream sequence in FVIII BDD with 746-1639 deletion






















































































































In another aspect, the invention provides libraries of components and methods to create the libraries derived from nucleotides encoding FVIII segments, XTEN, and FVIII segments linked to XTEN that are useful in the preparation of genes encoding the subject CFXTEN. In a first step, a library of genes encoding FVIII and XTEN inserted into the various single sites at or within 1-6 amino acids of an insertion site identified in Table 5 or illustrated in FIGS. 8-9 are created, expressed, and the CFXTEN recovered and evaluated for activity and pharmacokinetics as illustrated in FIG. 15 . Those CFXTEN showing enhanced properties are then used to create genes encoding a FVIII segment and the insertion site plus an XTEN, with components from each enhanced insertion represented in the library, as illustrated in FIG. 11 . In one embodiment, the library components are assembled using standard recombinant techniques in combinatorial fashion, as illustrated in FIG. 11 , resulting in permutations of CFXTEN with multiple internal and N- and C-terminus XTEN, that can include the insertion sites of or proximal to those Table 5, Table 6, Table 7, Table 8 and Table 9, or as illustrated in FIGS. 8-9 . The resulting constructs would then be evaluated for activity and enhanced pharmacokinetics, and those candidates resulting in CFXTEN with enhanced properties, e.g., reduced active clearance, resistance to proteases, reduced immunogenicity, and enhance pharmacokinetics, compared to FVIII not linked to XTEN, are evaluated further.
3. XTEN Permissive Loops
As described in detail elsewhere herein and as illustrated in FIGS. 33-36 , the inventors have recognized that each FVIII “A” domain comprise at least two “XTEN permissive loops” into which XTEN sequences can be inserted without eliminating procoagulant activity of the recombinant protein, or the ability of the recombinant proteins to be expressed in vivo or in vitro in a host cell. The inventors have identified the XTEN permissive loops as regions with, among other attributes, high surface or solvent exposure and high conformational flexibility. The A1 domain comprises an XTEN permissive loop-1 (A1-1) region and an XTEN permissive loop-2 (A1-2) region, the A2 domain comprises an XTEN permissive loop-1 (A2-1) region and an XTEN permissive loop-2 (A2-2) region, the A3 domain comprises an XTEN permissive loop-1 (A3-1) region and an XTEN permissive loop-2 (A3-2) region.
In certain aspects a recombinant FVIII protein as described above comprises at least one XTEN sequence inserted into at least one of the XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. In certain aspects a recombinant FVIII protein as described above comprises at least two XTEN sequences inserted into FVIII, e.g., into two different XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. Alternatively, a recombinant FVIII protein as described above can comprise two or more XTEN sequences inserted into a single XTEN permissive loop either with our without XTEN sequences inserted into other XTEN permissive loops, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. In certain aspects a recombinant FVIII protein as described above can comprise at least one XTEN sequence inserted into at least one of the XTEN permissive loops as described above, and can further comprise one or more XTEN sequences inserted into a3, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. In certain aspects, a recombinant FVIII protein of the invention can comprise three, four, five, six or more XTEN sequences inserted into one or more XTEN permissive loops or into a3, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell.
In certain aspects a recombinant FVIII protein as described above comprises at least one XTEN sequence inserted into a3, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. In certain aspects a recombinant FVIII protein of the invention comprises at least one XTEN sequence inserted into a3, and further comprises one or more XTEN sequences inserted into one or more XTEN permissive loops as described above, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell.
The inventors have recognized that a recombinant FVIII protein of the invention comprises at least two XTEN permissive loops in each of the FVIII A domain regions which allows for insertion of an XTEN sequence while having procoagulant activity and still being able to be expressed in vivo or in vitro by a host cell. Various crystal structures of FVIII have been determined, of varying degrees of resolution. These structures of FVIII and FVIIIa, determined by X-ray crystallography and molecular dynamic simulation, were used to generate models of accessible surface area and conformational flexibility for FVIII. For example, the crystal structure of human FVIII has been determined by Shen et al. Blood 111: 1240-1247 (2008) and Ngo et al. Structure 16: 597-606 (2008). The data for these structures is available from the Protein Data Bank (pdb.org) under Accession Numbers 2R7E and 3CDZ, respectively.
The predicted secondary structure of the heavy and light chains of human FVIII according to the Shen et al. crystal structure is reproduced in FIGS. 37A and 37B . The various beta strands predicted from the Shen et al. crystal structure are numbered consecutively in FIGS. 8A and 8B . In certain embodiments, the XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1, and A3-2 are contained within surface-exposed, flexible loop structures in the A domains of FVIII. A1-1 is located between beta strand 1 and beta strand 2, A1-2 is located between beta strand 11 and beta strand 12, A2-1 is located between beta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33, A3-1 is located between beta strand 38 and beta strand 39 and A3-2 is located between beta strand 45 and beta strand 46, according to the secondary structure of mature FVIII stored as Accession Number 2R7E of the PDB database (PDB:2R7E) and as shown in FIGS. 8A and 8B . The secondary structure of PDB Accession Number 2R7E shown in FIGS. 8A and 8B corresponds to the standardized secondary structure assignment according to the DSSP program (Kabsch and Sander, Biopolymers, 22:2577-2637 (1983)). The DSSP secondary structure of the mature FVIII stored as PDB Accession Number 2R7E can be accessed at the DSSP database, available at the world wide web site swift.cmbi.ru.nl/gv/dssp/ (last accessed Feb. 9, 2012) (Joosten et al., 39 (Suppl. 1): D411-D419 (2010)).
In certain aspects, a surface-exposed, flexible loop structure comprising A1-1 corresponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 of FIG. 30 . In certain aspects, A1-1 corresponds to a region in native mature human FVIII from about amino acid 18 to about amino acid 41 of FIG. 30 . In certain aspects, the surface-exposed, flexible loop structure comprising A1-2 corresponds to a region in native mature human FVIII from about amino acid 201 to about amino acid 232 of FIG. 30 . In certain aspects, A1-2 corresponds to a region in native mature human FVIII from about amino acid 218 to about amino acid 229 of FIG. 30 . In certain aspects, the surface-exposed, flexible loop structure comprising A2-1 corresponds to a region in native mature human FVIII from about amino acid 395 to about amino acid 421 of FIG. 30 . In certain aspects, A2-1 corresponds to a region in native mature human FVIII from about amino acid 397 to about amino acid 418 of FIG. 30 . In certain aspects, the surface-exposed, flexible loop structure comprising A2-2 corresponds to a region in native mature human FVIII from about amino acid 577 to about amino acid 635 of FIG. 30 . In certain aspects, A2-2 corresponds to a region in native mature human FVIII from about amino acid 595 to about amino acid 607 of FIG. 30 . In certain aspects, the surface-exposed, flexible loop structure comprising A3-1 corresponds to a region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 of FIG. 30 . In certain aspects, A3-1 corresponds to a region in native mature human FVIII from about amino acid 1711 to about amino acid 1725 of FIG. 30 . In certain aspects, the surface-exposed, flexible loop structure comprising A3-2 corresponds to a region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 of FIG. 3 . In certain aspects, A3-2 corresponds to a region in native mature human FVIII from about amino acid 1899 to about amino acid 1911 of FIG. 30 .
In certain aspects a recombinant FVIII protein of the invention comprises one or more XTEN sequences inserted into one or more XTEN permissive loops of FVIII, or into the a3 region, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. XTEN sequences to be inserted include those that increase the in vivo half-life or the in vivo or in vitro stability of FVIII.
In certain aspects, a recombinant FVIII protein of the invention comprises an XTEN sequences inserted immediately downstream of one or more amino acids corresponding to one or more amino acids in mature native human FVIII including, but not limited to: amino acid 18 of FIG. 30 , amino acid 26 of FIG. 30 , amino acid 40 of FIG. 30 , amino acid 220 of FIG. 30 , amino acid 224 of FIG. 30 , amino acid 399 of FIG. 30 , amino acid 403 of FIG. 30 , amino acid 599 of FIG. 30 , amino acid 603 of FIG. 30 , amino acid 1711 of FIG. 30 , amino acid 1720 of FIG. 30 , amino acid 1725 of FIG. 30 , amino acid 1900 of FIG. 30 , amino acid 1905 of FIG. 30 , amino acid 1910 of FIG. 30 , or any combination thereof, including corresponding insertions in BDD-variants of FVIII described herein.
In certain aspects, a recombinant FVIII protein of the invention comprises at least one XTEN sequence inserted into the a3 region of FVIII, either alone or in combination with one or more XTEN sequences being inserted into the XTEN permissive loops of the A domains (e.g., A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2 as described above), wherein the recombinant FVIII protein has procoagulant activity and can be expressed in vivo or in vitro in a host cell. In certain aspects, at least one XTEN sequence is inserted into the a3 region immediately downstream of an amino acid which corresponds to amino acid 1656 of FIG. 30 . In certain aspects, a recombinant FVIII protein of the invention comprises an XTEN sequence inserted into the a3 region as described, and further includes one or more XTEN sequences inserted immediately downstream of one or more amino acids corresponding to one or more amino acids in mature native human FVIII including, but not limited to: amino acid 18 of FIG. 30 , amino acid 26 of FIG. 30 , amino acid 40 of FIG. 30 , amino acid 220 of FIG. 30 , amino acid 224 of FIG. 30 , amino acid 399 of FIG. 30 , amino acid 403 of FIG. 30 , amino acid 599 of FIG. 30 , amino acid 603 of FIG. 30 , amino acid 1711 of FIG. 30 , amino acid 1720 of FIG. 30 , amino acid 1725 of FIG. 30 , amino acid 1900 of FIG. 30 , amino acid 1905 of FIG. 30 , amino acid 1910 of FIG. 30 , or any combination thereof.
It will be understood by one of skill in the art that the foregoing aspects of permissive loops of a native FVIII protein into which a heterologous protein can be inserted are also applicable to the B-domain deleted FVIII variants described herein; e.g., sequences set forth in Table 1. In practicing the present invention, it will be understood that a BDD-FVIII sequence of Table 1 can be substituted for the recombinant FVIII protein of the various embodiments described above, and it is believed that the resulting constructs will similarly retain procoagulant activity.
4. Interference with FVIII Binding Agents
It is an object of the present invention to provide procoagulant CFXTEN fusion protein compositions for use in human patients suffering from coagulopathies, such as haemophilia A, who have native or acquired antibodies, inhibitors, or other proteins or molecules that bind to FVIII that affect the activity or half-life of CFXTEN fusion proteins, wherein the CFXTEN retain a greater amount of procoagulant activity compared to the corresponding FVIII not linked to XTEN. As used herein, “FVIII binding agent” means any molecule capable of binding to native FVIII or to a recombinant factor VIII fusion protein of the invention comprising factor VIII or a fragment thereof, whether native, derived, or produced recombinantly. It is specifically contemplated that FVIII binding agent includes anti-FVIII antibodies and FVIII inhibitors, amongst other proteins capable of specifically binding to FVIII. In one aspect, the invention provides procoagulant CFXTEN fusion proteins that exhibit reduced binding to an anti-FVIII antibody or FVIII inhibitor that interferes with the procoagulant activity of FVIII. As used herein, “anti-FVIII antibody” or “anti-factor VIII antibody” means an antibody capable of binding FVIII or a FVIII component of a CFXTEN of the invention, said antibody including but not limited to the antibodies of Table 10 or polyclonal antibody from a hemophilia A patient with FVIII inhibitors. The term antibody includes monoclonal antibodies, polyclonal antibodies, antibody fragments and antibody fragment clones. As used herein, “FVIII inhibitor” or “anti-FVIII inhibitor antibody” means an antibody capable of binding FVIII or a FVIII component of a CFXTEN of the invention and that reduces by any means the procoagulant activity of FVIII or the FVIII component of a CFXTEN. In another aspect, the invention provides CFXTEN fusion proteins that retain procoagulant activity in the presence of a FVIII inhibitor. In another aspect, the invention provides CFXTEN fusion proteins comprising FVIII that exhibit increased terminal half-life in the presence of a FVIII binding agent compared to the FVIII not linked to XTEN.
The majority of inhibitory antibodies to human factor VIII act by binding to epitopes located in the A2 domain or the C2 domain of factor VIII, disrupting specific functions associated with these domains, (U.S. Pat. No. 6,770,744; Fulcher et al. Localization of human factor FVIII inhibitor epitopes to two polypeptide fragments. Proc. Natl. Acad. Sci. USA (1985) 82:7728-7732; Scandella et al. Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of fVIII fragments expressed in Escherichia coli . Proc. Natl. Acad. Sci. USA (1988) 85:6152-6156). While 68% percent of inhibitory antibodies are reported to be directed against the A2 and/or C2 domain, 3% act against the A1 domain and 46% against the a3 acidic region (Lavigne-Lissalde, G., et al. Characteristics, mechanisms of action, and epitope mapping of anti-factor VIII antibodies. Clin Rev Allergy Immunol (2009) 37:67-79). For example, certain heavy chain-specific inhibitors react with the 18.3-kD amino-terminal segment of the A2 domain (Scandella D, et al. 1988); Lollar P et al. Inhibition of human factor VIIIa by anti-A2 subunit antibodies. J Clin Invest 1994; 93:2497). FVIII contains a phospholipid binding site in the C2 domain between amino acids 2302 and 2332, and there is also a von Willebrand factor binding site in the C2 domain that acts in conjunction with amino acids 1649-1689 in the A3 domain. The C2 domain also has epitopes that, when bound by inhibitors, block the activation of FVIII by thrombin or factor Xa. Inhibitors binding specifically to the light chain recognize epitopes in the A3 domain or a major antigenic region in the C2 domain and can result in reduced procoagulant activity by preventing the binding of FVIII to phospholipid or reducing the dissociation rate of FVIII from von Willebrand factor (Gilles J G, et al. Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction. Blood (1993) 82:2452; Shima M, et al. A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost (1993) 69:240). Non-limiting examples of monoclonal FVIII inhibitors are listed in Table 9. In patients with high-titer inhibitors, there is an increased risk of developing recurrent bleeding in particular joints, which may ultimately result in decreased quality of life, disability, or death from excessive blood loss (U.S. Pat. Application No. 20120065077; Zhang et al., Clinic. Rev. Allerg. Immunol., 37:114-124 (2009); Gouw and van den Berg, Semin. Thromb. Hemost., 35:723-734 (2009))
While not intending to be bound by any particular theory, it is believed that the unstructured characteristic of the XTEN incorporated into the CFXTEN fusion proteins permits the XTEN to adopt conformations that result in steric hindrance to inhibitors that would otherwise bind to FVIII epitopes. As illustrated in FIG. 6 , as the incorporated XTEN assumes various random coil conformations, it spatially covers regions of the FVIII component of the fusion protein and sterically interferes with the ability of an inhibitor to bind to a FVIII epitope.
In one embodiment, the invention provides CFXTEN exhibiting procoagulant activity and reduced binding in the presence of an antibody binding to the C2 domain of factor VIII compared to the corresponding factor VIII not linked to XTEN and/or to native FVIII. In another embodiment, the invention provides CFXTEN exhibiting procoagulant activity and reduced binding in the presence of an antibody binding to the A2 domain of Factor VIII compared to the corresponding factor VIII not linked to XTEN or to native FVIII. In another embodiment, the invention provides CFXTEN exhibiting procoagulant activity and reduced binding in the presence of antibodies binding to the A2 and the C2 domain of Factor VIII, compared to the corresponding factor VIII not linked to XTEN or to native FVIII.
In one embodiment, the invention provides CFXTEN exhibiting procoagulant activity and reduced binding, compared to the corresponding FVIII not linked to XTEN, in the presence of an antibody selected from the group consisting of the antibodies of Table 10. In one embodiment, the CFXTEN fusion protein exhibits reduced binding to the antibody GMA8021. In another embodiment, the CFXTEN fusion protein exhibits reduced binding to the antibody GMA8008. In another embodiment, the CFXTEN fusion protein exhibits reduced binding to the antibody ESH4. In another embodiment, the CFXTEN fusion protein exhibits reduced binding to the antibody ESH8. In another embodiment, the CFXTEN fusion protein exhibits reduced binding to the antibody B02C11. In another embodiment, the CFXTEN fusion protein exhibits reduced binding and a greater degree of procoagulant activity, compared to the corresponding FVIII not linked to XTEN, in the presence of plasma from a hemophilia A subject with polyclonal antibody FVIII inhibitors, wherein the greater degree of procoagulant activity is determined by an in vitro assay such as a Bethesda assay or other assay described herein.
The CFXTEN exhibiting reduced binding by FVIII inhibitors can have one, or two, or three, or four, or five, or six or more individual XTEN, embodiments of which are disclosed herein. In the foregoing embodiments of this paragraph, a CFXTEN exhibits at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or less binding to the antibody when assessed in vitro in an assay capable of assaying the binding of an antibody to FVIII, such as assays described herein below or those known in the art. Alternatively, the reduced binding of the subject CFXTEN to the FVIII-binding antibodies can be assessed by retention of a higher degree of procoagulant activity in the presence of the antibody compared to FVIII not linked to XTEN, as described in the Examples. Thus, in the embodiments pertaining to reduced binding by FVIII inhibitors described herein, a CFXTEN exhibits, when reacted with the anti-FVIII antibody, at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 100%, or 200%, or 300%, or 400%, or 500% or more activity in a coagulation assay (such as described herein below) compared to the corresponding FVIII not linked to XTEN and reacted with the antibody. In the foregoing, the anti-FVIII antibody can be an antibody from Table 9 or a circulating anti-FVIII antibody from a hemophilia A subject. In another embodiment, the invention provides CFXTEN in which the assayed fusion protein, when assayed utilizing the Bethesda assay and an anti-FVIII antibody selected from Table 10 or a polyclonal anti-FVIII antibody preparation such as, but not limited to, plasma from a hemophilia A subject with FVIII inhibitors, results in a Bethesda titer with at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 fewer Bethesda units compared to a FVIII not linked to XTEN and assayed under comparable conditions. In another embodiment, the invention provides CFXTEN in which the assayed fusion protein results in less than 50%, or less than 40%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 14%, or less than 13%, or less than 12%, or less than 11%, or less than 10% of the Bethesda Units compared to a FVIII not linked to XTEN when assayed under comparable conditions utilizing the Bethesda assay and a polyclonal anti-FVIII antibody preparation such as, but not limited to, plasma from a hemophilia A subject with FVIII inhibitors.
TABLE 10 Anti-factor VIII antibodies Antibody Inhibitor Titer Designation Epitope BU/mg Reference BO2C11 C2 Domain 20000 U.S. Pat. No. 6,770,744 Met2199/Phe2200 Blood (2007) 110: 4234-4242 NMC VIII-5 C2 Domain U.S. Pat. No. 6,770,744 Glu2181-Val2243 ESH2 Light Chain ADI ESH4 Light Chain 39 U.S. Pat. No. 6,770,744 2303-2332 Blood (2007) 110: 4234-4242 ESH8 C2 Domain U.S. Pat. No. 6,770,744 2248-2285 10000 Blood (2007) 110: 4234-4242 RHD5 C1 Domain WO 2005/016455 (LMBP U.S. Pat. Application 6165CB) 20090263380 LE2E9 C1 Domain U.S. Pat. Application 20090263380 Blood (2000) 95: 156-163 I54 C2 Domain 1300 Blood (2007) 110: 4234-4242 F85 C2 Domain 6 Blood (2007) 110: 4234-4242 F100 C2 Domain 5 Blood (2007) 110: 4234-4242 F137 C2 Domain 6 Blood (2007) 110: 4234-4242 I89 C2 Domain 1900 Blood (2007) 110: 4234-4242 I117 C2 Domain 1800 Blood (2007) 110: 4234-4242 I109 C2 Domain 1500 Blood (2007) 110: 4234-4242 Met2199/Phe2200 1B5 C2 Domain 930 Blood (2007) 110: 4234-4242 3C6 C2 Domain 71 Blood (2007) 110: 4234-4242 3D12 C2 Domain 2600 Blood (2007) 110: 4234-4242 Phe2196 D102 C2 Domain 3800 Blood (2007) 110: 4234-4242 3G6 C2 Domain 25000 Blood (2007) 110: 4234-4242 2-77 C2 Domain 25000 Blood (2007) 110: 4234-4242 B45 C2 Domain 21000 Blood (2007) 110: 4234-4242 B9 C2 Domain 31000 Blood (2007) 110: 4234-4242 B11 C2 Domain 3300 Blood (2007) 110: 4234-4242 B75 C2 Domain Indeterminate Blood (2007) 110: 4234-4242 D105 C2 Domain 0.8 Blood (2007) 110: 4234-4242 Val2223/Lys2227 F77 C2 Domain 26000 Blood (2007) 110: 4234-4242 F178 C2 Domain 18000 Blood (2007) 110: 4234-4242 F67 C2 Domain 21000 Blood (2007) 110: 4234-4242 G99 C2 Domain 15000 Blood (2007) 110: 4234-4242 Val2223/Lys2227 G86 C2 Domain 4300 Blood (2007) 110: 4234-4242 I14 C2 Domain 44000 Blood (2007) 110: 4234-4242 I55 C2 Domain 10000 Blood (2007) 110: 4234-4242 2-117 C2 Domain >0.4 Blood (2007) 110: 4234-4242 GMA012 A2 domain GMA 497-510; 584-593 GMA8001 A3 Domain 156 GMA GMA8002 A1 Domain <1 GMA GMA8003 C2 Domain GMA GMA8004 A1 Domain GMA GMA8005 A1 A3/A1 Domain GMA GMA8006 C2 Domain GMA GMA8008 C2 Domain 1047 GMA GMA8009 A2 Domain 7923 GMA GMA8010 LC Domain GMA GMA8011 C1 Domain 97 GMA GMA8012 A1A3 Domain 204 GMA GMA8013 A3C2 Domain 30 GMA GMA8014 C2 Domain 7799 GMA GMA8015 A2 Domain 17079 GMA GMA8016 A2 Domain <1 GMA GMA8017 A2 Domain 334 GMA GMA8018 LC Domain 242 GMA GMA8019 CR-LC Domain GMA GMA8020 A1 A3 Domain 196 GMA GMA8021 A2 Domain 33928 GMA 4A4 A2 Domain 40000 J Thromb Haemost (2009) 7: 658- 664 3E6 C2 Domain 41 Blood (2007) 110: 4234-4242
American Diagnostica Inc. internet site, URL located on the World Wide Web at americandiagnostica.com/html/Product_Detail. asp?idCategory=5&idSubCategory=104&idpro=ESH-8 as it existed on Jan. 12, 2012

Green Mountain Antibodies internet site, URL located on the World Wide Web at greenmoab.com/product_details/16316/21582.html as it existed on Jan. 12, 2012

Assays for Inhibitor and Antibody Binding
The fusion proteins of the invention may be assayed to confirm reduced binding by FVIII inhibitors using methods known in the art. The assays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, immunoradiometric assays, fluorescent immunoassays, clotting assays, factor VIII inhibitor assays to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary are described briefly below but are not intended by way of limitation.
The Bethesda assay and the Nijmegen modification of the Bethesda assay are factor VIII inhibitor assays well-known as methods to detect FVIII inhibitors (Kasper C K, et al. Proceedings: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. (1975) 34(2):612). However, the assays can be modified to assay binding of inhibitors to FVIII compositions using inhibitors such as polyclonal or monoclonal anti-FVIII antibodies, including the antibodies of Table 10, and methods such as described in Example 52. Briefly, the modified Bethesda assay involves mixing titered volumes of the test sample with an equal volume of an inhibitor at a set concentration. The mixtures are incubated for 2 hours at 37° C. prior to analysis of the factor concentration by a coagulation assay such as a chromogenic assay. Similarly, a reference plasma with native factor VIII level is incubated that then assayed as the positive control. The endpoint is the titer resulting in 50% of the FVIII activity of the positive control, reported as Bethesda units. In the Nijimegen modification of the Bethesda assay, the assay samples are stabilized with imidazole buffer and the control sample is mixed with deficient plasma instead of buffer (Verbruggen B, et al. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost. (1995) 73(2):247-251).
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISA assays can detect antibodies to FVIII independent of their ability to block the procoagulant activity of FVIII, and have been utilized for the detection of anti-FVIII developing in hemophilia A patients. In a population of 131 patients with hemophilia A with inhibitors, the ELISA technique resulted in 97.7% sensitivity and 78.8% specificity, and had a high negative predictive value (98.6%) [Martin, P. G., et al. Evaluation of a novel ELISA screening test for detection of factor VIII inhibitory antibodies in haemophiliacs. Clin Lab Haematol (1999) 21:125-128]. Other investigators have found a highly significant correlation between the Bethesda titer and the absorbance values in an ELISA assay for detecting anti-FVIII Abs (Towfighi, F., et al. Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity. Acta Haematol (2005) 114:84-90), with the added advantage of the ability to detect non-inhibitory anti-FVIII antibodies. Assay protocols comprise preparing the binding ligand, which may include a sample comprising either factor VIII polypeptide or the CFXTEN fusion protein, coating the well of a 96 well microtiter plate with the antibody, adding the ligand test sample and incubating, then adding a detection antibody and incubating prior to washing and adding a alkaline phosphatase- or peroxidase-conjugated secondary antibody and incubating for an additional period before the addition of TMB substrate and processing for reading by spectrophotometer at 450 nm. In ELISAs the antibody or inhibitor of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody or inhibitor of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antibody, the ligand may be coated to the well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).
Standard or modified coagulation assays are used to measure reduced binding of FVIII binding agents. In one exemplary method (further described in Example 28), the optimal concentration of a given FVIII inhibitor to utilize in the assay is first determined by a titration experiment using varying amounts of the inhibitory antibody incubated at 37° C. for 2 hrs with the base vector expressing wild-type FVIII containing a His/Myc double tag. The FVIII activity is measured by the Coatest assay procedure described herein. The lowest concentration that results in optimal inhibition of FVIII activity is employed in the assay. In the assay, the FVIII inhibitor antibody at the optimal concentration is mixed with individual test samples and incubated at 37° C. for 2 hrs. The resulting test samples are then collected and utilized in the Coatest activity assay, along with untreated aliquots of the CFXTEN and positive control in order to assess the residual and baseline FVIII activity for each test sample.
The invention provides methods of making CFXTEN that exhibit reduced binding to FVIII binding agents, including FVIII inhibitors, and retention of procoagulant activity. In one embodiment, the method to make a CFXTEN with reduced binding to FVIII inhibitors comprises the steps of selecting a FVIII sequence with at least 90% sequence identity to a sequence of Table 1, selecting one, two, three, four, five, or six or more XTEN each with at least 70%, or at least 80%, or at least 90%, or at least 95-99% sequence identity to XTEN sequences of comparable length from Table 4, creating expression constructs designed to locate said XTEN at or proximal to locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9, expressing and recovering the resulting CFXTEN, and assaying the resulting fusion proteins in an assay described herein in order to confirm the reduced binding of the CFXTEN fusion protein. By the inventive method, a CFXTEN exhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to a FVIII binding agent including, but not limited to the antibodies of Table 10 or anti-FVIII antibodies from a hemophilia A subject, and retains at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% procoagulant activity compared to the corresponding FVIII not linked to XTEN.
Up to 8-10% of hemophilia A patients have antibodies that bind FVIII without affecting its procoagulant properties; they are not, therefore categorized as FVIII inhibitors. However, the binding of antibodies to FVIII is believed to lead to immune complexes that are cleared by the innate immune response or are more susceptible to proteolytic degradation (Kazatchkine M D. Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with haemophilia A. Clin Exp Immunol. (1980) 39(2):315-320). Accordingly, it is an object of the invention to provide CFXTEN fusion proteins comprising one or more XTEN that exhibit reduced binding of antibodies to FVIII that are not inhibitors, wherein the degradation or clearance of the CFXTEN is reduced at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or less compared to a corresponding FVIII not linked to XTEN or to native FVIII bound by such antibodies. The reduced binding of antibodies to CFXTEN compared to FVIII not linked to XTEN or to native FVIII can be assayed by in vitro and in vivo methods. In vitro methods include the aforementioned ELISA and Western blot methods. The reduced degradation or clearance of CFXTEN can be assessed in vivo by use of animal models or in human clinical trials. In one type of trial, factor VIII or CFXTEN are administered separately, preferably by intravenous infusion, to cohorts of patients having factor VIII deficiency who have antibodies that promote degradation or clearance of therapeutic human factor VIII. The dosage of the administered test article is in a range between 5 and 50 IU/kg body weight, preferably 10-45 IU/kg, and most preferably 40 IU/kg body weight. Approximately 1 hour after each administration, the recovery of factor VIII or CFXTEN from blood samples is measured in a functional one-stage or chromogenic coagulation assay to assess activity and by ELISA, HPLC, or similar assay to qualify the amount of intact factor VIII equivalent. Samples are taken again approximately 5-10 hours after infusion, and recovery is measured. Total recovery and the rate of disappearance of factor VIII from the samples is predictive of the antibody titer, and the comparison of results from the factor VIII and CFXTEN indicates the degree of reduced clearance and/or degradation of the CFXTEN. In one embodiment, the CFXTEN fusion protein exhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to an anti-FVIII antibody that promotes clearance but does not otherwise inhibit the procoagulant activity of intact native FVIII. In another embodiment, the CFXTEN fusion protein exhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to an anti-FVIII antibody that promotes the degradation of FVIII. In the foregoing embodiments of this paragraph, the reduced binding of the anti-FVIII antibody is alternatively characterized by an increased K D value of the FVIII antibody to the fusion protein compared to the FVIII of at least two-fold, or three-fold, or four-fold, or five-fold, or 10-fold, or 33-fold, or 100-fold, or 330-fold, or at least 1000-fold compared to the binding to the corresponding FVIII not linked to XTEN. In one embodiment, the CFXTEN fusion proteins comprising one or more XTEN exhibiting reduced reactivity to an anti-FVIII antibody exhibits an increased terminal half-life when administered to a subject with anti-FVIII antibodies of at least 48 h, or at least 72 h, or at least 96 h, or at least 120 h, or at least 144 h, or at least 14 days, or at least 21 days compared to FVIII not linked to XTEN. In the foregoing embodiment, the subject can be a human hemophilia A subject or it can be a mouse hemophilia A subject with circulating anti-FVIII antibodies.
Another aspect of the present invention is the use of CFXTEN fusion protein for a specific therapy of a coagulopathy in a subject with a FVIII inhibitor. The invention provides a method of treating a subject with circulating FVIII inhibitor(s) comprising the step of administering a clotting-effective amount of a CFXTEN fusion protein to the subject wherein the fusion protein exhibits greater procoagulant activity and/or clotting-effective concentrations of longer duration compared to either a corresponding factor VIII not linked to XTEN or compared to native factor VIII administered to the subject using a comparable amount and route of administration. In one embodiment of the method, the FVIII inhibitor in the subject is an anti-FVIII antibody. In another embodiment, the FVIII inhibitor is a neutralizing anti-FVIII antibody. In one embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the A1 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the A2 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the A3 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the C1 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the C2 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to both the C2 and A2 domain of FVIII. In another embodiment, the FVIII inhibitor binds to a FVIII epitope capable of being bound by one or more antibodies of Table 10. In another embodiment, the FVIII inhibitor is a polyclonal antibody from a hemophilia A subject with FVIII inhibitor antibodies.
An object of the present invention is the creation of CFXTEN with XTEN inserted to maximize the steric interference of FVIII binding agents that would otherwise bind to FVIII and neutralize procoagulant activity or result in the clearance or degradation of FVIII. Accordingly, in one approach the invention provides CFXTEN comprising one or more XTEN wherein the XTEN are inserted proximal to a binding site of a FVIII inhibitor or anti-FVIII antibody. In one embodiment, an XTEN is linked to the FVIII at a location selected from Table 5, Table 6, Table 7, Table 8, and Table 9 that is within about 50, or about 100, or about 150, or about 200, or about 250, or about 300 amino acids of a FVIII epitope that is bound by an antibody of Table 10. In another embodiment, the XTEN is linked to the FVIII within about 50, or about 100, or about 150, or about 200, or about 250, or about 300 amino acids of a FVIII epitope in the A2 or C2 domain that is bound by an antibody of Table 10. Accordingly, the invention provides CFXTEN fusion proteins comprising one or more XTEN wherein binding by FVIII inhibitors to the FVIII component of the fusion protein is reduced compared to the corresponding FVIII not linked to XTEN or to native FVIII and the CFXTEN retains procoagulant activity. In the foregoing embodiments hereinabove described in this paragraph, the fusion proteins can be assayed by the assays described herein below, the assays of the Examples, or other assays known in the art, and the inhibitors can be an antibody of Table 10, can be polyclonal anti-FVIII, or can be blood or plasma from a hemophilia A subject with FVIII inhibitors.
In another aspect, CFXTEN are designed to maximize the regions over which XTEN can adopt random coil conformations covering the fusion protein, thereby resulting in steric hindrance for anti-FVIII antibodies that would otherwise bind epitopes on the FVIII component of the fusion protein. It is believed that the incorporation of multiple XTEN into a CFXTEN provides a higher total hydrodynamic radius of the XTEN component compared to CFXTEN with fewer XTEN yet having approximately the same total of XTEN amino acids. Empirically, the hydrodynamic radius for a protein can be calculated based on size exclusion chromatography, and results of several fusion proteins using such methods are described in the Examples. Alternatively, the radius for XTEN polypeptides, such as those incorporated in the embodiments disclosed herein, can be approximated by mathematical formulae because the limited types of amino acids utilized have known characteristics that can be quantified. In one embodiment, the maximum radius of a single XTEN polypeptide is calculated (hereinafter “XTEN Radius”) according to the formula given by Equation II:
XTEN Radius=(√XTEN length 0.2037)+3.4627 II

In another embodiment, the sum of the maximum of the XTEN Radii for all XTEN segments in a CFXTEN is calculated (hereinafter “Sum XTEN Radii”) according to the formula given by Equation III:








Sum





XTEN





Radii

=


∑

i
=
1

m



XTEN






Radius
i






III








wherein: n=the number of XTEN segments
and i is an iterator




In another embodiment, the ratio of the SUM XTEN Radii of a CFXTEN comprising multiple XTEN to that of an XTEN Radius for a single XTEN of an equivalent length (in total amino acid residues to that of the CFXTEN) is calculated (hereinafter “Ratio XTEN Radii”) according to the formula given by Equation IV:








Ratio





XTEN





Radii

=



∑

i
=
1

n



XTEN






Radius
i





(




∑

i
=
1

n



XTEN






Length
i




*
0.2037

)

+
3.4627





IV








wherein: n=the number of XTEN segments
and i is an iterator




In applying the Equations to the XTEN, it will be understood by one of skill in the art that the calculated values represent maximum values that could vary or be reduced depending on the host cell utilized for expression of the XTEN polypeptide. It is believed that while E. coli expression would result in XTEN that achieves the calculated values, expression in eukaryotic host cells in which XTEN may be glycosylated could result in a radius of the polypeptide less than the maximum calculated value. Such differences can be quantified by methods such as size exclusion chromatography, the methods of which are detailed in the Examples.
In order to design CFTEN that maximize the area over which XTEN can adopt random coil conformations, it was discovered that CFXTEN designs with Ratio XTEN Radii above 2 provide greater coverage over the fusion protein than designs with values <2. Accordingly, in one embodiment the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or greater. In some embodiments, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater comprise at least three XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least two of the XTEN are linked to the fusion protein with no less than about 100, or about 200, or about 300, or about 400, or about 500 amino acids of separation between the two XTEN. In other embodiments, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater comprise at least four XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least three of the XTEN are linked to the fusion protein with no less than about 100, or about 200, or about 300, or about 400 amino acids of separation between any two of the three XTEN.
In another embodiment, the invention provides a CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater, the CFXTEN comprises at least three XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least two of the three of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least 100, or about 200, or about 300 to about 400 amino acids, and the third XTEN is linked within the B domain (or fragment thereof) or within the C domain (or the terminus thereof). In another embodiment, the invention provides a CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater, the CFXTEN comprises at least four XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least three of the four of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least 300 to about 400 amino acids and the fourth XTEN is linked within the B domain (or fragment thereof) or within the C domain (or the terminus thereof).
In yet other embodiments, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater, the CFXTEN comprises at least five XTEN with four XTEN having at least 42 to about 144 amino acids wherein at least four of the XTEN are linked to the fusion protein with no less than about 100, 200, or about 300, or about 400 amino acids of separation between any two of the four XTEN and a fifth XTEN is linked within the B domain (or fragment thereof) or within the C domain (or the terminus thereof). In one embodiment, the invention provides a CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater, the CFXTEN comprises at least five XTEN with four XTEN having at least 42 to about 144 amino acids wherein at least three of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least 300 to about 400 amino acids, the fourth XTEN is linked within the B domain (or fragment thereof) and a fifth XTEN is linked within the C domain (or the terminus thereof).
In one aspect, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or greater, and the composition does not comprise certain sequences. In one embodiment of the foregoing, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the proviso that the fusion protein does not comprise a sequence from any one of Table 50 or Table 51. In another embodiment of the foregoing, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the proviso that the fusion protein does not comprise a sequence having an AG family XTEN sequence. In another embodiment of the foregoing, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the proviso that the fusion protein does not comprise a sequence selected from GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34). In another embodiment of the foregoing, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the proviso that the fusion protein does not comprise any one of the sequences selected from GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34) and GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the proviso that the fusion protein does not comprise a sequence selected from GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59), PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSS (SEQ ID NO: 71), or PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGS (SEQ ID NO: 80). In another embodiment of the foregoing, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the proviso that the fusion protein does not comprise an XTEN sequence consisting of GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59), PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSS (SEQ ID NO: 71), or PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGS (SEQ ID NO: 80).
In one aspect, the present invention provides methods to create CFXTEN with XTEN inserted to maximize the steric interference of FVIII binding agents that would otherwise bind to FVIII and neutralize procoagulant activity or result in the clearance or degradation of FVIII. Accordingly, in one embodiment, the invention provides a method comprising the steps of selecting a FVIII sequence with at least 90% sequence identity to a sequence of Table 1, selecting three or more XTEN from Table 4 in which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or greater, creating expression constructs designed to locate said XTEN at or proximal to locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein the three or more XTEN are at least 300 to 400 amino acids, expressing and recovering the resulting CFXTEN, and assaying the resulting fusion proteins in an assay described herein in order to confirm the reduced binding of the CFXTEN fusion protein. By the inventive method, a CFXTEN exhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to a FVIII binding agent including, but not limited to the antibodies of Table 10, and exhibits procoagulant activity.
5. CFXTEN Fusion Protein Configurations with Spacer and Cleavage Sequences
In another aspect, the invention provides CFXTEN configured with one or more spacer sequences incorporated into or adjacent to the XTEN that are designed to incorporate or enhance a functionality or property to the composition, or as an aid in the assembly or manufacture of the fusion protein compositions. Such properties include, but are not limited to, inclusion of cleavage sequence(s) to permit release of components, inclusion of amino acids compatible with nucleotide restrictions sites to permit linkage of XTEN-encoding nucleotides to FVIII-encoding nucleotides or that facilitate construction of expression vectors, and linkers designed to reduce steric hindrance in regions of CFXTEN fusion proteins.
In an embodiment, a spacer sequence can be introduced between an XTEN sequence and a FVIII component to decrease steric hindrance such that the FVIII component may assume its desired tertiary structure and/or interact appropriately with its target substrate or processing enzyme. For spacers and methods of identifying desirable spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, specifically incorporated by reference herein. In one embodiment, the spacer comprises one or more peptide sequences that are between 1-50 amino acid residues in length, or about 1-25 residues, or about 1-10 residues in length. Spacer sequences, exclusive of cleavage sites, can comprise any of the natural L amino acids, and will preferably have XTEN-like properties in that the majority of residues will be hydrophilic amino acids that are sterically unhindered such as, but not limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P) and aspartate (D). The spacer can be a single glycine residue, polyglycines or polyalanines, or is predominately a mixture of combinations of glycine, serine and alanine residues. In one embodiment, a spacer sequence, exclusive of cleavage site amino acids, has about 1 to 10 amino acids that consist of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) and are substantially devoid of secondary structure; e.g., less than about 10%, or less than about 5% as determined by the Chou-Fasman and/or GOR algorithms. In one embodiment, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment, the spacer sequence is GPEGPS (SEQ ID NO: 1612) linked to a cleavage sequence of Table 12. In addition, spacer sequences are designed to avoid the introduction of T-cell epitopes which can, in part, be achieved by avoiding or limiting the number of hydrophobic amino acids utilized in the spacer; the determination of epitopes is described above and in the Examples.
In a particular embodiment, the CFXTEN fusion protein comprises one or more spacer sequences linked at the junction(s) between the payload FVIII sequence and the one or more XTEN incorporated into the fusion protein, wherein the spacer sequences comprise amino acids that are compatible with nucleotides encoding restriction sites. In another embodiment, the CFXTEN fusion protein comprises one or more spacer sequences linked at the junction(s) between the payload FVIII sequence and the one more XTEN incorporated into the fusion protein wherein the spacer sequences comprise amino acids that are compatible with nucleotides encoding restriction sites and the amino acids and the one more spacer sequence amino acids are chosen from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). In another embodiment, the CFXTEN fusion protein comprises one or more spacer sequences linked at the junction(s) between the payload FVIII sequence and one more XTEN incorporated into the fusion protein wherein the spacer sequences comprise amino acids that are compatible with nucleotides encoding restriction sites and the one more spacer sequences are chosen from the sequences of Table 11. The exact sequence of each spacer sequence is chosen to be compatible with cloning sites in expression vectors that are used for a particular CFXTEN construct. In one embodiment, the spacer sequence has properties compatible with XTEN. In one embodiment, the spacer sequence is GAGSPGAETA (SEQ ID NO: 178). For XTEN sequences that are incorporated internal to the FVIII sequence, each XTEN would generally be flanked by two spacer sequences comprising amino acids compatible with restriction sites, while XTEN attached to the N- or C-terminus would only require a single spacer sequence at the junction of the two components and another at the opposite end for incorporation into the vector. As would be apparent to one of ordinary skill in the art, the spacer sequences comprising amino acids compatible with restriction sites that are internal to FVIII could be omitted from the construct when an entire CFXTEN gene is synthetically generated.





TABLE 11



Spacer Sequences Compatible with Restriction Sites




Spacer Sequence
Restriction Enzyme



GSPG (SEQ ID NO: 174)
BsaI



ETET (SEQ ID NO: 175)
BsaI



PGSSS (SEQ ID NO: 176)
BbsI



GAP
AscI



GPA
FseI



GPSGP (SEQ ID NO: 177)
SfiI



AAA
SacII



TG
Agel



GT
KpnI



GAGSPGAETA (SEQ ID
SfiI

NO: 178)




ASS
XhoI










































In another aspect, the present invention provides CFXTEN configurations with cleavage sequences incorporated into the spacer sequences. In some embodiments, spacer sequences in a CFXTEN fusion protein composition comprise one or more cleavage sequences, which are identical or different, wherein the cleavage sequence may be acted on by a protease, as shown in FIG. 12 , to release FVIII, a FVIII component (e.g., the B domain) or XTEN sequence(s) from the fusion protein. In one embodiment, the incorporation of the cleavage sequence into the CFXTEN is designed to permit release of the FVIII component that becomes active or more active (with respect to its ability serve as a membrane binding site for factors IXa and X) upon its release from the XTEN. In the foregoing embodiment, the procoagulant activity of FVIII component of the CFXTEN is increased after cleavage by at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% compared to the intact CFXTEN. The cleavage sequences are located sufficiently close to the FVIII sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the FVIII sequence, such that any remaining residues attached to the FVIII after cleavage do not appreciably interfere with the activity (e.g., such as binding to a clotting protein) of the FVIII, yet provide sufficient access to the protease to be able to effect cleavage of the cleavage sequence. In some cases, the CFXTEN comprising the cleavage sequences will also have one or more spacer sequence amino acids between the FVIII and the cleavage sequence or the XTEN and the cleavage sequence to facilitate access of the protease; the spacer amino acids comprising any natural amino acid, including glycine, serine and alanine as preferred amino acids. In one embodiment, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that the CFXTEN can be cleaved after administration to a subject. In such case, the CFXTEN can serve as a prodrug or a circulating depot for the FVIII. In a particular construct of the foregoing, the CFXTEN would have one or two XTEN linked to the N- and/or the C-terminus of a FVIII-BDD via a cleavage sequence that can be acted upon by an activated coagulation factor, and would have an additional XTEN located between the processing amino acids at position R740 and R1689 such that the XTEN could be released, leaving a form of FVIII similar to native activated FVIII. In one embodiment of the foregoing construct, the FVIII that is released from the fusion protein by cleavage of the cleavage sequence exhibits at least about a two-fold, or at least about a three-fold, or at least about a four-fold, or at least about a five-fold, or at least about a six-fold, or at least about a eight-fold, or at least about a ten-fold, or at least about a 20-fold increase in activity compared to the intact CFXTEN fusion protein.
Examples of cleavage sites contemplated by the invention include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease selected from FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), Elastase-2, granzyme B, MMP-12, MMP-13, MMP-17 or MMP-20, or by non-mammalian proteases such as TEV, enterokinase, PreScission™ protease (rhinovirus 3C protease), and sortase A. Sequences known to be cleaved by the foregoing proteases and others are known in the art. Exemplary cleavage sequences contemplated by the invention and the respective cut sites within the sequences are presented in Table 12, as well as sequence variants thereof. For CFXTEN comprising incorporated cleavage sequence(s), it is generally preferred that the one or more cleavage sequences are substrates for activated clotting proteins. For example, thrombin (activated clotting factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 1618) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], which is cut after the arginine at position 4 in the sequence. Active FIIa is produced by cleavage of FII by FXa in the presence of phospholipids and calcium and is down stream from factor VIII in the coagulation pathway. Once activated, its natural role in coagulation is to cleave fibrinogen, which then in turn, begins clot formation. FIIa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. By incorporation of the LTPRSLLV sequence (SEQ ID NO: 1618) into the CFXTEN between and linking the FVIII and the XTEN components, the XTEN is removed from the adjoining FVIII concurrent with activation of either the extrinsic or intrinsic coagulation pathways when coagulation is required physiologically, thereby selectively releasing FVIII. In another embodiment, the invention provides CFXTEN with incorporated FXIa cleavage sequences between the FVIII and XTEN component(s) that are acted upon only by initiation of the intrinsic coagulation system, wherein a procoagulant form of FVIII is released from XTEN by FXIa to participate in the coagulation cascade. While not intending to be bound by any particular theory, it is believed that the CFXTEN of the foregoing embodiment would sequester the FVIII away from the other coagulation factors except at the site of active clotting, thus allowing for larger doses (and therefore longer dosing intervals) with minimal safety concerns.
Thus, cleavage sequences, particularly those susceptible to the procoagulant activated clotting proteins listed in Table 12, would provide for sustained release of FVIII that, in certain embodiments of the CFXTEN, can provide a higher degree of activity for the FVIII component released from the intact form of the CFXTEN, as well as additional safety margin for high doses of CFXTEN administered to a subject. In one embodiment, the invention provides CFXTEN comprising one or more cleavage sequences operably positioned to release the FVIII from the fusion protein upon cleavage, wherein the one or more cleavage sequences has at least about 86%, or at least about 92%, or 100% sequence identity to a sequence selected from Table 12.
In some embodiments, only the two or three amino acids flanking both sides of the cut site (four to six amino acids total) are incorporated into the cleavage sequence that, in turn, is incorporated into the CFXTEN of the embodiments, providing, e.g., XTEN release sites. In other embodiments, the incorporated cleavage sequence of Table 12 can have one or more deletions or insertions or one or two or three amino acid substitutions for any one or two or three amino acids in the known sequence, wherein the deletions, insertions or substitutions result in reduced or enhanced susceptibility but not an absence of susceptibility to the protease, resulting in an ability to tailor the rate of release of the FVIII from the XTEN. Exemplary substitutions within cleavage sequences that are utilized in the CFXTEN of the





TABLE 12



Protease C1eavage Sequences








Exemplary




Protease Acting
Cleavage
SEQ ID

SEQ ID

Upon Sequence
Sequence
NO:
Minimal Cut Site
NO:



FXIa
KLTR↓AET
179
KD/FL/T/R↓VA/VE/GT/GV




FXIa
DFTR↓VVG
180
KD/FL/T/R↓VA/VE/GT/GV




FXIIa
TMTR↓IVGG
181
NA




Kallikrein
SPFR↓STGG
182
—/—/FL/RY↓SR/RT/—/—




FVIIa
LQVR↓IVGG
183
NA




FIXa
PLGR↓IVGG
184
—/—/G/R↓—/—/—/—




FXa
IEGR↓TVGG
185
IA/E/GFP/R↓STI/VFS/—/G




FIIa (thrombin)
LTPR↓SLLV
186
—/—/PLA/R↓SAG/—/—/—




Elastase-2
LGPV↓SGVP
187
—/—/—/VIAT↓—/—/—/—




Granzyme-B
VAGD↓SLEE
188
V/—/—/D↓—/—/—/—




MMP-12
GPAG↓LGGA
189
G/PA/—/G↓L/—/G/—
190



MMP-13
GPAG↓LRGA
191
G/P/—/G↓L/—/GA/—
192



MMP-17
APLG↓LRLR
193
—/PS/—/-↓LQ/—/LT/—




MMP-20
PALP↓LVAQ
194
NA




TEV
ENLYFQ↓G
195
ENLYFQ↓G/S
196



Enterokinase
DDDK↓IVGG
197
DDDK↓IVGG
198



Protease 3C
LEVLFQ↓GP
199
LEVLFQ↓GP
200

(PreScission ™)







Sortase A
LPKT↓GSES
201
L/P/KEAD/T↓G/—/EKS/S
202



↓indicates cleavage site NA: not applicab1e

the listing of multiple amino acids before, between, or after a slash indicate a1ternative amino acids that can be substituted at the position; “—” indicates that any amino acid may be substituted for the corresponding amino acid indicated in the middle column


























































6. Exemplary CFXTEN Fusion Protein Sequences
Non-limiting examples of sequences of fusion proteins containing a single FVIII linked to one or more XTEN are presented in Table 21. The exemplary amino acid sequences of Table 21 (and the DNA sequences that encode them) contain his tags for purification purposes that, as would be apparent to one of skill in the art, can be deleted from the sequence without having an effect on the procoagulant activity of the CFXTEN fusion protein. In one embodiment, the CFXTEN of Table 21 further comprise amino acids on the N-terminus corresponding to that of native human FVIII (namely, the sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611)) to aid in the expression and secretion of the CFXTEN fusion protein. In one embodiment, a CFXTEN composition comprises a fusion protein having at least about 80% sequence identity compared to a CFXTEN from Table 21, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as compared to a CFXTEN from Table 21, when optimally aligned. In another embodiment, a CFXTEN composition comprises a fusion protein from Table 21 in which the C-terminal his-his-his-his-his-his sequence (SEQ ID NO: 1700) deleted. However, the invention also contemplates substitution of any of the FVIII sequences of Table 1 for a FVIII component of the CFXTEN of Table 21, and/or substitution of any sequence of any one of Tables 3, 4, and 13-17 for an XTEN component of the CFXTEN of Table 21. Generally, the resulting CFXTEN of the foregoing examples retain at least a portion of the procoagulant activity of the corresponding FVIII not linked to the XTEN. In the foregoing fusion proteins hereinabove described in this paragraph, the CFXTEN fusion protein can further comprise one or more cleavage sequences; e.g., a sequence from Table 12, the cleavage sequence being located between the FVIII and the XTEN sequences or between adjacent FVIII domains linked by XTEN. In some embodiments comprising cleavage sequence(s), the intact CFXTEN composition has less activity but a longer half-life in its intact form compared to a corresponding FVIII not linked to the XTEN, but is designed such that upon administration to a subject, the FVIII component is gradually released from the fusion protein by cleavage at the cleavage sequence(s) by endogenous proteases, whereupon the FVIII component exhibits procoagulant activity.
The CFXTEN compositions of the embodiments can be evaluated for activity using assays or in vivo parameters as described herein (e.g., in vitro coagulation assays, assays of Table 49, or a pharmacodynamic effect in a preclinical hemophilia model or in clinical trials in humans, using methods as described in the Examples or other methods known in the art for assessing FVIII activity) to determine the suitability of the configuration or the FVIII sequence variant, and those CFXTEN compositions (including after cleavage of any incorporated XTEN-releasing cleavage sites) that retain at least about 30%, or about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more activity compared to native FVIII sequence are considered suitable for use in the treatment of FVIII-related conditions.
V). Properties of the CFXTEN Compositions of the Invention
(a) Pharmacokinetic Properties of CFXTEN
It is an object of the present invention to provide CFXTEN fusion proteins and pharmaceutical compositions comprising CFXTEN with enhanced pharmacokinetics compared to FVIII not linked to XTEN. The pharmacokinetic properties of a FVIII enhanced by linking a given XTEN to the FVIII include, but are not limited to, terminal half-life, area under the curve (AUC), C max , volume of distribution, maintaining the biologically active CFXTEN above a minimum effective blood unit concentration for a longer period of time compared to the FVIII not linked to XTEN. The enhanced properties permit less frequent dosing and/or a longer-lived procoagulant effect compared to a comparable dose of FVIII not linked to XTEN. Enhancement of one or more of these properties can resulting benefits in the treatment of factor VIII-related conditions.
Exogenously administered factor VIII has been reported to have a terminal half-life in humans of approximately 12-14 hours when complexed with normal von Willebrand factor protein, whereas in the absence of von Willebrand factor, the half-life of factor VIII is reduced to 2 hours (Tuddenham E G, et al., Br J Haematol. (1982) 52(2):259-267; Bjorkman, S., et al. Clin Pharmacokinet. (2001) 40:815). As a result of the enhanced properties conferred by XTEN, the CFXTEN, when used at the dose and dose regimen determined to be appropriate for the subject and its underlying condition, can achieve a circulating concentration resulting in a desired procoagulant or clinical effect for an extended period of time compared to a comparable dose of the corresponding FVIII not linked to XTEN. As used herein, a “comparable dose” means a dose with an equivalent moles/kg or International Units/kg (IU/kg) for the composition that is administered to a subject. It will be understood in the art that a “comparable dose” of FVIII not linked to XTEN would represent a lesser weight of drug but would have essentially the same IUs or mole-equivalents of CFXTEN in the dose.
An international unit (“IU”) of factor VIII is defined in the art as the coagulant activity present in 1 ml of normal human plasma. A normal, non-hemophilic individual human is expected to have about 100 IU/dL factor VIII activity. In hemophilia A, the doses required to treat are dependent on the condition. For minor bleeding, doses of native or recombinant factor VIII of 20 to 40 IU/kg are typically administered, as necessary. For moderate bleeding, doses of 30 to 60 IU/kg are administered as necessary, and for major bleeding, doses of 80 to 100 IU/kg may be required, with repeat doses of 20 to 25 IU/kg given every 8 to 12 hours until the bleeding is resolved. For prophylaxis against bleeding in patients with severe hemophilia A, the usual doses of native or recombinant FVIII preparations are 20 to 40 IU/kg body weight at intervals of about 2 to 3 days. A standard equation for estimating an appropriate dose of a composition comprising FVIII is:
Required units=body weight (kg)×desired factor VIII rise (IU/dL or % of normal)×0.5 (IU/kg per IU/dL).

In many cases, the therapeutic levels for FVIII in subjects of different ages or degree of disease have been established and are available in published literature or are stated on the drug label for approved products containing the FVIII. For example, the Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis posted, on the ISTH Website 29 Nov. 2000, that the most widely used measure of hemophilia A is established by determining the circulating concentrations of plasma FVIII procoagulant levels, with persons with <1% (<0.01 IU/ml) factor VIII defined as severe; 1-5% (0.01-0.05 IU/ml) as moderately severe; and >5-40% (0.05-<0.40 IU/ml) as mild, where normal is 1 IU/ml of factor VIIIC (100%). The therapeutic levels can be established for new compositions, including those CFXTEN and pharmaceutical compositions comprising CFXTEN of the disclosure, using standard methods. In practicing the present invention, it will be understood that any dosage of CFXTEN that is effective may be used for treating bleeding episodes or maintaining hemostasis. The methods for establishing the therapeutic levels and dosing schedules for a given composition are known to those of skill in the art (see, e.g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11 th Edition, McGraw-Hill (2005)). For example, by using dose-escalation studies in subjects with the target condition to determine efficacy or a desirable pharmacologic effect, appearance of adverse events, and determination of circulating blood levels, the therapeutic blood levels for a given subject or population of subjects can be determined for a given drug or biologic. The dose escalation studies would evaluate the activity of a CFXTEN through studies in a subject or group of hemophilia A subjects. The studies would monitor blood levels of procoagulant, as well as physiological or clinical parameters as known in the art or as described herein for one or more parameters associated with the factor VIII-related condition, or clinical parameters associated with a beneficial outcome, together with observations and/or measured parameters to determine the no effect dose, adverse events, minimum effective dose and the like, together with measurement of pharmacokinetic parameters that establish the determined or derived circulating blood levels. The results can then be correlated with the dose administered and the blood concentrations of the therapeutic that are coincident with the foregoing determined parameters or effect levels. By these methods, a range of doses and blood concentrations can be correlated to the minimum effective dose as well as the maximum dose and blood concentration at which a desired effect occurs or is maintained and the period for which it can be maintained, thereby establishing the therapeutic blood levels and dosing schedule for the composition. Thus, by the foregoing methods, a C min blood level is established, below which the CFXTEN fusion protein would not have the desired pharmacologic effect and a C max blood level, above which side effects such as thrombosis may occur (Brobrow, R S, JABFP (2005) 18(2):147-149), establishing the therapeutic window for the composition.
One of skill in the art can, by the means disclosed herein or by other methods known in the art, confirm that the administered CFXTEN remains at therapeutic blood levels to maintain hemostasis for the desired interval or requires adjustment in dose or length or sequence of XTEN. Further, the determination of the appropriate dose and dose frequency to keep the CFXTEN within the therapeutic window establishes the therapeutically effective dose regimen; the schedule for administration of multiple consecutive doses using a therapeutically effective dose of the fusion protein to a subject in need thereof resulting in consecutive C max peaks and/or C min troughs that remain above therapeutically-effective concentrations and result in an improvement in at least one measured parameter relevant for the target condition. In one embodiment, the CFXTEN or a pharmaceutical compositions comprising CFXTEN administered at an appropriate dose to a subject results in blood concentrations of the CFXTEN fusion protein that remains above the minimum effective concentration to maintain hemostasis for a period at least about two-fold longer compared to the corresponding FVIII not linked to XTEN and administered at a comparable dose; alternatively at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer, alternatively at least about ten-fold longer, or at least about twenty-fold longer or greater compared to the corresponding FVIII not linked to XTEN and administered at a comparable dose. As used herein, an “appropriate dose” means a dose of a drug or biologic that, when administered to a subject, would result in a desirable therapeutic or pharmacologic effect (e.g., hemostasis) and/or a blood concentration within the therapeutic window.
In practicing the invention, CFXTEN with longer terminal half-life are generally preferred, so as to improve patient convenience, to increase the interval between doses and to reduce the amount of drug required to achieve a sustained effect. The enhanced PK parameters allow for reduced dosing of the subject compositions, compared to FVIII not linked to XTEN, particularly for those hemophilia A subjects receiving routine prophylaxis.
As described more fully in the Examples pertaining to pharmacokinetic characteristics of fusion proteins comprising XTEN, it was observed that increasing the total length of the XTEN, singly or in combination, confers a disproportionate increase in the terminal half-life of a fusion protein comprising the XTEN. Accordingly, the invention provides CFXTEN fusion proteins and pharmaceutical compositions comprising CFXTEN wherein the CFXTEN exhibits an enhanced half-life when administered to a subject. In some embodiments, the invention provides monomeric CFXTEN fusion proteins comprising one or more XTEN wherein the number and location of the XTEN are selected to confer an increase in the terminal half-life for the CFXTEN administered to a subject compared to the corresponding FVIII not linked to the XTEN and administered at a comparable dose, wherein the increase is at least about two-fold longer, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at least a 40-fold or greater increase in terminal half-life compared to the FVIII not linked to the XTEN. In other embodiments, the invention provides CXTEN compositions and pharmaceutical compositions comprising CFXTEN wherein the administration of a composition to a subject in need thereof results in a terminal half-life that is at least 12 h greater, or at least about 24 h greater, or at least about 48 h greater, or at least about 96 h greater, or at least about 144 h greater, or at least about 7 days greater, or at least about 14 days greater, or at least about 21 days greater compared to a comparable dose of FVIII not linked to XTEN. In another embodiment, administration of a coagulation-effective dose of a CFXTEN fusion protein to a subject in need thereof can result in a gain in time between consecutive doses necessary to maintain blood levels of about 0.1 IU/ml of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days between consecutive doses compared to a FVIII not linked to XTEN and administered at a comparable dose.
In one embodiment, the present invention provides CFXTEN fusion proteins and pharmaceutical compositions comprising CFXTEN that exhibit, when administered to a subject in need thereof, an increase in AUC of at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about a 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about a 2000% compared to the corresponding FVIII not linked to the XTEN and administered to a subject at a comparable dose. The pharmacokinetic parameters of a CFXTEN can be determined by standard methods involving dosing, the taking of blood samples at timed intervals, and the assaying of the protein using ELISA, HPLC, radioassay, clotting assays, the assays of Table 49, or other methods known in the art or as described herein, followed by standard calculations of the data to derive the half-life and other PK parameters.
In one embodiment, a smaller IU amount of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about 10-fold less or greater of the fusion protein is administered in comparison to the corresponding FVIII not linked to the XTEN under a dose regimen needed to maintain hemostasis and the fusion protein achieves a comparable area under the curve as the corresponding IU amount of the FVIII not linked to the XTEN needed to maintain hemostasis. In another embodiment, the CFXTEN fusion protein or a pharmaceutical compositions comprising CFXTEN requires less frequent administration for routine prophylaxis of a hemophilia A subject, wherein the dose of fusion protein is administered about every four days, about every seven days, about every 10 days, about every 14 days, about every 21 days, or about monthly to the subject, and the fusion protein achieves a comparable area under the curve as the corresponding FVIII not linked to the XTEN and administered to the subject. In yet other embodiments, an accumulative smaller IU amount of about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the fusion protein is administered to a subject in comparison to the corresponding IU amount of the FVIII not linked to the XTEN under a dose regimen needed to maintain a blood concentration of 0.1 IU/ml, yet the fusion protein achieves at least a comparable area under the curve as the corresponding FVIII not linked to the XTEN. The accumulative smaller IU amount is measure for a period of at least about one week, or about 14 days, or about 21 days, or about one month.
In one aspect, the invention provides CFXTEN compositions designed to reduce binding by FVIII binding agents, thereby increasing the terminal half-life of CFXTEN administered to a subject, while still retaining procoagulant activity. It is believed that the CFXTEN of the present invention have comparatively higher and/or sustained activity achieved by reduced active clearance of the molecule by the addition of unstructured XTEN to the FVIII coagulation factor. The clearance mechanisms to remove FVIII from the circulation have yet to be fully elucidated. Uptake, elimination, and inactivation of coagulation proteins can occur in the circulatory system as well as in the extravascular space. Coagulation factors are complex proteins that interact with a large number of other proteins, lipids, and receptors, and many of these interactions can contribute to the elimination of CFs from the circulation. The protein von Willebrand factor is an example of a FVIII binding agent that binds to FVIII. Factor VIII and von Willebrand factor (VWF) circulate in the blood as a tight, non-covalently linked complex in which VWF serves as a carrier that likely contributes to the protection of FVIII from active cleavage mechanisms, yet nevertheless results in a limitation on the terminal half-life of FVIII. For example: (i) VWF stabilizes the heterodimeric structure of FVIII; (ii) VWF protects FVIII from proteolytic degradation by phospholipid-binding proteases like activated protein C and activated FX (FXa); (iii) VWF interferes with binding of FVIII to negatively charged phospholipid surfaces exposed within activated platelets; (iv) VWF inhibits binding of FVIII to activated FIX (FIXa), thereby denying FVIII access to the FX-activating complex; and (v) VWF prevents the cellular uptake of FVIII (Lenting, P. J., et al., J Thrombosis and Haemostasis (2007) 5(7):1353-1360). In addition, LDL receptor-related protein (LRP1, also known as α2-macrogobulin receptor or CD91) has been identified as a candidate clearance receptor for FVIII, with LRP1 binding sites identified on both chains of the heterodimer form of FVIII (Lenting P J, et al., J Biol Chem (1999) 274: 23734-23739; Saenko E L, et al., J Biol Chem (1999) 274: 37685-37692). LRPs are involved in the clearance of a diversity of ligands including proteases, inhibitors of the Kunitz type, protease serpin complexes, lipases and lipoproteins (Narita, et al., Blood (1998) 2:555-560). It has been shown that the light chain, but not the heavy chain, of factor VIII binds to surface-exposed LRP1 receptor protein (Lentig et al. (J Biol Chem (1999) 274(34):23734-23739; and U.S. Pat. No. 6,919,311), which suggests that LRP1 may play an essential role in the active clearance of proteins like FVIII. While the VWF-FVIII interaction is of high affinity (<1 nM), the complex is nevertheless in a dynamic equilibrium, such that a small but significant portion of the FVIII molecules (5-8%) circulate as a free protein (Leyte A, et al., Biochem J (1989) 257: 679-683; Noe D A. Haemostasis (1996) 26: 289-303). As such, a portion of native FVIII is unprotected by VWF, allowing active clearance mechanisms to remove the unprotected FVIII from the circulation.
In one embodiment, the invention provides CFXTEN that associate with VWF but have enhanced protection from active clearance receptors conferred by the incorporation of two more XTEN at one or more locations within the FVIII molecule (e.g., locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 ), wherein the XTEN interfere with the interaction of the resulting CFXTEN with those clearance receptors with the result that the pharmacokinetic properties of the CFXTEN is enhanced compared to the corresponding FVIII not linked to XTEN. In another embodiment, the invention provides CFXTEN that have reduced binding affinity with VWF of at least 5% less, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70% less, but are nevertheless configured to have enhanced protection from active clearance receptors conferred by the incorporation of XTEN at one or more locations within the FVIII molecule, wherein the XTEN interfere with the interaction of factor VIII with those receptors. In the foregoing embodiments, the CFXTEN have an increased terminal half-life of at least about 12 h, or 24 h, or 48 h, or 72 h, or 96 h, or 120 h, or 144 h, or 7 days, or 10 days, or 14 days, or 21 days compared to the FVIII not linked to XTEN. The invention provides a method to create CFXTEN with reduced clearance wherein the CFXTEN fusion proteins created with the multiple insertions are evaluated for inhibition of binding to clearance receptors, compared to FVIII not linked to XTEN, using in vitro binding assays or in vivo pharmacokinetic models described herein or other assays known in the art, and selecting those that demonstrate reduced binding yet retain procoagulant FVIII activity. In addition, the foregoing fusion proteins can be optimized to have increased Ratio XTEN Radii of at least 2.0-3.5 in order to achieve pharmacokinetic properties that are further enhanced. Table 5, Table 6, Table 7, Table 8, and Table 9 and FIGS. 8-9 provide non-limiting examples of XTEN insertion points within the factor VIII sequence. Using such insertion points, the invention contemplates CFXTEN compositions that have configurations with multiple XTEN inserted with about 100, or about 200, or about 300, or about 400, or about 500 amino acids separating at least three XTEN to further increase the protection against active clearance mechanisms and, hence, increase the terminal half-life of the CFXTEN. Not to be bound by a particular theory, the XTEN of the CFXTEN compositions with high net charge (e.g., CFXTEN comprising AE family XTEN) are expected, as described above, to have less non-specific interactions with various negatively-charged surfaces such as blood vessels, tissues, or various receptors, which would further contribute to reduced active clearance. Conversely, the XTEN of the CFXTEN compositions with a low (or no) net charge (e.g., CFXTEN comprising AG family XTEN) are expected to have a higher degree of interaction with surfaces that, while contributing to active clearance, can potentiate the activity of the associated coagulation factor, given the known contribution of cell (e.g., platelets) and vascular surfaces to the coagulation process and the intensity of activation of coagulation factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry (2000) 39(32):9850-9858). The invention, in part, takes advantage of the fact that certain ligands wherein reduced binding to a clearance receptor, either as a result of a decreased on-rate or an increased off-rate, may be effected by the obstruction of a receptor site by an inserted XTEN forming random coil, resulting in the reduced binding. The choice of the particular configuration of the CFXTEN fusion protein can be tested by methods disclosed herein to confirm those configurations that reduce the degree of binding to a clearance receptor such that a reduced rate of active clearance is achieved. In one embodiment, the CFXTEN comprises a FVIII-XTEN sequence that has one or more XTEN inserted at locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 wherein the terminal half-life of the CFXTEN is increased at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about twenty-fold compared to a FVIII not linked to an XTEN. In another embodiment, the CFXTEN comprises a FVIII-XTEN sequence that has a first and at least a second XTEN inserted at a first and second location selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 wherein the terminal half-life of the CFXTEN is increased at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about twenty-fold compared to a FVIII not linked to an XTEN. In yet another embodiment, the CFXTEN comprises a FVIII-XTEN sequence that incorporates multiple XTEN sequences using three of more XTEN insertion locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 separated by about 100, or about 200, or about 300, or about 400, or about 500 amino acids, wherein the terminal half-life of the CFXTEN is increased at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about twenty-fold compared to a FVIII not linked to an XTEN. In the foregoing embodiments hereinabove described in this paragraph, the XTEN incorporated into the CFXTEN configurations can be identical or they can be different, and can have at least about 80%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, sequence identity to a sequence from any one of Tables 3, 4, and 13-17, and can optionally include one or more cleavage sequences from Table 12, facilitating release of one or more of the XTEN from the CFXTEN fusion protein.
In one embodiment, the invention provides CFXTEN that enhance the pharmacokinetics of the fusion protein by linking one or more XTEN to the FVIII component of the fusion protein wherein the fusion protein has an increase in apparent molecular weight factor of at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about ten-fold, or at least about twelve-fold, or at least about fifteen-fold, and wherein the terminal half-life of the CFXTEN when administered to a subject is increased at least about two-fold, or at least about four-fold, or at least about eight-fold, or at least about 10-fold or more compared to the corresponding FVIII not linked to XTEN. In the foregoing embodiment, wherein at least two XTEN molecules are incorporated into the CFXTEN, the XTEN can be identical or they can be of a different sequence composition, net charge, or length. The XTEN can have at least about 80%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, sequence identity to a sequence from any one of Tables 3, 4, and 13-17, and can optionally include one or more cleavage sequences from Table 12, facilitating release of one or more of the XTEN from the CFXTEN fusion protein.
Thus, the invention provides CFXTEN compositions in which the degree of activity, bioavailability, half-life or physicochemical characteristic of the fusion protein can be tailored by the selection and placement of the type and length of the XTEN in the CFXTEN compositions. Accordingly, the invention contemplates compositions in which a FVIII from Table 1 and XTEN or XTEN fragment from any one of Tables 3, 4, or 13-17 are produced, for example, in a configuration selected from any one of formulae I-VIII or the XTEN are inserted at locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 such that the construct has the desired property.
The invention provides methods to produce the CFXTEN compositions that can maintain the FVIII component at therapeutic levels in a subject in need thereof for at least a two-fold, or at least a three-fold, or at least a four-fold, or at least a five-fold greater period of time compared to comparable dosages of the corresponding FVIII not linked to XTEN. In one embodiment of the method, the subject is receiving routine prophylaxis to prevent bleeding episodes. In another embodiment of the method, the subject is receiving treatment for a bleeding episode. In another embodiment of the method, the subject is receiving treatment to raise the circulating blood concentration of procoagulant FVIII above 1%, or above 1-5%, or above 5-40% relative to FVIII concentrations in normal plasma. “Procoagulant” as used herein has its general meaning in the art and generally refers to an activity that promotes clot formation, either in an in vitro assay or in vivo. The method to produce the compositions that can maintain the FVIII component at therapeutic levels includes the steps of selecting one or more XTEN appropriate for conjugation to a FVIII to provide the desired pharmacokinetic properties in view of a given dose and dose regimen, creating a gene construct that encodes the CFXTEN in one of the configurations disclosed herein, transforming an appropriate host cell with an expression vector comprising the encoding gene, expressing the fusion protein under suitable culture conditions, recovering the CFXTEN, administration of the CFXTEN to a mammal followed by assays to verify the pharmacokinetic properties and the activity of the CFXTEN fusion protein (e.g., the ability to maintain hemostasis or serve as a procoagulant) and the safety of the administered composition. Those compositions exhibiting the desired properties are selected for further use. CFXTEN created by the methods provided herein can result in increased efficacy of the administered composition by, amongst other properties, maintaining the circulating concentrations of the procoagulant FVIII component at therapeutic levels for an enhanced period of time.
The invention provides methods to assay the CFXTEN fusion proteins of differing composition or configuration in order to provide CFXTEN with the desired degree of procoagulant and therapeutic activity and pharmacokinetic properties, as well as a sufficient safety profile. Specific in vitro and in vivo assays or animal models are used to assess the activity and functional characteristics of each configured CFXTEN and/or FVIII component to be incorporated into CFXTEN, including but not limited to the assays of the Examples, those assays of Table 49, as well as the following assays or other such assays known in the art for assaying the properties and effects of FVIII. Functional assays can be conducted that allow determination of coagulation activity, such as one-stage clotting assay and two-stage clotting assay (Barrowcliffe T W, Semin Thromb Hemost. (2002) 28(3):247-256), activated partial prothrombin (aPTT) assays (Belaaouaj A A et al., J. Biol. Chem. (2000) 275:27123-8; Diaz-Collier J A. Haemost (1994) 71:339-46), chromogenic FVIII assays (Lethagen, S., et al., Scandinavian J Haematology (1986) 37:448-453), or animal model pharmacodynamic assays including bleeding time or thrombelastography (TEG or ROTEM), among others. Other assays include determining the binding affinity of a CFXTEN for the target substrate using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in U.S. Pat. No. 5,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art. Other assays to determine the binding of FVIII inhibitors to CFXTEN include the Bethesda assay or the Nijmegen modification of the Bethesda assay. The foregoing assays can also be used to assess FVIII sequence variants (assayed as single components or as CFXTEN fusion proteins) and can be compared to the native FVIII to determine whether they have the same degree of procoagulant activity as the native CF, or some fraction thereof such that they are suitable for inclusion in CFXTEN; e.g., at least about 10%, or at least about 20$, or about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native FVIII.
Dose optimization is important for all drugs. A therapeutically effective dose or amount of the CFXTEN varies according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the administered fusion protein to elicit a desired response in the individual. For example, a standardized single dose of FVIII for all patients presenting with diverse bleeding conditions or abnormal clinical parameters (e.g., neutralizing antibodies) may not always be effective. Hemophilia A patients with trauma, who have undergone surgery, or that have high titers of FVIII inhibitory antibodies generally will require higher and more frequent dosing. Generally, dosage level is adjusted in frequency, duration, and units in keeping with the severity and duration of each patient's bleeding episode. Accordingly, the CFXTEN is included in the pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the fusion protein to stop bleeding, as measured by standard clotting assays. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically or pharmacologically effective amount of the CFXTEN and the appropriated dosing schedule, versus that amount that would result in insufficient potency such that clinical improvement or the arrest of bleeding is not achieved.
The invention provides methods to establish a dose regimen for the CFXTEN pharmaceutical compositions of the invention. The methods include administration of consecutive doses of a therapeutically effective amount of the CFXTEN pharmaceutical composition using variable periods of time between doses to determine that interval of dosing sufficient to achieve and/or maintain the desired parameter, blood level or clinical effect; such consecutive doses of a therapeutically effective amount at the effective interval establishes the therapeutically effective dose regimen for the CFXTEN for a factor VIII-related disease state or condition. A prophylactically effective amount refers to an amount of CFXTEN required for the period of time necessary to prevent a physiologic or clinical result or event; e.g., delayed onset of a bleeding episode or maintaining blood concentrations of procoagulant FVIII or equivalent above a threshold level (e.g., 1-5% to 5-40% of normal). In the methods of treatment, the dosage amount of the CFXTEN that is administered to a subject ranges from about 5 to 300 IU/kg/dose, or from about 10 to 100 IU/kg/dose, or from about 20 to about 65 IU/kg/dose, or from about 20 to about 40 IU/kg/dose for a subject. A suitable dosage may also depend on other factors that may influence the response to the drug; e.g., bleeding episodes generally requiring higher doses at more frequent intervals compared to prophylaxis.
In some embodiments, the method comprises administering a therapeutically-effective amount of a pharmaceutical composition comprising a CFXTEN fusion protein composition and at least one pharmaceutically acceptable carrier to a subject in need thereof, wherein the administration results in a greater improvement in at least one parameter or physiologic condition associated with a FVIII deficiency or coagulopathy, or results in a more favorable clinical outcome mediated by the FVIII component of the CFXTEN compared to the effect on the parameter, condition or clinical outcome mediated by administration of a pharmaceutical composition comprising a FVIII not linked to XTEN and administered at a comparable dose. Non-limiting examples of parameters that are improved include blood concentration of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a reduced bleeding time, resolution of a bleeding event, or a reduced Bethesda titer to the CFXTEN relative to native FVIII. In one embodiment of the foregoing, the improvement is achieved by administration of the CFXTEN pharmaceutical composition at a dose that achieves a circulating concentration of procoagulant FVIII (or equivalent) above a threshold level (e.g., 1-5% to 5-40% of normal FVIII levels), thereby establishing the therapeutically effective dose. In another embodiment of the foregoing, the improvement is achieved by administration of multiple consecutive doses of the CFXTEN pharmaceutical composition using a therapeutically effective dose regimen that maintains a circulating concentration of procoagulant FVIII (or equivalent) above a threshold level (e.g., 1-5% to 5-40% of normal FVIII levels) for the length of the dosing period. In another embodiment of the method, the administration of at least two consecutive doses of the CFXTEN pharmaceutical composition using a therapeutically effective dose regimen maintains a circulating concentration of procoagulant FVIII (or equivalent) above about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, or 40% of normal FVIII levels for a period that is at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer compared to a FVIII not linked to XTEN and administered using a therapeutically effective dose regimen
In one embodiment, the CFXTEN or a pharmaceutical compositions comprising CFXTEN administered at a therapeutically effective dose regimen results in a gain in time of at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about six-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer between at least two consecutive C max peaks and/or C min troughs for blood levels of the fusion protein compared to the corresponding biologically active protein of the fusion protein not linked to the XTEN and administered at a comparable dose regimen to a subject. In another embodiment, the CFXTEN administered at a therapeutically effective dose regimen results in a comparable improvement in one, or two, or three or more measured parameters using less frequent dosing or a lower total dosage in IUs of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the XTEN and administered to a subject using a therapeutically effective dose regimen for the FVIII. The measured parameters include any of the clinical, biochemical, or physiological parameters disclosed herein, or others known in the art for assessing subjects with factor VIII-related conditions.
(b) Pharmacology and Pharmaceutical Properties of CFXTEN
The present invention provides CFXTEN compositions comprising FVIII covalently linked to XTEN that have enhanced pharmaceutical and pharmacology properties compared to FVIII not linked to XTEN, as well as methods to enhance the therapeutic and/or procoagulant effect of the FVIII components of the compositions. In addition, the invention provides CFXTEN compositions with enhanced properties compared to those art-known fusion proteins of factor VIII containing albumin, immunoglobulin polypeptide partners, polypeptides of shorter length and/or polypeptide partners with repetitive sequences. In addition, CFXTEN fusion proteins provide significant advantages over chemical conjugates, such as pegylated constructs of FVIII, notably the fact that recombinant CFXTEN fusion proteins can be made in host cell expression systems, which can reduce time and cost at both the research and development and manufacturing stages of a product, as well as result in a more homogeneous, defined product with less toxicity from both the product and metabolites of the CFXTEN compared to pegylated conjugates.
As therapeutic agents, the CFXTEN possesses a number of advantages over therapeutics not comprising XTEN, including one or more of the following non-limiting properties: increased solubility, increased thermal stability, reduced immunogenicity, increased apparent molecular weight, reduced renal clearance, reduced proteolysis, reduced metabolism, enhanced therapeutic efficiency, less frequent dosage regimen with increased time between doses capable of maintaining hemostasis in a subject with hemophilia A, the ability to administer the CFXTEN composition subcutaneously or intramuscularly, a “tailored” rate of absorption when administered subcutaneously or intramuscularly, enhanced lyophilization stability, enhanced serum/plasma stability, increased terminal half-life, increased solubility in blood stream, decreased binding by neutralizing antibodies, decreased active clearance, tailored substrate binding affinity, stability to degradation, stability to freeze-thaw, stability to proteases, stability to ubiquitination, ease of administration, compatibility with other pharmaceutical excipients or carriers, persistence in the subject, increased stability in storage (e.g., increased shelf-life), and the like. The net effect of the enhanced properties is that the use of a CFXTEN composition can result in an overall enhanced therapeutic effect compared to a FVIII not linked to XTEN, result in economic benefits associated with less frequent dosing, and/or result in improved patient compliance when administered to a subject with a factor VIII-related condition.
The invention provides CFXTEN compositions and pharmaceutical compositions comprising CFXTEN wherein the administration of the composition results in an improvement in at least one of the clinical or biochemical parameters disclosed herein as being useful for assessing the subject diseases, conditions or disorders. Non-limiting examples of parameters that are improved include blood concentrations of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a reduced bleeding time, resolution of a bleeding event, or a reduced Bethesda titer to the CFXTEN relative to native FVIII. The enhanced pharmacokinetic properties of the subject CFXTEN permits using an accumulatively lower IU dose of fusion protein to maintain the parameter compared to the corresponding FVIII component not linked to the XTEN. In one embodiment, the total dose in IUs of an CFXTEN of the embodiments needed to achieve and maintain the improvement in the at least one parameter for about 2-7 days is at least about three-fold lower, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about 10-fold lower compared to the corresponding FVIII component not linked to the XTEN. In another embodiment, the total dose in IUs of a subject CFXTEN needed to achieve and maintain the improvement in the at least one parameter over two, three or four consecutive doses is at least about three-fold lower, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about 10-fold lower compared to the corresponding FVIII component not linked to the XTEN. Alternatively, the invention provides certain embodiments of CFXTEN wherein the period between consecutive administrations that results in achieving and maintaining the improvement in at least one parameter is at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about 10-fold longer compared to the corresponding FVIII component not linked to the XTEN and administered at a comparable IU dose. Alternatively, the invention provides certain embodiments of CFXTEN wherein administration of 25 IU/kg results in a 30% improvement in a aPTT assay (or similar coagulation assay) time in a hemophilia A subject compared to 25 IU/kg of the corresponding FVIII not linked to XTEN when assayed at about 2-7 days after administration. In yet another embodiment, the invention provides CFXTEN wherein administration of 25 IU/kg results in a 30% improvement in a bleeding time assay time in a hemophilia A subject compared to 25 IU/kg of the corresponding FVIII not linked to XTEN when assayed at about 2-7 days after administration.
In one embodiment, XTEN as a fusion partner increases the solubility of the FVIII payload. Accordingly, where enhancement of the pharmaceutical or physicochemical properties of the FVIII is desirable, such as the degree of aqueous solubility or stability, the length and/or the motif family composition of the XTEN sequences incorporated into the fusion protein may each be selected to confer a different degree of solubility and/or stability on the respective fusion proteins such that the overall pharmaceutical properties of the CFXTEN composition are enhanced. The CFXTEN fusion proteins can be constructed and assayed, using methods described herein, to confirm the physicochemical properties and the choice of the XTEN length sequence or location adjusted, as needed, to result in the desired properties. In one embodiment, the CFXTEN has an aqueous solubility that is at least about 25% greater compared to a FVIII not linked to the XTEN, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or at least about 1000% greater than the corresponding FVIII not linked to XTEN.
The invention provides methods to produce and recover expressed CFXTEN from a host cell with enhanced solubility and ease of recovery compared to FVIII not linked to XTEN. In one embodiment, the method includes the steps of transforming a eukaryotic host cell with a polynucleotide encoding a CFXTEN with one or more XTEN components of cumulative sequence length greater than about 100, or greater than about 200, or greater than about 400, or greater than about 600, or greater than about 800, or greater than about 1000, or greater than about 2000, or greater than about 3000 amino acid residues, expressing the CFXTEN fusion protein in the host cell under suitable culture and induction conditions, and recovering the expressed fusion protein in soluble form. In one embodiment, the one or more XTEN of the CFXTEN fusion proteins each have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN selected from any one of Tables 4, and 13-17, or fragments thereof, and the FVIII have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100% sequence identity compared to a FVIII selected from Table 1, and the CFXTEN components are in an N- to C-terminus configuration selected from any one of the configuration embodiments disclosed herein.
VI). Uses of the CFXTEN Compositions
The invention provides methods and regimens for achieving a beneficial effect in a factor VIII-related condition by the administration of compositions comprising CFXTEN. As used herein, “factor VIII-related condition” is intended to include, but is not limited to factor VIII deficiencies, bleeding disorders related to factor VIII deficiency, hemophilia A, neutralization of factor VIII by anti-FVIII antibodies or other factor VIII inhibitors, and bleeding episodes resulting from trauma or surgery or vascular injury and other such conditions that can be ameliorated or corrected by administration of FVIII to a subject. The inventive methods achieve a beneficial effect while addressing disadvantages and/or limitations of other methods of treatment using factor VIII preparations that have a relatively short terminal half-life, require frequent administrations, are neutralized by inhibitors or have unfavorable pharmacoeconomics.
Hemostasis is regulated by multiple protein factors, and such proteins, as well as analogues thereof, have found utility in the treatment of factor VIII-related conditions. However, the use of commercially-available FVIII has met with less than optimal success in the management of subjects afflicted with such conditions. In particular, dose optimization and frequency of dosing is important for FVIII used in maintaining circulating FVIII concentrations above threshold levels needed for hemostasis, as well as the treatment or prevention of bleeding episodes in hemophilia A subjects. The fact that commercially-available FVIII products have a short half-life necessitates frequent dosing in order to achieve clinical benefit, which results in difficulties in the management of such patients.
As established by the Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (posted on the ISTH Website 29 Nov. 2000), the most widely used measure of the severity of hemophilia A is established by determining the circulating concentrations of plasma FVIII procoagulant levels, with persons with <1% (<0.01 IU/ml) factor VIII defined as severe; 1-5% (0.01-0.05 IU/ml) as moderately severe; and >5-40% (0.05-<0.40 IU/ml) as mild, where normal is 1 IU/ml of factor VIIIC (100%).
The invention provides methods of treating a subject suffering from or at risk of developing a factor VIII-related condition. More particularly, the invention provides methods for treating or preventing controlling bleeding in subject. The subject can be any animal but preferably is a human. In one embodiment, the method comprises administering a coagulation-effective amount of a CFXTEN composition to the subject in need thereof. In another embodiment, the method comprises the step of administering to the subject with a bleed a coagulation-effective amount of a pharmaceutical composition that includes a CFXTEN, wherein the administration results in an arrest or attenuation of the bleeding. As used herein, “coagulation-effective amount” is an amount of a FVIII composition that, when administered to a subject, is sufficient to effect hemostasis or other beneficial or desired therapeutic (including preventative) result. In practicing the present invention, it will be understood that a coagulation-effective amount can be administered in one or more administrations. Precise coagulation-effective amounts of the pharmaceutical composition to be administered will be guided by the judgment of the practitioner, however, the unit dose will generally depend on the severity or cause of the bleeding and the amount of pre-existing FVIII in the subject. In a particular embodiment of the method of treating a bleed, a coagulation-effective amount of a pharmaceutical compositions comprising CFXTEN is administered to a subject suffering from a bleeding episode, wherein the administration results in the resolution of the bleeding for a duration at least two-fold, or at least three-fold, or at least four-fold longer compared to a FVIII not linked to XTEN and administered to a comparable subject with a comparable bleed at a comparable dose.
In another embodiment, the administration of a coagulation-effective amount of a CFXTEN composition to a subject with a factor VIII-related condition results in a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or greater improvement of one or more biochemical, physiological or clinical parameters associated with the FVIII condition, compared to the FVIII not linked to XTEN, when measured at between 2 and 7 days after administration. In another embodiment, the administration of a coagulation-effective amount of a CFXTEN composition to the subject in need thereof results in an improvement of one or more biochemical, physiological or clinical parameters associated with the FVIII condition for a period at least two-fold longer, or at least four-fold longer, or at least five-fold longer, or at least six-fold longer compared to period achieved by a FVIII not linked to XTEN and administered at a comparable dose. Non-limiting examples of parameters that are improved for a longer duration include blood concentrations of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a reduced bleeding time, among other FVIII-related parameters known in the art. In the foregoing embodiments of the paragraph, the administered CFXTEN comprises a FVIII with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a factor VIII of Table 1 and one or more XTEN sequences with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to an XTEN of Table 4 inserted into the FVIII at one or more locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9, or as depicted in FIGS. 8-9 . In certain embodiments, at least one XTEN insertion site of the CFXTEN is selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered relative to mature native human FVIII).
In a particular embodiment of the method of treatment, a coagulation-effective amount of CFXTEN fusion protein administered to a subject suffering from hemophilia A is sufficient to increase the circulating FVIII procoagulant concentration to greater than 0.05 IU/ml and to maintain hemostasis for at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, or at least about 168 h, or greater. In another embodiment, the administration of a coagulation-effective amount of a pharmaceutical composition comprising CFXTEN to a subject in need thereof results in a greater reduction in a one-stage clotting assay time of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in a blood sample from the subject at 2-7 days after the administration compared to the assay time in a subject after administration of a comparable amount of the corresponding FVIII not linked to XTEN. In another embodiment, the administration of a therapeutically effective amount of a CFXTEN or a pharmaceutical compositions comprising CFXTEN to a subject in need thereof results in a greater reduction in the activated partial prothrombin time of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in a blood sample from the subject 2-7 days after administration compared to the activated partial prothrombin time in a subject after administration of a comparable amount of the corresponding FVIII not linked to XTEN. In another embodiment, the administration of a CFXTEN or a pharmaceutical compositions comprising CFXTEN to a subject in need thereof using a therapeutically effective amount results in maintenance of activated partial prothrombin times within 30% of normal in a blood sample from the subject for a period of time that is at least two-fold, or at least about three-fold, or at least about four-fold longer compared to that of a FVIII not linked to XTEN and administered to a subject using a comparable dose.
In one embodiment of the method of treatment, the CFXTEN fusion protein is formulated and administered as a pharmaceutical composition comprising the CFXTEN in admixture with a pharmaceutically acceptable excipient. Methods for making pharmaceutical formulations are well known in the art. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. 1990 (See, also, Wang and Hanson, Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42-2S (1988)).
In another aspect, the invention provides a regimen for treating a hemophilia A patient, said regimen comprising a composition comprising a CFXTEN fusion protein. In one embodiment of the regimen for treating a hemophilia A patient, the regimen further comprises the step of determining the amount of pharmaceutical composition comprising the CFXTEN needed to achieve hemostasis in the patient. In some embodiments of the regimen, (i) a smaller IU amount of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about 10-fold less of the pharmaceutical composition comprising CFXTEN is administered to a subject in need thereof in comparison to the corresponding coagulation factor not linked to the XTEN under an otherwise same dose regimen, and the fusion protein achieves a comparable area under the curve (based on IU/ml) and/or a comparable therapeutic effect as the corresponding FVIII not linked to the XTEN; (ii) the pharmaceutical composition is administered less frequently (e.g., every three days, about every seven days, about every 10 days, about every 14 days, about every 21 days, or about monthly) in comparison to the corresponding FVIII not linked to the XTEN under an otherwise same dose amount, and the fusion protein achieves a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; or (iii) an accumulative smaller IU amount of at least about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the pharmaceutical composition is administered in comparison to the corresponding FVIII not linked to the XTEN under an otherwise same dose schedule and the CFXTEN fusion protein achieves a comparable therapeutic effect as the corresponding FVIII not linked to the XTEN. The accumulative smaller IU amount is measured for a period of at least about one week, or about 14 days, or about 21 days, or about one month. In the foregoing embodiments, the therapeutic effect can be determined by any of the measured parameters described herein, including but not limited to blood concentration of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a reduced bleeding time, resolution of a bleeding event, or a reduced Bethesda titer to the CFXTEN relative to native FVIII, fibrinogen levels, or other assays known in the art for assessing coagulopathies of FVIII. In another embodiment, the invention provides CFXTEN for use in a regimen for a treating a hemophilia A subject comprising administering an CFXTEN composition in two or more successive doses to the subject at an effective amount, wherein the administration results in at least a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the disease compared to a FVIII not linked to XTEN and administered using a comparable dose.
In one aspect, the present invention relates to a method of preventing or treating the bleeding in a patient, optionally a haemophilia A patient, having pre-existing inhibitor(s) against FVIII. Inhibitory antibodies against FVIII commonly develop in hemophiliacs, where the overall incidence of developing an inhibitor is 15-30%, particularly in haemophiliacs who are heavily exposed to FVIII concentrates (Algiman et al. Natural antibodies to factor VIII (anti-hemophilic factor) in healthy individuals. PNAS USA (1992) 89: 3795-3799). However, inhibitory antibodies also occur in patients in auto-immune disorders, malignancies (such as lymphoproliferative disorders, lymphomas and solid tumors), during pregnancy and in the post-partum state. Inhibition can also occur when antibodies interfere with the binding of FVIII to FIX and FX. Simultaneously or alternatively, anti-FVIII antibodies can interfere with the binding of von Willebrand factor and/or phospholipids to FVIII, affecting coagulation and/or half-life of FVIII. The presence of inhibitory antibodies is often first detected with symptoms such as easy bruising and uncontrolled bleeding, and is usually referred to as acquired hemophilia. Anti-FVIII antibodies can be determined by different methods including quantitation of anti-FVIII activity in coagulation assays, ELISA for FVIII inhibitors and purification using chromatography and immunoadsorption (Algiman et al., 1992). Accordingly, the inventive methods are used in the treatment or prevention of any condition associated with or characterized by the presence of inhibitory antibodies to FVIII. In one embodiment, the invention provides a method of treating a patient having a pre-existing inhibitor against FVIII, the method comprising the step of administering to the patient a coagulation-effective amount of a CFXTEN fusion protein that must be administered to achieve hemostasis, wherein the coagulation-effective amount of fusion protein administered is reduced in comparison to the amount of FVIII not linked to XTEN (or native FVIII) that must be administered to achieve hemostasis. In the method, the reduced amount of CFXTEN is about two-fold, or three-fold, or four-fold, or five-fold less in IU/kg compared to the corresponding FVIII not linked to XTEN. In another embodiment of the method, the amount of CFXTEN that is administered as a dose to achieve hemostasis is at least 20 to 40 IU/kg less, or 30 to 60 IU/kg less, or 40 to 80 IU/kg less, or 60 to 100 IU/kg less, or 100 to 140 IU/kg less, or 120 to 180 IU/kg less, or 140 to 200 IU/kg less compared to the corresponding FVIII not linked to XTEN or to native FVIII required to achieve hemostasis. In another embodiment, the invention provides a method of treating a bleeding episode in a hemophilia A subject having a titer of at least 10, or 20, or 30, or 40, or 50, or 75, or 100, or 150, or 200 or more Bethesda units against a FVIII not linked to XTEN, wherein the dose of CFXTEN fusion protein required to arrest the bleeding epidose is at least two-fold, or three-fold, or four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or 10-fold less in comparison to the amount of FVIII not linked to XTEN (or native FVIII) that must be administered to achieve hemostasis in a comparable subject. It will be understood by one of skill in the art that the amount of procoagulant administered to maintain hemostasis will depend on the severity of FVIII deficiency and/or the frequency or duration of bleeding.
A particular object of the present invention relates to use of CFXTEN with reduced binding by FVIII inhibitors that bind the A2 and/or C2 domains of Factor VIII as a drug. Such a drug is advantageously used for maintaining hemostasis in a patient suffering from haemophilia, wherein such patient has circulating FVIII inhibitors directed against the A2 domain and/or C2 domain of Factor VIII. In one embodiment, the invention provides a method of treatment, the method comprising the step of administering to the patient with a A2 domain-binding inhibitor a coagulation-effective amount of a CFXTEN fusion protein, wherein the CFXTEN exhibits at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% or less binding to an inhibitor that binds the A2 domain of FVIII, compared to the FVIII not linked to XTEN or to native FVIII, and wherein the administration results in hemostasis. In another embodiment, the invention provides a method of treatment, the method comprising the step of administering to the patient with a C2 domain-binding inhibitor a coagulation-effective amount of a CFXTEN fusion protein, wherein the CFXTEN exhibits at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% or less binding to an inhibitor that binds the C2 domain of FVIII, compared to the FVIII not linked to XTEN or to native FVIII, and wherein the administration results in hemostasis. The reduced binding of the subject CFXTEN can be assayed directly by ELISA that detects FVIII inhibitors, or measured indirectly by demonstration of reduced inhibition of FVIII activity of the CFXTEN compared to native FVIII in the presence of an inhibitor as measured by a factor VIII chromogenic test or one-step assay as described herein, or other suitable coagulation methods known in the art. Alternatively, the subject CFXTEN can be measured for reduced (or absence of) inhibition in the presence of known inhibitors by use of a modified Bethesda assay. According to a particular aspect of the present invention, a CFXTEN useful in the methods has reduced reactivity to one or more antibodies from Table 10, as well as naturally-occurring antibodies found in hemophilia patients. For testing purposes, such and other inhibitory antibodies can be obtained from humans (i.e. from the serum of patients which have inhibitory antibodies) or can be obtained from mice, guinea pigs, horses, goats, non-human primates and other mammals by immunization with FVIII, or fragments thereof, more particularly with a fragment comprising the all or part of the A2 or C2 domain, whether in polyclonal or monoclonal form.
The invention further contemplates that the CFXTEN used in accordance with the methods provided herein can be administered in conjunction with other treatment methods and compositions (e.g., other coagulation proteins) useful for treating factor VIII-related conditions, or conditions for which coagulation factor is adjunctive therapy; e.g., bleeding episodes due to injury or surgery. In another aspect, the invention provides methods of preparing a drug for a factor VIII-related condition, comprising combining a factor VIII sequence selected from Table 1 with one or more XTEN selected from Table 4 inserted in one or more insertion sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9 to result in a drug that retains at least a portion of the activity of the native FVIII. The invention provides a method of preparing a pharmaceutical composition, comprising the step of combining the drug of the foregoing embodiment with at least one pharmaceutically acceptable carrier. In one embodiment of the method of preparing a drug for a factor VIII-related condition, the factor VIII has a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity compared to a sequence selected from Table 1 and the one or more XTEN has a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity compared to a sequence selected from any one of Tables 3, 4, and 13-17, or a fragment thereof, wherein the one or more XTEN are inserted in one or more locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In a particular embodiment of the foregoing, at least one XTEN insertion site is selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered relative to mature native human FVIII). In another embodiment of the method, the CFXTEN comprises a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity compared to a sequence selected from any one of Table 21.
In another aspect, the invention provides a method of making the CFXTEN compositions to achieve desired pharmacokinetic, pharmacologic or pharmaceutical properties. In general, the steps in the design and production of the inventive fusion protein compositions, as illustrated in FIGS. 11-13 , include: (1) the selection of a FVIII (e.g., native proteins, sequences of Table 1, analogs or derivatives with activity) to treat the particular condition; (2) selecting one or more XTEN (e.g., sequences with at least 80% identity to sequences set forth in Table 4) that will confer the desired pharmacokinetic and physicochemical characteristics on the resulting CFXTEN (e.g., the administration of the CFXTEN composition to a subject results in the fusion protein being maintained above 0.05-0.4 IU/ml for a greater period compared to FVIII not linked to XTEN); (3) establishing a desired N- to C-terminus configuration of the CFXTEN to achieve the desired efficacy or PK parameters (e.g., selecting one or more insertion sites from Table 5, Table 6, Table 7, Table 8, and Table 9); (4) establishing the design of the expression vector encoding the configured CFXTEN; (5) transforming a suitable host with the expression vector; and (6) expressing and recovering the resultant isolated CFXTEN fusion protein. In one embodiment of the method of making CFXTEN, the XTEN for insertion are evaluated by the application of Equation IV to maximize the Ratio XTEN Radii for the fusion protein construct, with the XTEN resulting in values greater than 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0 being preferred. For those CFXTEN for which an increase in half-life or an increased period of time spent above the minimum coagulation-effective concentration is desired, the XTEN chosen for incorporation generally have at least about 144, or about 288, or about 432, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid residues where a single XTEN is to be incorporated into the CFXTEN. In another embodiment, the CFXTEN comprises a first XTEN of the foregoing lengths, and at least a second XTEN of about 36, or about 42, or about 72, or about 144, or about 288, or about 576, or about 864, or about 875, or about 912, or about 923, or about 1000 or more amino acid residues. The location of the XTEN within the fusion protein can include one, two, three, four, five or more locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 . In one embodiment, the method of design includes an insertion of XTEN into the FVIII of at least one site selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered relative to mature native human FVIII).
In another aspect, the invention provides methods of making CFXTEN compositions to improve ease of manufacture, result in increased stability, increased water solubility, and/or ease of formulation, as compared to the native FVIII. In one embodiment, the invention includes a method of increasing the water solubility of a FVIII comprising the step of linking the FVIII with at least about 80%, or about 90%, or about 95% identity to a sequence from Table 1 to one or more XTEN at one, two, three, four, five or more locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIG. 8-9 wherein the XTEN is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from any one of Tables 3, 4, and 13-17 such that a higher concentration in soluble form of the resulting CFXTEN can be achieved, under physiologic conditions, compared to the FVIII in an un-fused state. In a particular embodiment, the CFXTEN comprises a FVIII linked to two, three, four, or five XTEN having at least about 24, or about 36, or about 48, or about 60, or about 72, or about 84, or about 96, or about 144, or about 288 amino acid residues inserted at sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 , in which the solubility of the fusion protein under physiologic conditions is at least three-fold greater than the corresponding FVIII not linked to XTEN, or alternatively, at least four-fold, or five-fold, or six-fold, or seven-fold, or eight-fold, or nine-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 60-fold or greater than FVIII not linked to XTEN. Factors that contribute to the property of XTEN to confer increased water solubility of CFs when incorporated into a fusion protein include the high solubility of the XTEN fusion partner and the low degree of self-aggregation between molecules of XTEN in solution, as well as expanding the hydrophilicity of FVIII external loops into which the XTEN is inserted. In some embodiments, the method results in a CFXTEN fusion protein wherein the water solubility is at least about 20%, or at least about 30% greater, or at least about 50% greater, or at least about 75% greater, or at least about 90% greater, or at least about 100% greater, or at least about 150% greater, or at least about 200% greater, or at least about 400% greater, or at least about 600% greater, or at least about 800% greater, or at least about 1000% greater, or at least about 2000% greater under physiologic conditions, compared to the un-fused FVIII. In one embodiment, the XTEN of the CFXTEN fusion protein is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from any one of Tables 3, 4, and 13-17. In another embodiment, the invention includes a method of increasing the shelf-life of a FVIII comprising the step of linking the FVIII with one or more XTEN at one or more sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein the shelf-life of the resulting CFXTEN is extended compared to the FVIII in an un-fused state. As used herein, shelf-life refers to the period of time over which the procoagulant activity of a FVIII or CFXTEN that is in solution, lyophilized or in some other storage formulation remains stable without undue loss of activity or that remains within release specifications established for the pharmaceutical composition. A FVIII that degrades or aggregates generally has reduced functional activity or reduced bioavailability compared to one that remains in solution. Factors that contribute to the ability of the method to extend the shelf life of FVIII when incorporated into a fusion protein include increased water solubility, reduced self-aggregation in solution, and increased heat stability of the XTEN fusion partner. In particular, the low tendency of XTEN to aggregate facilitates methods of formulating pharmaceutical preparations containing higher drug concentrations of CFs, and the heat-stability of XTEN contributes to the property of CFXTEN fusion proteins to remain soluble and functionally active for extended periods. The method results in CFXTEN fusion proteins with prolonged or extended shelf-life that exhibit greater activity relative to a FVIII standard that has been subjected to the same storage and handling conditions. The standard may be the un-fused full-length FVIII or a commercially-available FVIII pharmaceutical composition. In one embodiment, the method includes the step of formulating the isolated CFXTEN with one or more pharmaceutically acceptable excipients that enhance the ability of the XTEN to retain its unstructured conformation and for the CFXTEN to remain soluble in the formulation for a time that is greater than that of the corresponding un-fused FVIII. In one embodiment, the method comprises linking a FVIII selected from Table 1 to one or more XTEN selected from any one of Tables 3, 4, and 13-17 inserted at one or more sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9 and admixing with at least one pharmaceutically acceptable excipient to create a pharmaceutical composition that retains greater than about 100% of the procoagulant activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the procoagulant activity of a FVIII standard subjected to the same storage and handling conditions when compared at a time point of at least 90 days, or at least 6 months, or at least 12 months. Shelf-life may also be assessed in terms of functional activity remaining after storage, normalized to functional activity when storage began. In some embodiments, CFXTEN pharmaceutical compositions of the invention retain about 50% more procoagulant activity, or about 60%, 70%, 80%, or 90% more of the procoagulant activity of a FVIII standard when subjected to the same conditions for the same period of up to 2 weeks, or 4 weeks, or 6 weeks or longer under various temperature conditions. In one embodiment, the CFXTEN pharmaceutical composition retains at least about 50%, or about 60%, or at least about 70%, or at least about 80%, and most preferably at least about 90% or more of its original activity in solution when heated at 80° C. for 10 min. In another embodiment, the CFXTEN pharmaceutical composition retains at least about 50%, preferably at least about 60%, or at least about 70%, or at least about 80%, or alternatively at least about 90% or more of its original activity in solution when heated or maintained at 37° C. for about 7 days. In another embodiment, CFXTEN pharmaceutical composition retains at least about 80% or more of its functional activity after exposure to a temperature of about 30° C. to about 70° C. over a period of time of about one hour to about 18 hours. In the foregoing embodiments hereinabove described in this paragraph, the retained activity of the CFXTEN pharmaceutical compositions is at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold greater at a given time point than that of a corresponding pharmaceutical composition comprising FVIII not linked to the XTEN.
VII). The Nucleic Acids Sequences of the Invention
The present invention provides isolated polynucleic acids encoding CFXTEN chimeric fusion proteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion proteins, including homologous variants thereof. In another aspect, the invention encompasses methods to produce polynucleic acids encoding CFXTEN chimeric fusion proteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion protein, including homologous variants thereof. In general, and as illustrated in FIGS. 11-13 , the methods of producing a polynucleotide sequence coding for a CFXTEN fusion protein and expressing the resulting gene product include assembling nucleotides encoding FVIII and XTEN, ligating the components in frame, incorporating the encoding gene into an expression vector appropriate for a host cell, transforming the appropriate host cell with the expression vector, and culturing the host cell under conditions causing or permitting the fusion protein to be expressed in the transformed host cell, thereby producing the biologically-active CFXTEN polypeptide, which is recovered as an isolated fusion protein by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology is used to make the polynucleotides and expression vectors of the present invention.
In accordance with the invention, nucleic acid sequences that encode CFXTEN (or its complement) are used to generate recombinant DNA molecules that direct the expression of CFXTEN fusion proteins in appropriate host cells. For the purposes of the invention, nucleic acid encoding a signal peptide corresponding to that of native human FVIII (encoding MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611)) can be added to any of the encoding constructs described herein to aid in the expression and secretion of the CFXTEN fusion protein. In one embodiment, the nucleic acid add is ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613), or the complement thereof.
Several cloning strategies are suitable for performing the present invention, many of which is used to generate a construct that comprises a gene coding for a fusion protein of the CFXTEN composition of the present invention, or its complement. In some embodiments, the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises at least a first FVIII and at least a first XTEN polypeptide, or their complement. In one embodiment of the foregoing, the gene comprises a sequence encoding a FVIII or sequence variant. In other embodiments, the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises nucleotides encoding at least a first molecule of FVIII or its complement and a first and at least a second XTEN or their complement that is used to transform a host cell for expression of the fusion protein of the CFXTEN composition. In the foregoing embodiments hereinabove described in this paragraph, the genes can further comprise nucleotides encoding spacer sequences that also encode cleavage sequence(s).
In designing a desired XTEN sequences, it was discovered that the non-repetitive nature of the XTEN of the inventive compositions is achieved despite use of a “building block” molecular approach in the creation of the XTEN-encoding sequences. This was achieved by the use of a library of polynucleotides encoding peptide sequence motifs, described above, that are then ligated and/or multimerized to create the genes encoding the XTEN sequences (see FIGS. 11 and 12 and Examples). Thus, while the XTEN(s) of the expressed fusion protein may consist of multiple units of as few as four different sequence motifs, because the motifs themselves consist of non-repetitive amino acid sequences, the overall XTEN sequence is rendered non-repetitive. Accordingly, in one embodiment, the XTEN-encoding polynucleotides comprise multiple polynucleotides that encode non-repetitive sequences, or motifs, operably linked in frame and in which the resulting expressed XTEN amino acid sequences are non-repetitive.
In one approach, a construct is first prepared containing the DNA sequence corresponding to CFXTEN fusion protein. DNA encoding the FVIII of the compositions is obtained synthetically, from a commercial source, or from a cDNA library prepared using standard methods from tissue or isolated cells believed to possess FVIII mRNA and to express it at a detectable level. If necessary, the coding sequence can be obtained using conventional primer extension procedures as described in Sambrook, et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA. One can then use polymerase chain reaction (PCR) methodology to amplify the target DNA or RNA coding sequence to obtain sufficient material for the preparation of the CFXTEN constructs containing the FVIII gene. Assays can then be conducted to confirm that the hybridizing full-length genes are the desired FVIII gene(s). By these conventional methods, DNA can be conveniently obtained from a cDNA library prepared from such sources. The FVIII encoding gene(s) can also created by standard synthetic procedures known in the art (e.g., automated nucleic acid synthesis using, for example one of the methods described in Engels et al. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNA sequences obtained from publicly available databases, patents, or literature references. Such procedures are well known in the art and well described in the scientific and patent literature. For example, sequences can be obtained from Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or GenBank Accession Numbers (e.g., Locus ID, NP_XXXXX, and XP_XXXXX) Model Protein identifiers available through the National Center for Biotechnology Information (NCBI) webpage, available on the world wide web at ncbi.nlm.nih.gov that correspond to entries in the CAS Registry or GenBank database that contain an amino acid sequence of the protein of interest or of a fragment or variant of the protein. In one embodiment, the FVIII encoding gene encodes a protein sequence from Table 1, or a fragment or variant thereof.
A gene or polynucleotide encoding the FVIII portion of the subject CFXTEN protein, in the case of an expressed fusion protein that comprises a single FVIII, is then cloned into a construct, which is a plasmid or other vector under control of appropriate transcription and translation sequences for high level protein expression in a biological system. In a later step, a second gene or polynucleotide coding for the XTEN is genetically fused to the nucleotides encoding the N- and/or C-terminus of the FVIII gene by cloning it into the construct adjacent and in frame with the gene(s) coding for the FVIII. This second step occurs through a ligation or multimerization step. In the foregoing embodiments hereinabove described in this paragraph, it is to be understood that the gene constructs that are created can alternatively be the complement of the respective genes that encode the respective fusion proteins.
The gene encoding for the XTEN can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to ligate short sequences of polynucleotides encoding XTEN into longer XTEN genes of a desired length and sequence. In one embodiment, the method ligates two or more codon-optimized oligonucleotides encoding XTEN motif or segment sequences of about 9 to 14 amino acids, or about 12 to 20 amino acids, or about 18 to 42 amino acids, or about 42 to about 144 amino acids, or about 144 to about 288 amino acids, or 288 to about 864 amino acids or longer, or any combination of the foregoing ranges of motif or segment lengths.
Alternatively, the disclosed method is used to multimerize XTEN-encoding sequences into longer sequences of a desired length; e.g., a gene encoding 36 amino acids of XTEN can be dimerized into a gene encoding 72 amino acids, then 144, then 288, etc. Even with multimerization, XTEN polypeptides can be constructed such that the XTEN-encoding gene has low or virtually no repetitiveness through design of the codons selected for the motifs of the shortest unit being used, which can reduce recombination and increase stability of the encoding gene in the transformed host.
Genes encoding XTEN with non-repetitive sequences are assembled from oligonucleotides using standard techniques of gene synthesis. The gene design can be performed using algorithms that optimize codon usage and amino acid composition. In one method of the invention, a library of relatively short XTEN-encoding polynucleotide constructs is created and then assembled, as described above. The resulting genes are then assembled with genes encoding FVIII or regions of FVIII, as illustrated in FIGS. 11 and 12 , and the resulting genes used to transform a host cell and produce and recover the CFXTEN for evaluation of its properties, as described herein.
In another aspect, the invention provides isolated nucleic acids comprising a polynucleotide sequence encoding the CFXTEN fusion protein embodiments described herein. In one embodiment, the isolated nucleic acid comprises a polynucleotide sequence selected from (a) a sequence having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a sequence of comparable length selected from Table 21, when optimally aligned, or (b) the complement of the polynucleotide of (a). In another embodiment, the isolated nucleic acid comprises the sequence ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613) linked to the 5′ end of the nucleic acid of (a) or the complement of the sequence linked to the 3′ end of (b).
Polynucleotide Libraries
In another aspect, the invention provides libraries of polynucleotides that encode XTEN sequences that are used to assemble genes that encode XTEN of a desired length and sequence.
In certain embodiments, the XTEN-encoding library constructs comprise polynucleotides that encode polypeptide segments of a fixed length. As an initial step, a library of oligonucleotides that encode motifs of 9-14 amino acid residues can be assembled. In a preferred embodiment, libraries of oligonucleotides that encode motifs of 12 amino acids are assembled.
The XTEN-encoding sequence segments can be dimerized or multimerized into longer encoding sequences, as depicted schematically in FIG. 13 . Dimerization or multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art. This process of can be repeated multiple times until the resulting XTEN-encoding sequences have reached the organization of sequence and desired length, providing the XTEN-encoding genes. As will be appreciated, a library of polynucleotides that encodes, e.g., 12 amino acid motifs can be dimerized and/or ligated into a library of polynucleotides that encode 36 amino acids. Libraries encoding motifs of different lengths; e.g., 9-14 amino acid motifs leading to libraries encoding 27 to 42 amino acids are contemplated by the invention. In turn, the library of polynucleotides that encode 27 to 42 amino acids, and preferably 36 amino acids (as described in the Examples) can be serially dimerized into a library containing successively longer lengths of polynucleotides that encode XTEN sequences of a desired length for incorporation into the gene encoding the CFXTEN fusion protein, as disclosed herein.
A more efficient way to optimize the DNA sequence encoding XTEN is based on combinatorial libraries. The gene encoding XTEN can be designed and synthesized in segment such that multiple codon versions are obtained for each segment. These segments can be randomly assembled into a library of genes such that each library member encodes the same amino acid sequences but library members comprise a large number of codon versions. Such libraries can be screened for genes that result in high-level expression and/or a low abundance of truncation products. The process of combinatorial gene assembly is illustrated in FIG. 18 . The genes in FIG. 18 are assembled from 6 base fragments and each fragment is available in 4 different codon versions. This allows for a theoretical diversity of 4096.
In some embodiments, libraries are assembled of polynucleotides that encode amino acids that are limited to specific sequence XTEN families; e.g., the AD, AE, AF, AG, AM, or AQ sequences of Table 4. In other embodiments, libraries comprise sequences that encode two or more of the motif family sequences from Table 3. The names and sequences of representative, non-limiting polynucleotide sequences of libraries that encode 36mers are presented in Tables 13-17, and the methods used to create them are described more fully in the respective Examples. In other embodiments, libraries that encode XTEN are constructed from segments of polynucleotide codons linked in a randomized sequence that encode amino acids wherein at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% of the codons are selected from the group consisting of condons for glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) amino acids. The libraries can be used, in turn, for serial dimerization or ligation to achieve polynucleotide sequence libraries that encode XTEN sequences, for example, of 42, 48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids, or up to a total length of about 3000 amino acids, as well as intermediate lengths, in which the encoded XTEN can have one or more of the properties disclosed herein, when expressed as a component of a CFXTEN fusion protein. In some cases, the polynucleotide library sequences may also include additional bases used as “sequencing islands,” described more fully below.
FIG. 14 is a schematic flowchart of representative, non-limiting steps in the assembly of a XTEN polynucleotide construct and a CFXTEN polynucleotide construct in the embodiments of the invention. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a library to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The resulting pool of ligation products is gel-purified and the band with the desired length of XTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene is cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is than performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, resulting in the gene 500 encoding an FVIII-XTEN fusion protein.
One may clone the library of XTEN-encoding genes into one or more expression vectors known in the art. To facilitate the identification of well-expressing library members, one can construct the library as fusion to a reporter protein. Non-limiting examples of suitable reporter genes are green fluorescent protein, luciferace, alkaline phosphatase, and beta-galactosidase. By screening, one can identify short XTEN sequences that can be expressed in high concentration in the host organism of choice. Subsequently, one can generate a library of random XTEN dimers and repeat the screen for high level of expression. Subsequently, one can screen the resulting constructs for a number of properties such as level of expression, protease stability, or binding to antiserum.
One aspect of the invention is to provide polynucleotide sequences encoding the components of the fusion protein wherein the creation of the sequence has undergone codon optimization. Of particular interest is codon optimization with the goal of improving expression of the polypeptide compositions and to improve the genetic stability of the encoding gene in the production hosts. For example, codon optimization is of particular importance for XTEN sequences that are rich in glycine or that have very repetitive amino acid sequences. Codon optimization is performed using computer programs (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53), some of which minimize ribosomal pausing (Coda Genomics Inc.). In one embodiment, one can perform codon optimization by constructing codon libraries where all members of the library encode the same amino acid sequence but where codon usage is varied. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production of XTEN-containing products. When designing XTEN sequences one can consider a number of properties. One can minimize the repetitiveness in the encoding DNA sequences. In addition, one can avoid or minimize the use of codons that are rarely used by the production host (e.g. the AGG and AGA arginine codons and one leucine codon in E. coli ). In the case of E. coli , two glycine codons, GGA and GGG, are rarely used in highly expressed proteins. Thus codon optimization of the gene encoding XTEN sequences can be very desirable. DNA sequences that have a high level of glycine tend to have a high GC content that can lead to instability or low expression levels. Thus, when possible, it is preferred to choose codons such that the GC-content of XTEN-encoding sequence is suitable for the production organism that will be used to manufacture the XTEN.
In one embodiment, polynucleotide libraries are constructed using the disclosed methods wherein all members of the library encode the same amino acid sequence but where codon usage for the respective amino acids in the sequence is varied or optimized for the intended host cell. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production of XTEN-containing products. In one embodiment, the libraries are optimized for expression in a eukaryotic host cell.
Optionally, one can sequence clones in the library to eliminate isolates that contain undesirable sequences. The initial library of short XTEN sequences allows some variation in amino acid sequence. For instance one can randomize some codons such that a number of hydrophilic amino acids can occur in a particular position. During the process of iterative multimerization one can screen the resulting library members for other characteristics like solubility or protease resistance in addition to a screen for high-level expression.
Once the gene that encodes the XTEN of desired length and properties is selected, it is genetically fused at the desired location to the nucleotides encoding the FVIII gene(s) by cloning it into the construct adjacent and in frame with the gene coding for FVIII, or alternatively between nucleotides encoding adjacent domains of the FVIII, or alternatively within a sequence encoding a given FVIII domain, or alternatively in frame with nucleotides encoding a spacer/cleavage sequence linked to a terminal XTEN. The invention provides various permutations of the foregoing, depending on the CFXTEN to be encoded. For example, a gene encoding a CFXTEN fusion protein comprising a FVIII and two XTEN, such as embodied by formula VI, as depicted above, the gene would have polynucleotides encoding FVIII, encoding two XTEN, which can be identical or different in composition and sequence length. In one non-limiting embodiment of the foregoing, the FVIII polynucleotides would encode factor VIII and the polynucleotides encoding the C-terminus XTEN would encode an XTEN of 288 amino acids and the polynucleotides encoding an internal XTEN adjacent to the C-terminus of the A2 domain would encode an XTEN of 144 amino acids. The step of cloning the FVIII genes into the XTEN construct can occur through a ligation or multimerization step, as shown in FIG. 14 . The constructs encoding CFXTEN fusion proteins can be designed in different configurations of the components XTEN, CF, and spacer sequences, such as the configurations of formulae I-VIII. In one embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5′ to 3′) FVIII, an XTEN internal to the B domain, and a C-terminal XTEN. In another embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5′ to 3′) FVIIII, spacer sequence linked to the C-terminus, and XTEN. The spacer polynucleotides can optionally comprise sequences encoding cleavage sequences. As will be apparent to those of skill in the art, multiple permutations of FVIII domains and inserted XTEN are possible.
Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences may also be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics. 1981. 2: 482-489), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, (Journal of Molecular Biology. 1970. 48:443-453). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores.
Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term “complementary sequences” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the polynucleotides that encode the CFXTEN sequences under stringent conditions, such as those described herein.
The resulting polynucleotides encoding the CFXTEN chimeric fusion proteins can then be individually cloned into an expression vector. The nucleic acid sequence is inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Such techniques are well known in the art and well described in the scientific and patent literature.
Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Representative plasmids are illustrated in FIG. 17 , with encoding regions for different configurations of FVIII and XTEN components portrayed.
The invention provides for the use of plasmid vectors containing replication and control sequences that are compatible with and recognized by the host cell, and are operably linked to the CFXTEN gene for controlled expression of the CFXTEN fusion proteins. The vector ordinarily carries a replication site, as well as sequences that encode proteins that are capable of providing phenotypic selection in transformed cells. Such vector sequences are well known for a variety of bacteria, yeast, and viruses. Useful expression vectors that can be used include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. “Expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA encoding the fusion protein in a suitable host. The requirements are that the vectors are replicable and viable in the host cell of choice. Low- or high-copy number vectors may be used as desired.
Other suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col E1, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of the 2 m plasmid, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or the expression control sequences; and the like. Yeast expression systems that can also be used in the present invention include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like.
The control sequences of the vector include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control termination of transcription and translation. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the DNA encoding the FVIII polypeptide variant in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell 41:521-530, 1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences such as UCOE (ubiquitous chromatin opening elements).
Examples of suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter or the tpiA promoter. Examples of other useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase. Preferred are the TAKA-amylase and gluA promoters.
Promoters suitable for use in expression vectors with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)], all is operably linked to the DNA encoding CFXTEN polypeptides. Promoters for use in bacterial systems can also contain a Shine-Dalgarno (S.D.) sequence, operably linked to the DNA encoding CFXTEN polypeptides.
The invention contemplates use of other expression systems including, for example, a baculovirus expression system with both non-fusion transfer vectors, such as, but not limited to pVL941 Summers, et al., Virology 84:390-402 (1978)), pVL1393 (Invitrogen), pVL1392 (Summers, et al., Virology 84:390-402 (1978) and Invitrogen) and pBlueBacIII (Invitrogen), and fusion transfer vectors such as, but not limited to, pAc7 00 (Summers, et al., Virology 84:390-402 (1978)), pAc701 and pAc70-2 (same as pAc700, with different reading frames), pAc360 Invitrogen) and pBlueBacHisA, B, C (Invitrogen) can be used.
Examples of suitable promoters for directing the transcription of the DNA encoding the FVIII polypeptide variant in mammalian cells are the CMV promoter (Boshart et al., Cell 41:521-530, 1985), the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences such as UCOE (ubiquitous chromatin opening elements).
The DNA sequences encoding the CFXTEN may also, if necessary, be operably connected to a suitable terminator, such as the hGH terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or the TPI1 terminators (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099). Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the CFXTEN sequence itself, including splice sites obtained from adenovirus. Also contained in the expression vectors is a polyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 Elb region, the hGH terminator (DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981). The expression vectors may also include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites; and enhancer sequences, such as the SV40 enhancer.
To direct the CFXTEN of the present invention into the secretory pathway of the host cells, a secretory signal sequence (a.k.a., a leader sequence, a prepro sequence, or a pre sequence) may be included in the recombinant vector. The secretory signal sequence is operably linked to the DNA sequences encoding the CFXTEN, usually positioned 5′ to the DNA sequence encoding the CFXTEN fusion protein. The secretory signal sequence may be that, normally associated with the native FVIII protein or may be from a gene encoding another secreted protein. Non-limiting examples include OmpA, PhoA, and DsbA for E. coli expression, ppL-alpha, DEX4, invertase signal peptide, acid phosphatase signal peptide, CPY, or INU1 for yeast expression, and IL2L, SV40, IgG kappa and IgG lambda for mammalian expression. Signal sequences are typically proteolytically removed from the protein during the translocation and secretion process, generating a defined N-terminus. Methods are disclosed in Arnau, et al., Protein Expression and Purification 48: 1-13 (2006).
The procedures used to ligate the DNA sequences coding for the CFXTEN, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3 rd edition, Cold Spring Harbor Laboratory Press, 2001). In this manner, a chimeric DNA molecule coding for a monomeric CFXTEN fusion protein is generated within the construct. Optionally, this chimeric DNA molecule may be transferred or cloned into another construct that is a more appropriate expression vector. At this point, a host cell capable of expressing the chimeric DNA molecule can be transformed with the chimeric DNA molecule.
Non-limiting examples of mammalian cell lines for use in the present invention are the COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), BHK-570 cells (ATCC CRL 10314), CHO-K1 (ATCC CCL 61), CHO-S (Invitrogen 11619-012), and 293-F (Invitrogen R790-7), and the parental and derivative cell lines known in the art useful for expression of FVIII. A tk-ts 13 BHK cell line is also available from the ATCC under accession number CRL 1632. In addition, a number of other cell lines may be used within the present invention, including Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
Examples of suitable yeasts cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri . Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides there from are described, e.g. in U.S. Pat. No. 4,599,311, U.S. Pat. No. 4,931,373, U.S. Pat. Nos. 4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine. A preferred vector for use in yeast is the POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequences encoding the CFXTEN may be preceded by a signal sequence and optionally a leader sequence, e.g. as described above. Further examples of suitable yeast cells are strains of Kluyveromyces , such as K. lactis, Hansenula , e.g. H. polymorpha , or Pichia , e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279). Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger . The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023, EP 184 438 The transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989, Gene 78: 147-156. The transformation of Trichoderma spp. may be performed for instance as described in EP 244 234.
Other suitable cells that can be used in the present invention include, but are not limited to, prokaryotic host cells strains such as Escherichia coli , (e.g., strain DH5-a), Bacillus subtilis, Salmonella typhimurium , or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus . Non-limiting examples of suitable prokaryotes include those from the genera: Actinoplanes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroflexus; Enterococcus; Escherichia; Lactobacillus; Listeria; Oceanobacillus; Paracoccus; Pseudomonas; Staphylococcus; Streptococcus; Streptomyces; Thermoplasma ; and Vibrio.
Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g., Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.
Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973), transfection with many commercially available reagents such as FuGENEG Roche Diagnostics, Mannheim, Germany) or lipofectamine (Invitrogen) or by electroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identify and select cells that express the exogenous DNA, a gene that confers a selectable phenotype (a selectable marker) is generally introduced into cells along with the gene or cDNA of interest. Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, puromycin, zeocin, and methotrexate. The selectable marker may be an amplifiable selectable marker. A preferred amplifiable selectable marker is a dihydrofolate reductase (DHFR) sequence. Further examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., incorporated herein by reference). The person skilled in the art will easily be able to choose suitable selectable markers. Any known selectable marker may be employed so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.
Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If, on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to add additional DNA, known as “carrier DNA,” to the mixture that is introduced into the cells.
After the cells have taken up the DNA, they are grown in an appropriate growth medium, typically 1-2 days, to begin expressing the gene of interest. As used herein the term “appropriate growth medium” means a medium containing nutrients and other components required for the growth of cells and the expression of the CFXTEN of interest. Media generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, protein and growth factors. For production of gamma-carboxylated proteins, the medium will contain vitamin K, preferably at a concentration of about 0.1 μg/ml to about 5 μg/ml. Drug selection is then applied to select for the growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased to select for an increased copy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of the FVIII polypeptide variant of interest.
The transformed or transfected host cell is then cultured in a suitable nutrient medium under conditions permitting expression of the CFXTEN polypeptide after which the resulting peptide may be recovered from the culture as an isolated fusion protein. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
Gene expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological of fluorescent methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids or the detection of selectable markers, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence FVIII polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to FVIII and encoding a specific antibody epitope. Examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT).
Expressed CFXTEN polypeptide product(s) may be purified via methods known in the art or by methods disclosed herein. Procedures such as gel filtration, affinity purification (e.g., using an anti-FVIII antibody column), salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography and gel electrophoresis may be used; each tailored to recover and purify the fusion protein produced by the respective host cells. Additional purification may be achieved by conventional chemical purification means, such as high performance liquid chromatography. Some expressed CFXTEN may require refolding during isolation and purification. Methods of purification are described in Robert K. Scopes,
Protein Purification Principles and Practice, Charles R. Castor (ed.), Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step purification separations are also described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83 (1994). For therapeutic purposes it is preferred that the CFXTEN fusion proteins of the invention are substantially pure. Thus, in a preferred embodiment of the invention the CFXTEN of the invention is purified to at least about 90 to 95% homogeneity, preferably to at least about 98% homogeneity. Purity may be assessed by, e.g., gel electrophoresis, HPLC, and amino-terminal amino acid sequencing.
VIII). Pharmaceutical Compositions
The present invention provides pharmaceutical compositions comprising CFXTEN. In one embodiment, the pharmaceutical composition comprises a CFXTEN fusion protein disclosed herein admixed with at least one pharmaceutically acceptable carrier. CFXTEN polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the polypeptide is combined in admixture with a pharmaceutically acceptable carrier vehicle, such as aqueous solutions, buffers, solvents and/or pharmaceutically acceptable suspensions, emulsions, stabilizers or excipients. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. Formulations of the pharmaceutical compositions are prepared for storage by mixing the active CFXTEN ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients (e.g., sodium chloride, a calcium salt, sucrose, or polysorbate) or stabilizers (e.g., sucrose, trehalose, raffinose, arginine, a calcium salt, glycine or histidine), as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilized formulations or aqueous solutions.
The pharmaceutical composition may be supplied as a lyophilized powder to be reconstituted prior to administration. In another embodiment, the pharmaceutical composition may be supplied in a liquid form in a vial, the contents of which can be administered directly to a patient. Alternatively, the composition is supplied as a liquid in a pre-filled syringe for administration of the composition. In another embodiment, the composition is supplied as a liquid in a pre-filled vial that can be incorporated into a pump.
The pharmaceutical compositions can be administered by any suitable means or route, including subcutaneously, subcutaneously by infusion pump, intramuscularly, and intravenously. It will be appreciated that the preferred route will vary with the disease and age of the recipient, and the severity of the condition being treated.
In one embodiment, the CFXTEN pharmaceutical composition in liquid form or after reconstitution (when supplied as a lyophilized powder) comprises coagulation factor VIII with an activity of at least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at least 300 IU/ml, or at least 400 IU/ml, or an activity of at least 500 IU/ml, or an activity of at least 600 IU/ml, which composition is capable of increasing factor VIII activity to at least 1.5% of the normal plasma level in the blood for at least about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours after administration of the factor VIII pharmaceutical composition to a subject in need of routine prophylaxis. In another embodiment, the CFXTEN pharmaceutical composition in liquid form or after reconstitution (when supplied as a lyophilized powder) comprises coagulation factor VIII with an activity of at least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at least 300 IU/ml, or at least 400 IU/ml, or at least 500 IU/ml, or an activity of at least 600 IU/ml, which composition is capable of increasing factor VIII activity to at least 2.5% of the normal plasma level in the blood for at least about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours after administration to a subject in need of routine prophylaxis. It is specifically contemplated that the pharmaceutical compositions of the foregoing can be formulated to include one or more excipients, buffers or other ingredients known in the art to be compatible with administration by the intravenous route or the subcutaneous route or the intramuscular route. Thus, in the embodiments hereinabove described in this paragraph, the pharmaceutical composition is administered subcutaneously, intramuscularly or intravenously.
The compositions of the invention may be formulated using a variety of excipients. Suitable excipients include microcrystalline cellulose (e.g. Avicel PH102, Avicel PH101), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (such as Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K100M, Premium CR Methocel K100M, Methocel E5, Opadry®), magnesium stearate, talc, triethyl citrate, aqueous ethylcellulose dispersion (Surelease®), and protamine sulfate. The slow release agent may also comprise a carrier, which can comprise, for example, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Pharmaceutically acceptable salts can also be used in these slow release agents, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The composition may also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes may also be used as a carrier.
In another embodiment, the compositions of the present invention are encapsulated in liposomes, which have demonstrated utility in delivering beneficial active agents in a controlled manner over prolonged periods of time. Liposomes are closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be unilamellar vesicles possessing a single membrane bilayer or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting membrane bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase. In one embodiment, the liposome may be coated with a flexible water soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding the liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences as described in U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094, the contents of which are incorporated by reference in their entirety. Additional liposomal technologies are described in U.S. Pat. Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024; 6,294,191; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and 4,684,479, the contents of which are incorporated herein by reference. These describe liposomes and lipid-coated microbubbles, and methods for their manufacture. Thus, one skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a liposome for the extended release of the polypeptides of the present invention.
For liquid formulations, a desired property is that the formulation be supplied in a form that can pass through a 25, 28, 30, 31, 32 gauge needle for intravenous, intramuscular, intraarticular, or subcutaneous administration.
Syringe pumps may also be used as slow release agents. Such devices are described in U.S. Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908; 6,572,585; 5,298,022; 5,176,502; 5,492,534; 5,318,540; and 4,988,337, the contents of which are incorporated herein by reference. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a syringe pump for the extended release of the compositions of the present invention.
IX). Pharmaceutical Kits
In another aspect, the invention provides a kit to facilitate the use of the CFXTEN polypeptides. The kit comprises the pharmaceutical composition provided herein, a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc., formed from a variety of materials such as glass or plastic. The container holds a pharmaceutical composition as a formulation that is effective for treating the FVIII-related condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The package insert can list the approved indications for the drug, instructions for the reconstitution and/or administration of the drug for the use for the approved indication, appropriate dosage and safety information, and information identifying the lot and expiration of the drug. In another embodiment of the foregoing, the kit can comprise a second container that can carry a suitable diluent for the pharmaceutical composition, the use of which will provide the user with the appropriate concentration to be delivered to the subject.
EXAMPLES
Example 1
Construction of XTEN_AD36 Motif Segments
The following example describes the construction of a collection of codon-optimized genes encoding motif sequences of 36 amino acids. As a first step, a stuffer vector pCW0359 was constructed based on a pET vector and that includes a T7 promoter. pCW0359 encodes a cellulose binding domain (CBD) and a TEV protease recognition site followed by a stuffer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were inserted such that they generate compatible overhangs after digestion. The stuffer sequence is followed by a truncated version of the GFP gene and a His tag. The stuffer sequence contains stop codons and thus E. coli cells carrying the stuffer plasmid pCW0359 form non-fluorescent colonies. The stuffer vector pCW0359 was digested with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification. The sequences were designated XTEN_AD36, reflecting the AD family of motifs. Its segments have the amino acid sequence [X] 3 where X is a 12mer peptide with the sequences: GESPGGSSGSES (SEQ ID NO: 19), GSEGSSGPGESS (SEQ ID NO: 20), GSSESGSSEGGP (SEQ ID NO: 21), or GSGGEPSESGSS (SEQ ID NO: 22). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:







(SEQ ID NO: 1619)


AD1for: AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC





(SEQ ID NO: 1620)


AD1rev: ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC





(SEQ ID NO: 1621)


AD2for: AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC





(SEQ ID NO: 1622)


AD2rev: ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT





(SEQ ID NO: 1623)


AD3for: AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC





(SEQ ID NO: 1624)


AD3rev: ACCTGGACCRCCYTCRGAAGAACCGCTTTCRGARGA





(SEQ ID NO: 1625)


AD4for: AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC



























We also annealed the phosphorylated oligonucleotide 3 KpnI stopper For: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylated oligonucleotide pr — 3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in the resulting library designated LCW0401 showed green fluorescence after induction, which shows that the sequence of XTEN_AD36 had been ligated in frame with the GFP gene and that most sequences of XTEN_AD36 had good expression levels.
We screened 96 isolates from library LCW0401 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 39 clones were identified that contained correct XTEN_AD36 segments. The file names of the nucleotide and amino acid constructs and the sequences for these segments are listed in Table 13.





TABLE 13



DNA and Amino Acid Sequences for AD 36-mer motifs (SEQ ID NOS 203-278,

respectively, in order of appearance)





File name
Amino acid sequence
Nucleotide sequence



LCW0401_001_
GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCGTCCGAGTCTGGTAGCTCA

GFP-N_A01.ab1
SSGSESGESPGGSSGSES
GGTGAATCTCCGGGTGGCTCTAGCGGTTCCGAGTCA



GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA



LCW0401_002_
GSEGSSGPGESSGESPGG
GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCA

GFP-N_B01.ab1
SSGSESGSSESGSSEGGP
GGTGAATCTCCTGGTGGTTCCAGCGGTTCTGAATCA



GGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA



LCW0401_003_
GSSESGSSEGGPGSSESG
GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTGGTCCA

GFP-N_C01.ab1
SSEGGPGESPGGSSGSES
GGTTCCTCTGAAAGCGGTTCTTCTGAGGGTGGTCCA



GGTGAATCTCCGGGTGGCTCCAGCGGTTCCGAGTCA



LCW0401_004_
GSGGEPSESGSSGSSESG
GGTTCCGGTGGCGAACCGTCTGAATCTGGTAGCTCA

GFP-N_D01.ab1
SSEGGPGSGGEPSESGSS
GGTTCTTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA



GGTTCTGGTGGTGAACCTTCCGAGTCTGGTAGCTCA



LCW0401_007_
GSSESGSSEGGPGSEGSS
GGTTCTTCCGAAAGCGGTTCTTCTGAGGGTGGTCCA

GFP-N_F01.ab1
GPGESSGSEGSSGPGESS
GGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTCTTCA



GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCA



LCW0401_008_
GSSESGSSEGGPGESPGG
GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA

GFP-N_G01.ab1
SSGSESGSEGSSGPGESS
GGTGAATCTCCAGGTGGTTCCAGCGGTTCTGAGTCA



GGTAGCGAAGGTTCTTCTGGTCCAGGTGAATCCTCA



LCW0401_012_
GSGGEPSESGSSGSGGEP
GGTTCTGGTGGTGAACCGTCTGAGTCTGGTAGCTCA

GFP-N_H01.ab1
SESGSSGSEGSSGPGESS
GGTTCCGGTGGCGAACCATCCGAATCTGGTAGCTCA



GGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTCTTCA



LCW0401_015_
GSSESGSSEGGPGSEGSS
GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCA

GFP-N_A02.ab1
GPGESSGESPGGSSGSES
GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCA



GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCA



LCW0401_016_
GSSESGSSEGGPGSSESG
GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA

GFP-N_B02.ab1
SSEGGPGSSESGSSEGGP
GGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA



GGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCA



LCW0401_020_
GSGGEPSESGSSGSEGSS
GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCA

GFP-N_E02.ab1
GPGESSGSSESGSSEGGP
GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCA



GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA



LCW0401_022_
GSGGEPSESGSSGSSESG
GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA

GFP-N_F02.ab1
SSEGGPGSGGEPSESGSS
GGTTCTTCCGAAAGCGGTTCTTCTGAAGGTGGTCCA



GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCA



LCW0401_024_
GSGGEPSESGSSGSSESG
GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA

GFP-N_G02.ab1
SSEGGPGESPGGSSGSES
GGTTCCTCCGAAAGCGGTTCTTCTGAAGGTGGTCCA



GGTGAATCTCCAGGTGGTTCTAGCGGTTCTGAATCA



LCW0401_026_
GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCGTCTGAGTCTGGTAGCTCA

GFP-N_H02.ab1
SSGSESGSEGSSGPGESS
GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAATCA



GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCA



LCW0401_027_
GSGGEPSESGSSGESPGG
GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCA

GFP-N_A03.ab1
SSGSESGSGGEPSESGSS
GGTGAATCTCCGGGTGGTTCTAGCGGTTCTGAGTCA



GGTTCTGGTGGTGAACCTTCCGAGTCTGGTAGCTCA



LCW0401_028_
GSSESGSSEGGPGSSESG
GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA

GFP-N_B03.ab1
SSEGGPGSSESGSSEGGP
GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA



GGTTCTTCCGAAAGCGGTTCTTCTGAAGGCGGTCCA



LCW0401_030_
GESPGGSSGSESGSEGSS
GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAGTCA

GFP-N_C03.ab1
GPGESSGSEGSSGPGESS
GGTAGCGAAGGTTCTTCCGGTCCGGGTGAGTCCTCA



GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCTTCA



LCW0401_031_
GSGGEPSESGSSGSGGEP
GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCA

GFP-N_D03.ab1
SESGSSGSSESGSSEGGP
GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCA



GGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCA



LCW0401_033_
GSGGEPSESGSSGSGGEP
GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCA

GFP-N_E03.ab1
SESGSSGSGGEPSESGSS
GGTTCCGGTGGCGAACCATCCGAGTCTGGTAGCTCA



GGTTCCGGTGGTGAACCATCCGAGTCTGGTAGCTCA



LCW0401_037_
GSGGEPSESGSSGSSESG
GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCA

GFP-N_F03.ab1
SSEGGPGSEGSSGPGESS
GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA



GGTAGCGAAGGTTCTTCTGGTCCGGGCGAGTCTTCA



LCW0401_038_
GSGGEPSESGSSGSEGSS
GGTTCCGGTGGTGAACCGTCCGAGTCTGGTAGCTCA

GFP-N_G03.ab1
GPGESSGSGGEPSESGSS
GGTAGCGAAGGTTCTTCTGGTCCGGGTGAGTCTTCA



GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA



LCW0401_039_
GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA

GFP-N_H03.ab1
SSGSESGSGGEPSESGSS
GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA



GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCA



LCW0401_040_
GSSESGSSEGGPGSGGEP
GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA

GFP-N_A04.ab1
SESGSSGSSESGSSEGGP
GGTTCCGGTGGTGAACCATCTGAATCTGGTAGCTCA



GGTTCTTCTGAAAGCGGTTCTTCTGAAGGTGGTCCA



LCW0401_042_
GSEGSSGPGESSGESPGG
GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCTTCA

GFP-N_C04.ab1
SSGSESGSEGSSGPGESS
GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCA



GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCCTCA



LCW0401_046_
GSSESGSSEGGPGSSESG
GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCGGTCCA

GFP-N_D04.ab1
SSEGGPGSSESGSSEGGP
GGTTCTTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA



GGTTCCTCCGAAAGCGGTTCTTCTGAGGGTGGTCCA



LCW0401_047_
GSGGEPSESGSSGESPGG
GGTTCTGGTGGCGAACCTTCCGAGTCTGGTAGCTCA

GFP-N_E04.ab1
SSGSESGESPGGSSGSES
GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAGTCA



GGTGAATCTCCGGGTGGTTCCAGCGGTTCTGAGTCA



LCW0401_051_
GSGGEPSESGSSGSEGSS
GGTTCTGGTGGCGAACCATCTGAGTCTGGTAGCTCA

GFP-N_F04.ab1
GPGESSGESPGGSSGSES
GGTAGCGAAGGTTCTTCCGGTCCAGGCGAGTCTTCA



GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCA



LCW0401_053_
GESPGGSSGSESGESPGG
GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA

GFP-N_H04.ab1
SSGSESGESPGGSSGSES
GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCA



GGTGAATCTCCTGGTGGTTCTAGCGGTTCTGAATCA



LCW0401_054_
GSEGSSGPGESSGSEGSS
GGTAGCGAAGGTTCTTCCGGTCCAGGTGAATCTTCA

GFP-N_A05.ab1
GPGESSGSGGEPSESGSS
GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCCTCA



GGTTCCGGTGGCGAACCATCTGAATCTGGTAGCTCA



LCW0401_059_
GSGGEPSESGSSGSEGSS
GGTTCTGGTGGCGAACCATCCGAATCTGGTAGCTCA

GFP-N_D05.ab1
GPGESSGESPGGSSGSES
GGTAGCGAAGGTTCTTCTGGTCCTGGCGAATCTTCA



GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAATCA



LCW0401_060_
GSGGEPSESGSSGSSESG
GGTTCCGGTGGTGAACCGTCCGAATCTGGTAGCTCA

GFP-N_E05.ab1
SSEGGPGSGGEPSESGSS
GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA



GGTTCCGGTGGTGAACCTTCTGAGTCTGGTAGCTCA



LCW0401_061_
GSSESGSSEGGPGSGGEP
GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA

GFP-N_F05.ab1
SESGSSGSEGSSGPGESS
GGTTCTGGTGGCGAACCATCTGAATCTGGTAGCTCA



GGTAGCGAAGGTTCTTCCGGTCCGGGTGAATCTTCA



LCW0401_063_
GSGGEPSESGSSGSEGSS
GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA

GFP-N_H05.ab1
GPGESSGSEGSSGPGESS
GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCA



GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCA



LCW0401_066_
GSGGEPSESGSSGSSESG
GGTTCTGGTGGCGAACCATCCGAGTCTGGTAGCTCA

GFP-N_B06.ab1
SSEGGPGSGGEPSESGSS
GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCA



GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA



LCW0401_067_
GSGGEPSESGSSGESPGG
GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCA

GFP-N_C06.ab1
SSGSESGESPGGSSGSES
GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAATCA



GGTGAATCTCCAGGTGGTTCTAGCGGTTCCGAATCA



LCW0401_069_
GSGGEPSESGSSGSGGEP
GGTTCCGGTGGTGAACCATCTGAGTCTGGTAGCTCA

GFP-N_D06.ab1
SESGSSGESPGGSSGSES
GGTTCCGGTGGCGAACCGTCCGAGTCTGGTAGCTCA



GGTGAATCTCCGGGTGGTTCCAGCGGTTCCGAATCA



LCW0401_070_
GSEGSSGPGESSGSSESG
GGTAGCGAAGGTTCTTCTGGTCCGGGCGAATCCTCA

GFP-N_E06.ab1
SSEGGPGSEGSSGPGESS
GGTTCCTCCGAAAGCGGTTCTTCCGAAGGTGGTCCA



GGTAGCGAAGGTTCTTCCGGTCCTGGTGAATCTTCA



LCW0401_078_
GSSESGSSEGGPGESPGG
GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCGGTCCA

GFP-N_F06.ab1
SSGSESGESPGGSSGSES
GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAATCA



GGTGAATCTCCTGGTGGCTCCAGCGGTTCCGAGTCA



LCW0401_079_
GSEGSSGPGESSGSEGSS
GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGTCTTCA

GFP-N_G06.ab1
GPGESSGSGGEPSESGSS
GGTAGCGAAGGTTCTTCCGGTCCTGGCGAGTCTTCA



GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCA












































































































































































Example 2
Construction of XTEN_AE36 Segments
A codon library encoding XTEN sequences of 36 amino acid length was constructed. The XTEN sequence was designated XTEN_AE36. Its segments have the amino acid sequence [α] 3 where X is a 12mer peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 23), GSEPATSGSETP (SEQ ID NO: 24), GTSESATPESGP (SEQ ID NO: 25), or GTSTEPSEGSAP (SEQ ID NO: 26). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:







(SEQ ID NO: 1628)


AE1for: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA





(SEQ ID NO: 1629)


AE1rev: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT





(SEQ ID NO: 1630)


AE2for: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC





(SEQ ID NO: 1631)


AE2rev: ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT





(SEQ ID NO: 1632)


AE3for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC





(SEQ ID NO: 1633)


AE3rev: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT





(SEQ ID NO: 1634)


AE4for: AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC





(SEQ ID NO: 1635)


AE4rev: ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT






























We also annealed the phosphorylated oligonucleotide 3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylated oligonucleotide pr — 3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in the resulting library designated LCW0402 showed green fluorescence after induction which shows that the sequence of XTEN_AE36 had been ligated in frame with the GFP gene and most sequences of XTEN_AE36 show good expression.
We screened 96 isolates from library LCW0402 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 37 clones were identified that contained correct XTEN_AE36 segments. The file names of the nucleotide and amino acid constructs and the sequences for these segments are listed in Table 14.





TABLE 14



DNA and Amino Acid Sequences for AE 36-mer motifs (SEQ ID NOS 279-352,

respectively, in order of appearance)





File name
Amino acid sequence
Nucleotide sequence



LCW0402_002_
GSPAGSPTSTEEGTSE
GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA

GFP-N_A07.ab1
SATPESGPGTSTEPSE
GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCA


GSAP
GGTACCTCTACCGAACCGTCTGAGGGCAGCGCACCA



LCW0402_003_
GTSTEPSEGSAPGTST
GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCA

GFP-N_B07.ab1
EPSEGSAPGTSTEPSE
GGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCA


GSAP
GGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA



LCW0402_004_
GTSTEPSEGSAPGTSE
GGTACCTCTACCGAACCGTCTGAAGGTAGCGCACCA

GFP-N_C07.ab1
SATPESGPGTSESATP
GGTACCTCTGAAAGCGCAACTCCTGAGTCCGGTCCA


ESGP
GGTACTTCTGAAAGCGCAACCCCGGAGTCTGGCCCA



LCW0402_005_
GTSTEPSEGSAPGTSE
GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCA

GFP-N_D07.ab1
SATPESGPGTSESATP
GGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCA


ESGP
GGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA



LCW0402_006_
GSEPATSGSETPGTSE
GGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCA

GFP-N_E07.ab1
SATPESGPGSPAGSPT
GGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCA


STEE
GGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAA



LCW0402_008_
GTSESATPESGPGSEP
GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA

GFP-N_F07.ab1
ATSGSETPGTSTEPSE
GGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCA


GSAP
GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA



LCW0402_009_
GSPAGSPTSTEEGSPA
GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAGGAA

GFP-N_G07.ab1
GSPTSTEEGSEPATSG
GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAAGAA


SETP
GGTAGCGAACCGGCTACCTCCGGCTCTGAAACTCCA



LCW0402_011_
GSPAGSPTSTEEGTSE
GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAA

GFP-N_A08.ab1
SATPESGPGTSTEPSE
GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCA


GSAP
GGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA



LCW0402_012_
GSPAGSPTSTEEGSPA
GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAA

GFP-N_B08.ab1
GSPTSTEEGTSTEPSE
GGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAA


GSAP
GGTACTTCTACCGAACCTTCCGAAGGTAGCGCTCCA



LCW0402_013_
GTSESATPESGPGTST
GGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCA

GFP-N_C08.ab1
EPSEGSAPGTSTEPSE
GGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCA


GSAP
GGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCA



LCW0402_014_
GTSTEPSEGSAPGSPA
GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCA

GFP-N_D08.ab1
GSPTSTEEGTSTEPSE
GGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAA


GSAP
GGTACTTCTACCGAACCTTCTGAGGGTAGCGCACCA



LCW0402_015_
GSEPATSGSETPGSPA
GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA

GFP-N_E08.ab1
GSPTSTEEGTSESATP
GGTAGCCCTGCTGGCTCTCCGACCTCTACCGAAGAA


ESGP
GGTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCA



LCW0402_016_
GTSTEPSEGSAPGTSE
GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA

GFP-N_F08.ab1
SATPESGPGTSESATP
GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA


ESGP
GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA



LCW0402_020_
GTSTEPSEGSAPGSEP
GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCA

GFP-N_G08.ab1
ATSGSETPGSPAGSPT
GGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCA


STEE
GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA



LCW0402_023_
GSPAGSPTSTEEGTSE
GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAA

GFP-N_A09.ab1
SATPESGPGSEPATSG
GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCA


SETP
GGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA



LCW0402_024_
GTSESATPESGPGSPA
GGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCA

GFP-N_B09.ab1
GSPTSTEEGSPAGSPT
GGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAA


STEE
GGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA



LCW0402_025_
GTSTEPSEGSAPGTSE
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA

GFP-N_C09.ab1
SATPESGPGTSTEPSE
GGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCA


GSAP
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA



LCW0402_026_
GSPAGSPTSTEEGTST
GGTAGCCCGGCAGGCTCTCCGACTTCCACCGAGGAA

GFP-N_D09.ab1
EPSEGSAPGSEPATSG
GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA


SETP
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA



LCW0402_027_
GSPAGSPTSTEEGTST
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAA

GFP-N_E09.ab1
EPSEGSAPGTSTEPSE
GGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA


GSAP
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA



LCW0402_032_
GSEPATSGSETPGTSE
GGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCA

GFP-N_H09.ab1
SATPESGPGSPAGSPT
GGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCA


STEE
GGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAA



LCW0402_034_
GTSESATPESGPGTST
GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCA

GFP-N_A10.ab1
EPSEGSAPGTSTEPSE
GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA


GSAP
GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA



LCW0402_036_
GSPAGSPTSTEEGTST
GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA

GFP-N_C10.ab1
EPSEGSAPGTSTEPSE
GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA


GSAP
GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA



LCW0402_039_
GTSTEPSEGSAPGTST
GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCA

GFP-N_E10.ab1
EPSEGSAPGTSTEPSE
GGTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCA


GSAP
GGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCA



LCW0402_040_
GSEPATSGSETPGTSE
GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA

GFP-N_F10.ab1
SATPESGPGTSTEPSE
GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA


GSAP
GGTACTTCTACTGAACCGTCCGAGGGCAGCGCACCA



LCW0402_041_
GTSTEPSEGSAPGSPA
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA

GFP-N_G10.ab1
GSPTSTEEGTSTEPSE
GGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA


GSAP
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA



LCW0402_050_
GSEPATSGSETPGTSE
GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCA

GFP-N_A11.ab1
SATPESGPGSEPATSG
GGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCA


SETP
GGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCA



LCW0402_051_
GSEPATSGSETPGTSE
GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCA

GFP-N_B11.ab1
SATPESGPGSEPATSG
GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGCCCA


SETP
GGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA



LCW0402_059_
GSEPATSGSETPGSEP
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCA

GFP-N_E11.ab1
ATSGSETPGTSTEPSE
GGTAGCGAACCTGCAACCTCCGGCTCTGAAACCCCA


GSAP
GGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCA



LCW0402_060_
GTSESATPESGPGSEP
GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCA

GFP-N_F11.ab1
ATSGSETPGSEPATSG
GGTAGCGAACCGGCTACTTCTGGTTCTGAAACCCCA


SETP
GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA



LCW0402_061_
GTSTEPSEGSAPGTST
GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA

GFP-N_G11.ab1
EPSEGSAPGTSESATP
GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCA


ESGP
GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA



LCW0402_065_
GSEPATSGSETPGTSE
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA

GFP-N_A12.ab1
SATPESGPGTSESATP
GGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCA


ESGP
GGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCA



LCW0402_066_
GSEPATSGSETPGSEP
GGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCA

GFP-N_B12.ab1
ATSGSETPGTSTEPSE
GGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCA


GSAP
GGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCA



LCW0402_067_
GSEPATSGSETPGTST
GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA

GFP-N_C12.ab1
EPSEGSAPGSEPATSG
GGTACTTCTACCGAACCGTCCGAGGGTAGCGCTCCA


SETP
GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA



LCW0402_069_
GTSTEPSEGSAPGTST
GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCA

GFP-N_D12.ab1
EPSEGSAPGSEPATSG
GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA


SETP
GGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCA



LCW0402_073_
GTSTEPSEGSAPGSEP
GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCA

GFP-N_F12.ab1
ATSGSETPGSPAGSPT
GGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCA


STEE
GGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAA



LCW0402_074_
GSEPATSGSETPGSPA
GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA

GFP-N_G12.ab1
GSPTSTEEGTSESATP
GGTAGCCCAGCTGGTTCTCCAACCTCTACTGAGGAA


ESGP
GGTACTTCTGAAAGCGCTACCCCTGAATCTGGTCCA



LCW0402_075_
GTSESATPESGPGSEP
GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA

GFP-N_H12.ab1
ATSGSETPGTSESATP
GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA


ESGP
GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA








































































































































































Example 3
Construction of XTEN_AF36 Segments
A codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN_AF36. Its segments have the amino acid sequence [X] 3 where X is a 12mer peptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 27), GTSTPESGSASP (SEQ ID NO: 28), GTSPSGESSTAP (SEQ ID NO: 29), or GSTSSTAESPGP (SEQ ID NO: 30). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:







(SEQ ID NO: 1636)


AF1for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC





(SEQ ID NO: 1637)


AF1rev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA





(SEQ ID NO: 1638)


AF2for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC





(SEQ ID NO: 1639)


AF2rev: ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT





(SEQ ID NO: 1640)


AF3for: AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCC





(SEQ ID NO: 1641)


AF3rev: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT





(SEQ ID NO: 1642)


AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC





(SEQ ID NO: 1643)


AF4rev: ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA






























We also annealed the phosphorylated oligonucleotide 3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylated oligonucleotide pr — 3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in the resulting library designated LCW0403 showed green fluorescence after induction which shows that the sequence of XTEN_AF36 had been ligated in frame with the GFP gene and most sequences of XTEN_AF36 show good expression.
We screened 96 isolates from library LCW0403 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 44 clones were identified that contained correct XTEN_AF36 segments. The file names of the nucleotide and amino acid constructs and the sequences for these segments are listed in Table 15.





TABLE 15



DNA and Amino Acid Sequences for AF 36-mer motifs (SEQ ID NOS 353-440,

respectively, in order of appearance)





File name
Amino acid sequence
Nucleotide sequence



LCW0403_004_
GTSTPESGSASPGTSP
GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCA

GFP-N_A01.ab1
SGESSTAPGTSPSGES
GGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAG


STAP
GTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCA



LCW0403_005_
GTSPSGESSTAPGSTS
GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCA

GFP-N_B01.ab1
STAESPGPGTSPSGES
GGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAG


STAP
GTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCA



LCW0403_006_
GSTSSTAESPGPGTSP
GGTTCCACCAGCTCTACTGCTGAATCTCCTGGTCCAG

GFP-N_C01.ab1
SGESSTAPGTSTPESG
GTACCTCTCCTAGCGGTGAATCTTCTACTGCTCCAGG


SASP
TACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCA



LCW0403_007_
GSTSSTAESPGPGSTS
GGTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAG

GFP-N_D01.ab1
STAESPGPGTSPSGES
GTTCCACCAGCTCTACCGCAGAATCTCCGGGTCCAG


STAP
GTACTTCCCCTAGCGGTGAATCTTCTACCGCACCA



LCW0403_008_
GSTSSTAESPGPGTSP
GGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAG

GFP-N_E01.ab1
SGESSTAPGTSTPESG
GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG


SASP
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA



LCW0403_010_
GSTSSTAESPGPGTST
GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAG

GFP-N_F01.ab1
PESGSASPGSTSESPS
GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG


GTAP
GTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA



LCW0403_011_
GSTSSTAESPGPGTST
GGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAG

GFP-N_G01.ab1
PESGSASPGTSTPESG
GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG


SASP
GTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA



LCW0403_012_
GSTSESPSGTAPGTSP
GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG

GFP-N_H01.ab1
SGESSTAPGSTSESPS
GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG


GTAP
TTCTACTAGCGAATCTCCTTCTGGCACTGCACCA



LCW0403_013_
GSTSSTAESPGPGSTS
GGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCA

GFP-N_A02.ab1
STAESPGPGTSPSGES
GGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG


STAP
GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCA



LCW0403_014_
GSTSSTAESPGPGTST
GGTTCCACTAGCTCTACTGCAGAATCTCCTGGCCCAG

GFP-N_B02.ab1
PESGSASPGSTSESPS
GTACCTCTACCCCTGAAAGCGGCTCTGCATCTCCAG


GTAP
GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA



LCW0403_015_
GSTSSTAESPGPGSTS
GGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAG

GFP-N_C02.ab1
STAESPGPGTSPSGES
GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGG


STAP
TACCTCCCCGAGCGGTGAATCTTCTACTGCACCA



LCW0403_017_
GSTSSTAESPGPGSTS
GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG

GFP-N_D02.ab1
ESPSGTAPGSTSSTAE
GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAG


SPGP
GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCA



LCW0403_018_
GSTSSTAESPGPGSTS
GGTTCTACCAGCTCTACCGCAGAATCTCCTGGCCCA

GFP-N_E02.ab1
STAESPGPGSTSSTAE
GGTTCCACTAGCTCTACCGCTGAATCTCCTGGTCCAG


SPGP
GTTCTACTAGCTCTACCGCTGAATCTCCTGGTCCA



LCW0403_019_
GSTSESPSGTAPGSTS
GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG

GFP-N_F02.ab1
STAESPGPGSTSSTAE
GTTCCACTAGCTCTACCGCTGAATCTCCTGGCCCAGG


SPGP
TTCCACTAGCTCTACTGCAGAATCTCCTGGTCCA



LCW0403_023_
GSTSESPSGTAPGSTS
GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG

GFP-N_H02.ab1
ESPSGTAPGSTSESPS
GTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGG


GTAP
TTCTACCAGCGAATCTCCTTCTGGTACTGCACCA



LCW0403_024_
GSTSSTAESPGPGSTS
GGTTCCACCAGCTCTACTGCTGAATCTCCTGGCCCAG

GFP-N_A03.ab1
STAESPGPGSTSSTAE
GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG


SPGP
TTCCACCAGCTCTACCGCTGAATCTCCGGGTCCA



LCW0403_025_
GSTSSTAESPGPGSTS
GGTTCCACTAGCTCTACCGCAGAATCTCCTGGTCCAG

GFP-N_B03.ab1
STAESPGPGTSPSGES
GTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGG


STAP
TACCTCCCCTAGCGGCGAATCTTCTACCGCTCCA



LCW0403_028_
GSSPSASTGTGPGSST
GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAG

GFP-N_D03.ab1
PSGATGSPGSSTPSGA
GTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGG


TGSP
TAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA



LCW0403_029_
GTSPSGESSTAPGTST
GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAG

GFP-N_E03.ab1
PESGSASPGSTSSTAE
GTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAG


SPGP
GTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCA



LCW0403_030_
GSTSSTAESPGPGSTS
GGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAG

GFP-N_F03.ab1
STAESPGPGTSTPESG
GTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAGG


SASP
TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA



LCW0403_031_
GTSPSGESSTAPGSTS
GGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAG

GFP-N_G03.ab1
STAESPGPGTSTPESG
GTTCTACCAGCTCTACTGCTGAATCTCCTGGCCCAGG


SASP
TACTTCTACCCCGGAAAGCGGCTCCGCTTCTCCA



LCW0403_033_
GSTSESPSGTAPGSTS
GGTTCTACTAGCGAATCCCCTTCTGGTACTGCACCAG

GFP-N_H03.ab1
STAESPGPGSTSSTAE
GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG


SPGP
TTCCACCAGCTCTACCGCAGAATCTCCTGGTCCA



LCW0403_035_
GSTSSTAESPGPGSTS
GGTTCCACCAGCTCTACCGCTGAATCTCCGGGCCCA

GFP-N_A04.ab1
ESPSGTAPGSTSSTAE
GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA


SPGP
GGTTCTACTAGCTCTACCGCAGAATCTCCGGGCCCA



LCW0403_036_
GSTSSTAESPGPGTSP
GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAG

GFP-N_B04.ab1
SGESSTAPGTSTPESG
GTACTTCCCCGAGCGGTGAATCTTCTACTGCACCAG


SASP
GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA



LCW0403_039_
GSTSESPSGTAPGSTS
GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG

GFP-N_C04.ab1
ESPSGTAPGTSPSGES
GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAG


STAP
GTACTTCTCCTAGCGGCGAATCTTCTACCGCACCA



LCW0403_041_
GSTSESPSGTAPGSTS
GGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAG

GFP-N_D04.ab1
ESPSGTAPGTSTPESG
GTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAG


SASP
GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA



LCW0403_044_
GTSTPESGSASPGSTS
GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAG

GFP-N_E04.ab1
STAESPGPGSTSSTAE
GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG


SPGP
GTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCA



LCW0403_046_
GSTSESPSGTAPGSTS
GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA

GFP-N_F04.ab1
ESPSGTAPGTSPSGES
GGTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAG


STAP
GTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCA



LCW0403_047_
GSTSSTAESPGPGSTS
GGTTCTACTAGCTCTACCGCTGAATCTCCTGGCCCAG

GFP-N_G04.ab1
STAESPGPGSTSESPS
GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG


GTAP
GTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCA



LCW0403_049_
GSTSSTAESPGPGSTS
GGTTCCACCAGCTCTACTGCAGAATCTCCTGGCCCA

GFP-N_H04.ab1
STAESPGPGTSTPESG
GGTTCTACTAGCTCTACCGCAGAATCTCCTGGTCCAG


SASP
GTACCTCTACTCCTGAAAGCGGTTCCGCATCTCCA



LCW0403_051_
GSTSSTAESPGPGSTS
GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAG

GFP-N_A05.ab1
STAESPGPGSTSESPS
GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGG


GTAP
TTCTACTAGCGAATCTCCTTCTGGTACCGCTCCA



LCW0403_053_
GTSPSGESSTAPGSTS
GGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCA

GFP-N_B05.ab1
ESPSGTAPGSTSSTAE
GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG


SPGP
GTTCCACCAGCTCTACTGCAGAATCTCCGGGTCCA



LCW0403_054_
GSTSESPSGTAPGTSP
GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG

GFP-N_C05.ab1
SGESSTAPGSTSSTAE
GTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGG


SPGP
TTCTACCAGCTCTACCGCAGAATCTCCGGGTCCA



LCW0403_057_
GSTSSTAESPGPGSTS
GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG

GFP-N_D05.ab1
ESPSGTAPGTSPSGES
GTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAG


STAP
GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCA



LCW0403_058_
GSTSESPSGTAPGSTS
GGTTCTACTAGCGAATCTCCTTCTGGCACTGCACCAG

GFP-N_E05.ab1
ESPSGTAPGTSTPESG
GTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAG


SASP
GTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA



LCW0403_060_
GTSTPESGSASPGSTS
GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCA

GFP-N_F05.ab1
ESPSGTAPGSTSSTAE
GGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA


SPGP
GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCA



LCW0403_063_
GSTSSTAESPGPGTSP
GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCA

GFP-N_G05.ab1
SGESSTAPGTSPSGES
GGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAG


STAP
GTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCA



LCW0403_064_
GTSPSGESSTAPGTSP
GGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAG

GFP-N_H05.ab1
SGESSTAPGTSPSGES
GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG


STAP
TACCTCCCCTAGCGGTGAATCTTCTACCGCACCA



LCW0403_065_
GSTSSTAESPGPGTST
GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG

GFP-N_A06.ab1
PESGSASPGSTSESPS
GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGG


GTAP
TTCTACTAGCGAATCTCCGTCTGGCACCGCACCA



LCW0403_066_
GSTSESPSGTAPGTSP
GGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAG

GFP-N_B06.ab1
SGESSTAPGTSPSGES
GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG


STAP
TACTTCCCCTAGCGGCGAATCTTCTACCGCTCCA



LCW0403_067_
GSTSESPSGTAPGTST
GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG

GFP-N_C06.ab1
PESGSASPGSTSSTAE
GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGG


SPGP
TTCCACTAGCTCTACCGCTGAATCTCCGGGTCCA



LCW0403_068_
GSTSSTAESPGPGSTS
GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG

GFP-N_D06.ab1
STAESPGPGSTSESPS
GTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGG


GTAP
TTCTACCAGCGAATCTCCGTCTGGCACCGCACCA



LCW0403_069_
GSTSESPSGTAPGTST
GGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCA

GFP-N_E06.ab1
PESGSASPGTSTPESG
GGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAG


SASP
GTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCA



LCW0403_070_
GSTSESPSGTAPGTST
GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG

GFP-N_F06.ab1
PESGSASPGTSTPESG
GTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG


SASP
TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA




































































































































































































Example 4
Construction of XTEN_AG36 Segments
A codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN_AG36. Its segments have the amino acid sequence [X] 3 where X is a 12mer peptide with the sequence: GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), or GASPGTSSTGSP (SEQ ID NO: 34). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:







AG1for:


(SEQ ID NO: 1644)


AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC





AG1rev:


(SEQ ID NO: 1645)


ACCTGGAGARGAAGAWGCRGTACCGCTRCCMGGRGT





AG2for:


(SEQ ID NO: 1646)


AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC





AG2rev:


(SEQ ID NO: 1647)


ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT





AG3for:


(SEQ ID NO: 1648)


AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC





AG3rev:


(SEQ ID NO: 1649)


ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA





AG4for:


(SEQ ID NO: 1650)


AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC





AG4rev:


(SEQ ID NO: 1651)


ACCTGGAGAACCRGTAGAGCTRGTRCCMGGRGAWGC






































We also annealed the phosphorylated oligonucleotide 3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylated oligonucleotide pr — 3 KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0359. Most of the clones in the resulting library designated LCW0404 showed green fluorescence after induction which shows that the sequence of XTEN_AG36 had been ligated in frame with the GFP gene and most sequences of XTEN_AG36 show good expression.
We screened 96 isolates from library LCW0404 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 44 clones were identified that contained correct XTEN_AG36 segments. The file names of the nucleotide and amino acid constructs and the sequences for these segments are listed in Table 16.





TABLE 16



DNA and Amino Acid Sequences for AG 36-mer motifs (SEQ ID NOS 441-528,

respectively, in order of appearance)





File name
Amino acid sequence
Nucleotide sequence



LCW0404_001_
GASPGTSSTGSPGTPGS
GGTGCATCCCCGGGCACTAGCTCTACCGGTTCTCCA

GFP-N_A07.ab1
GTASSSPGSSTPSGATG
GGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAG


SP
GTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCA



LCW0404_003_
GSSTPSGATGSPGSSPS
GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAG

GFP-N_B07.ab1
ASTGTGPGSSTPSGATG
GTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGG


SP
TAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCA



LCW0404_006_
GASPGTSSTGSPGSSPS
GGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG

GFP-N_C07.ab1
ASTGTGPGSSTPSGATG
GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGG


SP
TAGCTCTACCCCGTCTGGTGCTACTGGTTCCCCA



LCW0404_007_
GTPGSGTASSSPGSSTPS
GGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAG

GFP-N_D07.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACCCCTTCTGGTGCAACTGGTTCCCCAGG


P
TGCATCCCCTGGTACTAGCTCTACCGGTTCTCCA



LCW0404_009_
GTPGSGTASSSPGASPG
GGTACCCCTGGCAGCGGTACTGCTTCTTCTTCTCCAG

GFP-N_E07.ab1
TSSTGSPGSRPSASTGT
GTGCTTCCCCTGGTACCAGCTCTACCGGTTCTCCAGG


GP
TTCTAGACCTTCTGCATCCACCGGTACTGGTCCA



LCW0404_011_
GASPGTSSTGSPGSSTPS
GGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAG

GFP-N_F07.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGG


P
TGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCA



LCW0404_012_
GTPGSGTASSSPGSSTPS
GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCA

GFP-N_G07.ab1
GATGSPGSSTPSGATGS
GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG


P
GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA



LCW0404_014_
GASPGTSSTGSPGASPG
GGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAG

GFP-N_H07.ab1
TSSTGSPGASPGTSSTGS
GTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGG


P
TGCTTCTCCTGGTACCAGCTCTACTGGTTCTCCA



LCW0404_015_
GSSTPSGATGSPGSSPS
GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA

GFP-N_A08.ab1
ASTGTGPGASPGTSSTG
GGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAG


SP
GTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA



LCW0404_016_
GSSTPSGATGSPGSSTPS
GGTAGCTCTACTCCTTCTGGTGCTACCGGTTCCCCAG

GFP-N_B08.ab1
GATGSPGTPGSGTASSS
GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG


P
TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA



LCW0404_017_
GSSTPSGATGSPGSSTPS
GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG

GFP-N_C08.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACTCCTTCTGGTGCTACTGGCTCCCCAGG


P
TGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA



LCW0404_018_
GTPGSGTASSSPGSSPS
GGTACTCCTGGTAGCGGTACCGCATCTTCCTCTCCAG

GFP-N_D08.ab1
ASTGTGPGSSTPSGATG
GTTCTAGCCCTTCTGCATCTACCGGTACCGGTCCAGG


SP
TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCA



LCW0404_023_
GASPGTSSTGSPGSSPS
GGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAG

GFP-N_F08.ab1
ASTGTGPGTPGSGTASS
GTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGG


SP
TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA



LCW0404_025_
GSSTPSGATGSPGSSTPS
GGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG

GFP-N_G08.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG


P
TGCTTCTCCGGGTACCAGCTCTACTGGTTCTCCA



LCW0404_029_
GTPGSGTASSSPGSSTPS
GGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAG

GFP-N_A09.ab1
GATGSPGSSPSASTGTG
GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGG


P
TTCTAGCCCGTCTGCATCTACCGGTACCGGCCCA



LCW0404_030_
GSSTPSGATGSPGTPGS
GGTAGCTCTACTCCTTCTGGTGCAACCGGCTCCCCAG

GFP-N_B09.ab1
GTASSSPGTPGSGTASS
GTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAG


SP
GTACTCCGGGTAGCGGTACTGCTTCTTCTTCTCCA



LCW0404_031_
GTPGSGTASSSPGSSTPS
GGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG

GFP-N_C09.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGG


P
TGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCA



LCW0404_034_
GSSTPSGATGSPGSSTPS
GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG

GFP-N_D09.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAG


P
GTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCA



LCW0404_035_
GASPGTSSTGSPGTPGS
GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG

GFP-N_E09.ab1
GTASSSPGSSTPSGATG
GTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAG


SP
GTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCA



LCW0404_036_
GSSPSASTGTGPGSSTPS
GGTTCTAGCCCGTCTGCTTCCACCGGTACTGGCCCAG

GFP-N_F09.ab1
GATGSPGTPGSGTASSS
GTAGCTCTACCCCGTCTGGTGCAACTGGTTCCCCAGG


P
TACCCCTGGTAGCGGTACCGCTTCTTCTTCTCCA



LCW0404_037_
GASPGTSSTGSPGSSPS
GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG

GFP-N_G09.ab1
ASTGTGPGSSTPSGATG
GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG


SP
TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCA



LCW0404_040_
GASPGTSSTGSPGSSTPS
GGTGCATCCCCGGGCACCAGCTCTACCGGTTCTCCA

GFP-N_H09.ab1
GATGSPGSSTPSGATGS
GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG


P
GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA



LCW0404_041_
GTPGSGTASSSPGSSTPS
GGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAG

GFP-N_A10.ab1
GATGSPGTPGSGTASSS
GTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGG


P
TACCCCGGGTAGCGGTACCGCATCTTCTTCTCCA



LCW0404_043_
GSSPSASTGTGPGSSTPS
GGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAG

GFP-N_C10.ab1
GATGSPGSSTPSGATGS
GTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGG


P
TAGCTCTACTCCTTCTGGTGCAACTGGCTCTCCA



LCW0404_045_
GASPGTSSTGSPGSSPS
GGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTCCAG

GFP-N_D10.ab1
ASTGTGPGSSPSASTGT
GTTCTAGCCCTTCTGCTTCTACCGGTACTGGTCCAGG


GP
TTCTAGCCCTTCTGCATCCACTGGTACTGGTCCA



LCW0404_047_
GTPGSGTASSSPGASPG
GGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG

GFP-N_F10.ab1
TSSTGSPGASPGTSSTGS
GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGG


P
TGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCA



LCW0404_048_
GSSTPSGATGSPGASPG
GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG

GFP-N_G10.ab1
TSSTGSPGSSTPSGATGS
GTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGG


P
TAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA



LCW0404_049_
GSSTPSGATGSPGTPGS
GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAG

GFP-N_H10.ab1
GTASSSPGSSTPSGATG
GTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG


SP
TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCA



LCW0404_050_
GASPGTSSTGSPGSSPS
GGTGCATCTCCTGGTACCAGCTCTACTGGTTCTCCAG

GFP-N_A11.ab1
ASTGTGPGSSTPSGATG
GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGG


SP
TAGCTCTACTCCTTCTGGTGCTACCGGTTCTCCA



LCW0404_051_
GSSTPSGATGSPGSSTPS
GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAG

GFP-N_B11.ab1
GATGSPGSSTPSGATGS
GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG


P
TAGCTCTACCCCGTCTGGTGCAACTGGCTCTCCA



LCW0404_052_
GASPGTSSTGSPGTPGS
GGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCA

GFP-N_C11.ab1
GTASSSPGASPGTSSTG
GGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAG


SP
GTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCA



LCW0404_053_
GSSTPSGATGSPGSSPS
GGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAG

GFP-N_D11.ab1
ASTGTGPGASPGTSSTG
GTTCTAGCCCGTCTGCATCCACTGGTACCGGTCCAGG


SP
TGCTTCCCCTGGCACCAGCTCTACCGGTTCTCCA



LCW0404_057_
GASPGTSSTGSPGSSTPS
GGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAG

GFP-N_E11.ab1
GATGSPGSSPSASTGTG
GTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGG


P
TTCTAGCCCTTCTGCATCTACCGGTACTGGTCCA



LCW0404_060_
GTPGSGTASSSPGSSTPS
GGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG

GFP-N_F11.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGG


P
TGCTTCTCCGGGTACCAGCTCTACCGGTTCTCCA



LCW0404_062_
GSSTPSGATGSPGTPGS
GGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA

GFP-N_G11.ab1
GTASSSPGSSTPSGATG
GGTACTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAG


SP
GTAGCTCTACTCCGTCTGGTGCTACCGGCTCCCCA



LCW0404_066_
GSSPSASTGTGPGSSPS
GGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAG

GFP-N_H11.ab1
ASTGTGPGASPGTSSTG
GTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGG


SP
TGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCA



LCW0404_067_
GTPGSGTASSSPGSSTPS
GGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAG

GFP-N_A12.ab1
GATGSPGSNPSASTGTG
GTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGG


P
TTCTAACCCTTCTGCATCCACCGGTACCGGCCCA



LCW0404_068_
GSSPSASTGTGPGSSTPS
GGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAG

GFP-N_B12.ab1
GATGSPGASPGTSSTGS
GTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGG


P
TGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCA



LCW0404_069_
GSSTPSGATGSPGASPG
GGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAG

GFP-N_C12.ab1
TSSTGSPGTPGSGTASSS
GTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAG


P
GTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCA



LCW0404_070_
GSSTPSGATGSPGSSTPS
GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG

GFP-N_D12.ab1
GATGSPGSSTPSGATGS
GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG


P
TAGCTCTACCCCTTCTGGTGCAACTGGCTCTCCA



LCW0404_073_
GASPGTSSTGSPGTPGS
GGTGCTTCTCCTGGCACTAGCTCTACCGGTTCTCCAG

GFP-N_E12.ab1
GTASSSPGSSTPSGATG
GTACCCCTGGTAGCGGTACCGCATCTTCCTCTCCAGG


SP
TAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCA



LCW0404_075_
GSSTPSGATGSPGSSPS
GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAG

GFP-N_F12.ab1
ASTGTGPGSSPSASTGT
GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG


GP
TTCTAGCCCGTCTGCATCTACTGGTACTGGTCCA



LCW0404_080_
GASPGTSSTGSPGSSPS
GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAG

GFP-N_G12.ab1
ASTGTGPGSSPSASTGT
GTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGG


GP
TTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCA



LCW0404_081_
GASPGTSSTGSPGSSPS
GGTGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCAG

GFP-N_H12.ab1
ASTGTGPGTPGSGTASS
GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGG


SP
TACCCCTGGCAGCGGTACCGCATCTTCCTCTCCA




































































































































































































Example 5
Construction of XTEN_AE864
XTEN AE864 was constructed from serial dimerization of XTEN AE36 to AE72, 144, 288, 576 and 864. A collection of XTEN_AE72 segments was constructed from 37 different segments of XTEN_AE36. Cultures of E. coli harboring all 37 different 36-amino acid segments were mixed and plasmid was isolated. This plasmid pool was digested with BsaI/NcoI to generate the small fragment as the insert. The same plasmid pool was digested with BbsI/NcoI to generate the large fragment as the vector. The insert and vector fragments were ligated resulting in a doubling of the length and the ligation mixture was transformed into BL21Gold(DE3) cells to obtain colonies of XTEN_AE72.
This library of XTEN_AE72 segments was designated LCW0406. All clones from LCW0406 were combined and dimerized again using the same process as described above yielding library LCW0410 of XTEN_AE144. All clones from LCW0410 were combined and dimerized again using the same process as described above yielding library LCW0414 of XTEN_AE288. Two isolates LCW0414.001 and LCW0414.002 were randomly picked from the library and sequenced to verify the identities. All clones from LCW0414 were combined and dimerized again using the same process as described above yielding library LCW0418 of XTEN_AE576. We screened 96 isolates from library LCW0418 for high level of GFP fluorescence. 8 isolates with right sizes of inserts by PCR and strong fluorescence were sequenced and 2 isolates (LCW0418.018 and LCW0418.052) were chosen for future use based on sequencing and expression data.
The specific clone pCW0432 of XTEN_AE864 was constructed by combining LCW0418.018 of XTEN_AE576 and LCW0414.002 of XTEN_AE288 using the same dimerization process as described above.
Example 6
Construction of XTEN_AM144
A collection of XTEN_AM144 segments was constructed starting from 37 different segments of XTEN_AE36, 44 segments of XTEN_AF36, and 44 segments of XTEN_AG36.
Cultures of E. coli that harboring all 125 different 36-amino acid segments were mixed and plasmid was isolated. This plasmid pool was digested with BsaI/NcoI to generate the small fragment as the insert. The same plasmid pool was digested with BbsI/NcoI to generate the large fragment as the vector. The insert and vector fragments were ligated resulting in a doubling of the length and the ligation mixture was transformed into BL21Gold(DE3) cells to obtain colonies of XTEN_AM72.
This library of XTEN_AM72 segments was designated LCW0461. All clones from LCW0461 were combined and dimerized again using the same process as described above yielding library LCW0462. 1512 Isolates from library LCW0462 were screened for protein expression. Individual colonies were transferred into 96 well plates and cultured overnight as starter cultures. These starter cultures were diluted into fresh autoinduction medium and cultured for 20-30 h. Expression was measured using a fluorescence plate reader with excitation at 395 nm and emission at 510 nm. 192 isolates showed high level expression and were submitted for DNA sequencing. Most clones in library LCW0462 showed good expression and similar physicochemical properties suggesting that most combinations of XTEN_AM36 segments yield useful XTEN sequences. Thirty isolates from LCW0462 were chosen as a preferred collection of XTEN_AM144 segments for the construction of multifunctional proteins that contain multiple XTEN segments. The file names of the nucleotide and amino acid constructs and the sequences for these segments are listed in Table 17.





TABLE 17



DNA and amino acid sequences for AM144 segments (SEO ID NOS 529-594,

respectively, in order of appearance)





Clone
Sequence Trimmed
Protein Sequence



LCW462_r1
GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGC
GTPGSGTASSSPGS


TCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCC
STPSGATGSPGSSTP


CGTCTGGTGCAACCGGCTCCCCAGGTAGCCCGGCTGGCTCTC
SGATGSPGSPAGSP


CTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTG
TSTEEGTSESATPES


AGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCG
GPGTSTEPSEGSAP


CTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGG
GSSPSASTGTGPGS


TTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCT
SPSASTGTGPGASP


CCGGGTACTAGCTCTACTGGTTCTCCAGGTACCTCTACCGAAC
GTSSTGSPGTSTEPS


CGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTG
EGSAPGTSTEPSEG


AGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTG
SAPGSEPATSGSETP


AAACTCCA




LCW462_r5
GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAGGTTCTA
GSTSESPSGTAPGST


CTAGCGAATCCCCTTCTGGTACCGCACCAGGTACTTCTCCGAG
SESPSGTAPGTSPSG


CGGCGAATCTTCTACTGCTCCAGGTACCTCTACTGAACCTTCC
ESSTAPGTSTEPSEG


GAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGC
SAPGTSTEPSEGSAP


AGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGT
GTSESATPESGPGA


CCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTA
SPGTSSTGSPGSSTP


GCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCC
SGATGSPGASPGTS


GGGTACCAGCTCTACCGGTTCTCCAGGTTCTACTAGCGAATCT
STGSPGSTSESPSGT


CCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTG
APGSTSESPSGTAP


GCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTT
GTSTPESGSASP


CTCCA




LCW462_r9
GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACT
GTSTEPSEGSAPGT


TCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAA
SESATPESGPGTSES


AGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTT
ATPESGPGTSTEPSE


CTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG
GSAPGTSESATPES


AGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG
GPGTSTEPSEGSAP


CACCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAG
GTSTEPSEGSAPGS


GTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCC
EPATSGSETPGSPA


CGGCTGGCTCTCCGACCTCCACCGAGGAAGGTGCTTCTCCTG
GSPTSTEEGASPGT


GCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCTTC
SSTGSPGSSPSASTG


TACCGGTACTGGTCCAGGTTCTAGCCCTTCTGCATCCACTGGT
TGPGSSPSASTGTG


ACTGGTCCA
P



LCW462_r10
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC
GSEPATSGSETPGT


TCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA
SESATPESGPGTSES


AGCGCTACTCCGGAATCCGGTCCAGGTTCTACCAGCGAATCT
ATPESGPGSTSESPS


CCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTG
GTAPGSTSESPSGT


GTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGC
APGTSPSGESSTAP


ACCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGT
GASPGTSSTGSPGS


TCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCTA
SPSASTGTGPGSSTP


CCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCGTC
SGATGSPGSSTPSG


TGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCT
ATGSPGSSTPSGAT


ACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTT
GSPGASPGTSSTGS


CTCCA
P



LCW462_r15
GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTA
GASPGTSSTGSPGS


GCCCTTCTGCATCCACCGGTACCGGTCCAGGTAGCTCTACCCC
SPSASTGTGPGSSTP


TTCTGGTGCAACCGGCTCTCCAGGTACTTCTGAAAGCGCTACC
SGATGSPGTSESAT


CCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT
PESGPGSEPATSGSE


GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT
TPGSEPATSGSETP


CCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGT
GTSESATPESGPGT


ACCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCT
STEPSEGSAPGTSTE


ACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCTACCGAA
PSEGSAPGTSTEPSE


CCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCT
GSAPGTSTEPSEGS


GAGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCT
APGSEPATSGSETP


GAAACTCCA




LCW462_r16
GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC
GTSTEPSEGSAPGSP


CCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG
AGSPTSTEEGTSTEP


AACCTTCTGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAA
SEGSAPGTSESATP


CTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCT
ESGPGSEPATSGSE


CTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTG
TPGTSESATPESGP


GTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG
GSPAGSPTSTEEGT


GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC
SESATPESGPGTSTE


TACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGC
PSEGSAPGSEPATS


TACTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCC
GSETPGTSTEPSEGS


GAGGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCT
APGSEPATSGSETP


GAAACTCCA




LCW462_r20
GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC
GTSTEPSEGSAPGT


TCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC
STEPSEGSAPGTSTE


GAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCG
PSEGSAPGTSTEPSE


TCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAG
GSAPGTSTEPSEGS


GGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGC
APGTSTEPSEGSAP


GCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA
GTSTEPSEGSAPGT


GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACT
SESATPESGPGTSES


TCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTACTG
ATPESGPGTSTEPSE


AACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTT
GSAPGSEPATSGSE


CTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCT
TPGSPAGSPTSTEE


CCACCGAGGAA




LCW462_r23
GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT
GTSTEPSEGSAPGT


TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG
STEPSEGSAPGTSTE


AACCTTCCGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCC
PSEGSAPGSTSESPS


CTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGG
GTAPGSTSESPSGT


CACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCT
APGTSTPESGSASP


CCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT
GSEPATSGSETPGT


ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA
SESATPESGPGTSTE


CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTACTGAAC
PSEGSAPGTSTEPSE


CGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCC
GSAPGTSESATPES


CGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT
GPGTSESATPESGP


CCGGCCCA




LCW462_r24
GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAGGTTCTA
GSSTPSGATGSPGS


GCCCGTCTGCTTCTACCGGTACCGGTCCAGGTAGCTCTACCCC
SPSASTGTGPGSSTP


TTCTGGTGCTACTGGTTCTCCAGGTAGCCCTGCTGGCTCTCCG
SGATGSPGSPAGSP


ACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTA
TSTEEGSPAGSPTST


CTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTC
EEGTSTEPSEGSAP


CAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTC
GASPGTSSTGSPGS


TAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGC
SPSASTGTGPGTPG


AGCGGTACTGCTTCTTCCTCTCCAGGTTCTACTAGCTCTACTG
SGTASSSPGSTSSTA


CTGAATCTCCTGGCCCAGGTACTTCTCCTAGCGGTGAATCTTC
ESPGPGTSPSGESST


TACCGCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCT
APGTSTPESGSASP


CCA




LCW462_r27
GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACT
GTSTEPSEGSAPGT


TCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT
SESATPESGPGTSTE


GAACCGTCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCG
PSEGSAPGTSTEPSE


TCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCG
GSAPGTSESATPES


GAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCC
GPGTSESATPESGP


GGCCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG
GTPGSGTASSSPGA


GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTC
SPGTSSTGSPGASP


TCCGGGCACTAGCTCTACTGGTTCTCCAGGTAGCCCTGCTGGC
GTSSTGSPGSPAGS


TCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCG
PTSTEEGSPAGSPTS


ACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGT
TEEGTSTEPSEGSAP


AGCGCTCCA




LCW462_r28
GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACT
GSPAGSPTSTEEGT


TCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACT
STEPSEGSAPGTSTE


GAACCTTCTGAGGGCAGCGCTCCAGGTACCTCTACCGAACCG
PSEGSAPGTSTEPSE


TCTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCT
GSAPGTSESATPES


GAGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCT
GPGTSESATPESGP


GGCCCAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG
GTPGSGTASSSPGS


GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTC
STPSGATGSPGASP


TCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCTCTACTGAA
GTSSTGSPGTSTEPS


CCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC
EGSAPGTSESATPE


CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGT
SGPGTSTEPSEGSAP


AGCGCACCA




LCW462_r38
GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACT
GSEPATSGSETPGT


TCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCG
SESATPESGPGSEPA


GCTACTTCCGGCTCTGAAACCCCAGGTAGCTCTACCCCGTCTG
TSGSETPGSSTPSGA


GTGCAACCGGCTCCCCAGGTACTCCTGGTAGCGGTACCGCTT
TGSPGTPGSGTASS


CTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTC
SPGSSTPSGATGSP


CCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGT
GASPGTSSTGSPGS


AGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC
STPSGATGSPGASP


CGGGTACCAGCTCTACCGGTTCTCCAGGTAGCGAACCTGCTA
GTSSTGSPGSEPATS


CTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCCGA
GSETPGTSTEPSEGS


GGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA
APGSEPATSGSETP


AACTCCA




LCW462_r39
GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACC
GTSTEPSEGSAPGT


TCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAA
STEPSEGSAPGTSES


AGCGCAACCCCTGAATCCGGTCCAGGTAGCCCTGCTGGCTCT
ATPESGPGSPAGSP


CCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACT
TSTEEGSPAGSPTST


TCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGC
EEGTSTEPSEGSAP


GCTCCAGGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA
GSPAGSPTSTEEGT


GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC
STEPSEGSAPGTSTE


TCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTGCTTCCCCG
PSEGSAPGASPGTS


GGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTT
STGSPGSSPSASTGT


CTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGG
GPGSSPSASTGTGP


TACTGGTCCA




LCW462_r41
GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTT
GSSTPSGATGSPGA


CTCCTGGTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCC
SPGTSSTGSPGSSTP


GTCTGGTGCTACTGGCTCTCCAGGTAGCCCTGCTGGCTCTCCA
SGATGSPGSPAGSP


ACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA
TSTEEGTSESATPES


TCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC
GPGSEPATSGSETP


CCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTA
GASPGTSSTGSPGS


GCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCC
STPSGATGSPGSSPS


TTCTGCATCTACCGGTACTGGTCCAGGTTCTACCAGCGAATCC
ASTGTGPGSTSESPS


CCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTG
GTAPGSTSESPSGT


GCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTT
APGTSTPESGSASP


CTCCA




LCW462_r42
GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTA
GSTSESPSGTAPGST


CTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAG
SESPSGTAPGTSPSG


CGGCGAATCTTCTACCGCACCAGGTACCTCTGAAAGCGCTAC
ESSTAPGTSESATPE


TCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGG
SGPGTSTEPSEGSAP


TAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGC
GTSTEPSEGSAPGT


ACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG
STEPSEGSAPGTSES


TACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT
ATPESGPGTSTEPSE


ACTGAACCGTCCGAAGGTAGCGCACCAGGTAGCTCTACCCCG
GSAPGSSTPSGATG


TCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT
SPGASPGTSSTGSP


CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG
GSSTPSGATGSP


CTCTCCA




LCW462_r43
GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCT
GSTSSTAESPGPGTS


CTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAG
PSGESSTAPGTSPSG


CGGTGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACCGCT
ESSTAPGSTSSTAES


GAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTC
PGPGSTSSTAESPGP


CTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCC
GTSTPESGSASPGTS


AGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTTCT
PSGESSTAPGSTSST


ACCAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTACCC
AESPGPGTSTPESGS


CGGAAAGCGGCTCCGCTTCTCCAGGTTCTACCAGCTCTACCG
ASPGSTSSTAESPGP


CTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGG
GSTSESPSGTAPGTS


CACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCA
PSGESSTAP


CCA




LCW462_r45
GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTTCTA
GTSTPESGSASPGST


CCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCT
SESPSGTAPGSTSST


CTACTGCTGAATCTCCGGGCCCAGGTACCTCTACTGAACCTTC
AESPGPGTSTEPSE


CGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGG
GSAPGTSTEPSEGS


CAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGG
APGTSESATPESGP


TCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGG
GTSESATPESGPGT


TACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCT
STEPSEGSAPGTSTE


ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGC
PSEGSAPGTSESATP


GCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCC
ESGPGTSTEPSEGS


GAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGT
APGTSTEPSEGSAP


AGCGCTCCC




LCW462_r47
GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACC
GTSTEPSEGSAPGT


TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCG
STEPSEGSAPGSEPA


GCAACCTCCGGTTCTGAAACTCCAGGTACTTCTACTGAACCGT
TSGSETPGTSTEPSE


CTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG
GSAPGTSESATPES


AATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCG
GPGTSESATPESGP


GCCCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG
GASPGTSSTGSPGS


GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTC
SPSASTGTGPGSSTP


TACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCG
SGATGSPGSSTPSG


TCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTG
ATGSPGSSTPSGAT


CTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCG
GSPGASPGTSSTGS


GTTCTCCA
P



LCW462_r54
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGC
GSEPATSGSETPGS


GAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACT
EPATSGSETPGTSTE


GAACCTTCTGAGGGCAGCGCACCAGGTAGCGAACCTGCAACC
PSEGSAPGSEPATS


TCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT
GSETPGTSESATPES


GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC
GPGTSTEPSEGSAP


GCACCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG
GSSTPSGATGSPGS


GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTC
STPSGATGSPGASP


TCCGGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTACCCCG
GTSSTGSPGSSTPSG


TCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT
ATGSPGASPGTSST


CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG
GSPGSSTPSGATGS


CTCTCCA
P



LCW462_r55
GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT
GTSTEPSEGSAPGT


TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG
STEPSEGSAPGTSTE


AACCTTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCTA
PSEGSAPGTSESATP


CTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCCGAAG
ESGPGTSTEPSEGS


GCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGCG
APGTSTEPSEGSAP


CTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGG
GSTSESPSGTAPGTS


TACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCC
PSGESSTAPGTSPSG


CCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCCCGGCTGGC
ESSTAPGSPAGSPTS


TCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTC
TEEGTSESATPESGP


CTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTA
GTSTEPSEGSAP


GCGCTCCA




LCW462_r57
GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGC
GTSTEPSEGSAPGS


GAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCT
EPATSGSETPGSPA


GGCTCTCCGACCTCCACCGAGGAAGGTAGCCCGGCAGGCTCT
GSPTSTEEGSPAGSP


CCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG
TSTEEGTSESATPES


GAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGC
GPGTSTEPSEGSAP


GCACCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA
GTSTEPSEGSAPGT


GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACT
STEPSEGSAPGTSES


TCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCTCTACT
ATPESGPGSSTPSG


CCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTT
ATGSPGSSPSASTG


CCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTA
TGPGASPGTSSTGS


CTGGTTCTCCA
P



LCW462_r61
GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAGGTAGC
GSEPATSGSETPGSP


CCTGCTGGCTCTCCGACCTCTACCGAAGAAGGTACCTCTGAA
AGSPTSTEEGTSES


AGCGCTACCCCTGAGTCTGGCCCAGGTACCTCTACTGAACCTT
ATPESGPGTSTEPSE


CCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG
GSAPGTSTEPSEGS


GCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG
APGTSESATPESGP


GTCCAGGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAG
GTSTPESGSASPGST


GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTA
SESPSGTAPGSTSST


CTAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCTGAAA
AESPGPGTSESATP


GCGCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGT
ESGPGTSTEPSEGS


CCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG
APGTSTEPSEGSAP


GTAGCGCTCCA




LCW462_r64
GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT
GTSTEPSEGSAPGT


TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG
STEPSEGSAPGTSTE


AACCTTCCGAAGGTAGCGCACCAGGTACCTCTACCGAACCGT
PSEGSAPGTSTEPSE


CTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTG
GSAPGTSESATPES


AGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCTG
GPGTSESATPESGP


GCCCAGGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG
GTPGSGTASSSPGS


GTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGGTGCTTC
STPSGATGSPGASP


TCCGGGTACCAGCTCTACCGGTTCTCCAGGTTCCACCAGCTCT
GTSSTGSPGSTSSTA


ACTGCTGAATCTCCTGGTCCAGGTACCTCTCCTAGCGGTGAAT
ESPGPGTSPSGESST


CTTCTACTGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCTGC
APGTSTPESGSASP


TTCTCCA




LCW462_r67
GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACT
GSPAGSPTSTEEGT


TCTGAAAGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACC
SESATPESGPGTSTE


GAACCGTCTGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCA
PSEGSAPGTSESATP


ACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGC
ESGPGSEPATSGSE


TCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGC
TPGTSTEPSEGSAP


GCACCAGGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA
GSPAGSPTSTEEGT


GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC
STEPSEGSAPGTSTE


TCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACTTCTACC
PSEGSAPGTSTEPSE


GAACCGTCCGAGGGCAGCGCTCCAGGTACTTCTACTGAACCT
GSAPGTSTEPSEGS


TCTGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAA
APGTSTEPSEGSAP


GGTAGCGCACCA




LCW462_r69
GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTA
GTSPSGESSTAPGST


CTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAG
SSTAESPGPGTSPSG


CGGTGAATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACT
ESSTAPGTSESATPE


CCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGT
SGPGTSTEPSEGSAP


AGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCA
GTSTEPSEGSAPGSS


CCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTA
PSASTGTGPGSSTPS


GCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCC
GATGSPGASPGTSS


GGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTACTCCGGAA
TGSPGTSTPESGSAS


AGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTT
PGTSPSGESSTAPGT


CTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGC
SPSGESSTAP


TCCA




LCW462_r70
GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC
GTSESATPESGPGT


TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTG
STEPSEGSAPGTSTE


AACCGTCCGAAGGTAGCGCACCAGGTAGCCCTGCTGGCTCTC
PSEGSAPGSPAGSP


CGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTT
TSTEEGSPAGSPTST


CTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCG
EEGTSTEPSEGSAP


CTCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGG
GSSPSASTGTGPGS


TAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCT
STPSGATGSPGSSTP


ACTCCTTCTGGTGCAACTGGCTCTCCAGGTAGCGAACCGGCA
SGATGSPGSEPATS


ACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTACT
GSETPGTSESATPES


CCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCT
GPGSEPATSGSETP


GAAACCCCA




LCW462_r72
GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC
GTSTEPSEGSAPGT


TCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC
STEPSEGSAPGTSTE


GAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACCCCGTCT
PSEGSAPGSSTPSG


GGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTA
ATGSPGASPGTSST


CCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTC
GSPGSSTPSGATGS


TCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGG
PGTSESATPESGPGS


TAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCT
EPATSGSETPGTSTE


ACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACTAGCGAA
PSEGSAPGSTSESPS


TCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGT
GTAPGSTSESPSGT


CTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCG
APGTSTPESGSASP


CTTCTCCA




LCW462_r73
GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGTTCCA
GTSTPESGSASPGST


CTAGCTCTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCTC
SSTAESPGPGSTSST


TACTGCTGAATCTCCTGGCCCAGGTTCTAGCCCTTCTGCATCT
AESPGPGSSPSAST


ACTGGTACTGGCCCAGGTAGCTCTACTCCTTCTGGTGCTACCG
GTGPGSSTPSGATG


GCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCC
SPGASPGTSSTGSP


AGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTAC
GSEPATSGSETPGT


CTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGC
SESATPESGPGSPA


AGGTTCTCCGACTTCCACTGAGGAAGGTTCTACTAGCGAATC
GSPTSTEEGSTSESP


TCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCT
SGTAPGSTSESPSGT


GGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCT
APGTSTPESGSASP


TCTCCC




LCW462_r78
GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACT
GSPAGSPTSTEEGT


TCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG
SESATPESGPGTSTE


AACCGTCCGAAGGTAGCGCTCCAGGTTCTACCAGCGAATCTC
PSEGSAPGSTSESPS


CTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGG
GTAPGSTSESPSGT


TACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCA
APGTSPSGESSTAP


CCAGGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT
GTSTEPSEGSAPGSP


AGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCT
AGSPTSTEEGTSTEP


ACCGAACCTTCTGAGGGTAGCGCACCAGGTAGCGAACCTGCA
SEGSAPGSEPATSG


ACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT
SETPGTSESATPESG


CCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC
PGTSTEPSEGSAP


AGCGCACCA




LCW462_r79
GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC
GTSTEPSEGSAPGSP


CCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG
AGSPTSTEEGTSTEP


AACCTTCTGAGGGTAGCGCACCAGGTACCTCCCCTAGCGGCG
SEGSAPGTSPSGESS


AATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTC
TAPGTSPSGESSTAP


TACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCA
GTSPSGESSTAPGST


CCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTT
SESPSGTAPGSTSES


CTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTAC
PSGTAPGTSTPESGS


CCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCGAACCTGCAAC
ASPGSEPATSGSETP


CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT
GTSESATPESGPGT


GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC
STEPSEGSAP


GCACCA




LCW462_r87
GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC
GSEPATSGSETPGT


TCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA
SESATPESGPGTSES


AGCGCTACTCCGGAATCCGGTCCAGGTACTTCTCCGAGCGGT
ATPESGPGTSPSGES


GAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAAT
STAPGSTSSTAESPG


CTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGC
PGTSPSGESSTAPGS


TCCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGT
TSESPSGTAPGTSPS


ACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCA
GESSTAPGSTSSTA


GCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTACTCCGTC
ESPGPGSSTPSGAT


TGGTGCAACCGGTTCCCCAGGTAGCTCTACCCCTTCTGGTGCA
GSPGSSTPSGATGS


ACCGGCTCCCCAGGTAGCTCTACCCCTTCTGGTGCAAACTGG
PGSSTPSGANWLS


CTCTCC




LCW462_r88
GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGC
GSPAGSPTSTEEGSP


CCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCG
AGSPTSTEEGTSTEP


AACCTTCCGAAGGTAGCGCTCCAGGTACCTCTACTGAACCTT
SEGSAPGTSTEPSE


CCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG
GSAPGTSTEPSEGS


GCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG
APGTSESATPESGP


GTCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGG
GASPGTSSTGSPGS


TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCC
STPSGATGSPGASP


CCGGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGT
GTSSTGSPGSSTPSG


CTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG
ATGSPGTPGSGTAS


CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGG
SSPGSSTPSGATGSP


CTCTCCA




LCW462_r89
GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTC
GSSTPSGATGSPGT


CGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCC
PGSGTASSSPGSSTP


TTCTGGTGCTACTGGCTCTCCAGGTAGCCCGGCTGGCTCTCCT
SGATGSPGSPAGSP


ACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAG
TSTEEGTSESATPES


TCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT
GPGTSTEPSEGSAP


CCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGT
GTSESATPESGPGS


AGCGAACCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCT
EPATSGSETPGTSES


GAAAGCGCAACCCCGGAATCTGGTCCAGGTACTTCTACTGAA
ATPESGPGTSTEPSE


CCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACC
GSAPGTSESATPES


CCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAG
GPGTSESATPESGP


TCCGGCCCA
































































































































































































































































































































































































































Example 7
Construction of XTEN_AM288
The entire library LCW0462 was dimerized as described in Example 6 resulting in a library of XTEN_AM288 clones designated LCW0463. 1512 isolates from library LCW0463 were screened using the protocol described in Example 6. 176 highly expressing clones were sequenced and 40 preferred XTEN_AM288 segments were chosen for the construction of multifunctional proteins that contain multiple XTEN segments with 288 amino acid residues.
Example 8
Construction of XTEN_AM432
We generated a library of XTEN_AM432 segments by recombining segments from library LCW0462 of XTEN_AM144 segments and segments from library LCW0463 of XTENAM288 segments. This new library of XTEN_AM432 segment was designated LCW0464. Plasmids were isolated from cultures of E. coli harboring LCW0462 and LCW0463, respectively. 1512 isolates from library LCW0464 were screened using the protocol described in Example 6. 176 highly expressing clones were sequenced and 39 preferred XTEN_AM432 segment were chosen for the construction of longer XTENs and for the construction of multifunctional proteins that contain multiple XTEN segments with 432 amino acid residues.
In parallel we constructed library LMS0100 of XTENAM432 segments using preferred segments of XTEN_AM144 and XTEN_AM288. Screening this library yielded 4 isolates that were selected for further construction
Example 9
Construction of XTEN_AM875
The stuffer vector pCW0359 was digested with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification.
We annealed the phosphorylated oligonucleotide BsaI-AscI-KpnIfor P: AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1652) and the non-phosphorylated oligonucleotide BsaI-Ascl-KpnIrev: CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC (SEQ ID NO: 1653) for introducing the sequencing island A (SI-A) which encodes amino acids GASASGAPSTG (SEQ ID NO: 1654) and has the restriction enzyme AscI recognition nucleotide sequence GGCGCGCC inside. The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCW0359 prepared above to yield pCW0466 containing SI-A. We then generated a library of XTEN_AM443 segments by recombining 43 preferred XTEN_AM432 segments from Example 8 and SI-A segments from pCW0466 at C-terminus using the same dimerization process described in Example 5. This new library of XTEN_AM443 segments was designated LCW0479.
We generated a library of XTEN_AM875 segments by recombining segments from library LCW0479 of XTEN_AM443 segments and 43 preferred XTEN_AM432 segments from Example 8 using the same dimerization process described in example 5. This new library of XTEN_AM875 segment was designated LCW0481.
Example 10
Construction of XTEN_AM1318
We annealed the phosphorylated oligonucleotide BsaI-FseI-KpnIfor P: AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1655) and the non-phosphorylated oligonucleotide BsaI-FseI-KpnIrev: CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG (SEQ ID NO: 1656) for introducing the sequencing island B (SI-B) which encodes amino acids GPEPTGPAPSG (SEQ ID NO: 1657) and has the restriction enzyme FseI recognition nucleotide sequence GGCCGGCC inside. The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCW0359 as used in Example 9 to yield pCW0467 containing SI-B. We then generated a library of XTEN_AM443 segments by recombining 43 preferred XTEN_AM432 segments from Example 8 and SI-B segments from pCW0467 at C-terminus using the same dimerization process described in example 5. This new library of XTEN_AM443 segments was designated LCW0480.
We generated a library of XTEN_AM1318 segments by recombining segments from library LCW0480 of XTEN_AM443 segments and segments from library LCW0481 of XTENAM875 segments using the same dimerization process as in Example 5. This new library of XTEN_AM1318 segment was designated LCW0487.
Example 11
Construction of XTEN_AD864
Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AD864 sequences starting from segments of XTEN_AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN_AD864 were evaluated and found to show good expression and excellent solubility under physiological conditions. One intermediate construct of XTEN_AD576 was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20 h was measured.
Example 12
Construction of XTEN_AF864
Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AF864 sequences starting from segments of XTEN_AF36 listed in Example 3. These sequences were assembled as described in Example 5. Several isolates from XTEN_AF864 were evaluated and found to show good expression and excellent solubility under physiological conditions. One intermediate construct of XTEN_AF540 was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20 h was measured. A full length clone of XTEN_AF864 had excellent solubility and showed half-life exceeding 60 h in cynomolgus monkeys. A second set of XTEN_AF sequences was assembled including a sequencing island as described in Example 9.
Example 13
Construction of XTEN_AG864
Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AG864 sequences starting from segments of XTEN_AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN_AG864 were evaluated and found to show good expression and excellent solubility under physiological conditions. A full length clone of XTEN_AG864 had excellent solubility and showed half-life exceeding 60 h in cynomolgus monkeys.
Example 14
Methods of Producing and Evaluating CFXTEN with Internal and Terminal XTEN
The design, construction and evaluation of CFXTEN comprising FVIII and one or more XTEN is accomplished using a systematic approach. The regions suitable for XTEN insertion sites include, but are to limited to regions at or proximal to the known domain boundaries of FVIII, exon boundaries, known surface loops, regions with a low degree of order, and hydrophilic regions. By analysis of the foregoing, different regions across the sequence of the FVIII B domain deleted (BDD) sequence have been identified as insertion sites for XTEN, non-limiting examples of which are listed in Tables 5-8, and shown schematically in FIGS. 8 and 9 . Initially, individual constructs are created (using methods described, below) in which DNA encoding a single XTEN or XTEN fragment of a length ranging from 6 to 2004 amino acid residues is inserted into the FVIII sequence corresponding to or near (e.g., within 6 amino acids) each of the single insertion sites identified in Table 5, Table 6, Table 7, Table 8, and Table 9, and the resulting constructs are expressed and the recovered protein then evaluated for their effects on retention of procoagulant activity using, e.g., one of the in vitro assays of Table 49. For example, using the methods described below, constructs are made in which an XTEN sequence is inserted within the A1, A2, B, A3, C1 and C2 domain sequences of FVIII, as well as linked to the C-terminus, and the resulting expressed fusion proteins are evaluated in a chromogenic assay of Table 49, compared to a FVIII not linked to XTEN. CFXTEN fusion proteins can be further classified acting to high, intermediate and low categories based on the activities they exhibit. In those cases where the CFXTEN exhibits activity that is comparable or modestly reduced compared to FVIII, the insertion site is deemed favorable. In those cases where the activity is intermediate, the insertion site can be adjusted from 1-6 amino acids towards the N- or C-terminus of the insertion site and/or the length or net charge of the XTEN may be altered and the resulting construct(s) re-evaluated to determine whether the activity is improved. Alternatively, the XTEN is inserted into the construct with flanking cleavage sites; preferably sites that are susceptible to cleavage by proteases found in clotting assays, such that the XTEN is released during the activation of the FVIII component, thereby providing additional information about the suitability of the XTEN insertion site in the fusion protein.
Once all of the individual insertion sites are evaluated and the favorable insertion sites are identified, libraries of constructs are created with two, three, four, five or more XTEN inserted in the permutations of favorable sites. The length and net charge of the XTEN (e.g., XTEN of the AE versus AG family) are varied in order to ascertain the effects of these variables on FVIII activity and physicochemical properties of the fusion protein. CFXTEN constructs that retain a desired degree of in vitro procoagulant FVIII activity are then evaluated in vivo using mouse and/or dog models of hemophilia A, as described in Examples below, or other models known in the art. In addition, constructs are assayed in the presence of FVIII inhibitors and other anti-FVIII antibodies to determine constructs that retain activity. In addition, CFXTEN constructs are made that incorporate cleavage sequences at or near the junction(s) of FVIII and XTEN (e.g., sequences from Table 8) designed to release the XTEN and are evaluated for enhancement of FVIII activity and effects on terminal half-life. By the iterative process of making constructs combining different insertion sites, varying the length and composition qualities of the XTEN (e.g., different XTEN families), and evaluation, the skilled artisan obtains, by the foregoing methods, CFXTEN with desired properties, such as but not limited to of procoagulant FVIII activity, reduced binding with FVIII inhibitors, enhanced pharmacokinetic properties, ability to administer to a subject by different routes, and/or enhanced pharmaceutical properties.
Example 15
Methods of Producing and Evaluating CFXTEN Containing FVIII and AE_XTEN
A general scheme for producing and evaluating CFXTEN compositions is presented in FIG. 15 , and forms the basis for the general description of this Example. Using the disclosed methods and those known to one of ordinary skill in the art, together with guidance provided in the illustrative examples, a skilled artesian can create and evaluate CFXTEN fusion proteins comprising XTEN and FVIII or variants of FVIII known in the art. The Example is, therefore, to be construed as merely illustrative, and not limitative of the methods in any way whatsoever; numerous variations will be apparent to the ordinarily skilled artisan. In this Example, a CFXTEN of a factor VIII BDD linked to an XTEN of the AE family of motifs is created.
The general scheme for producing polynucleotides encoding XTEN is presented in FIGS. 11 and 12 . FIG. 14 is a schematic flowchart of representative steps in the assembly of an XTEN polynucleotide construct in one of the embodiments of the invention. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12-amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a library that can multimerize to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The motif libraries include specific sequence XTEN families; e.g., AD, AE, AF, AG, AM, or AQ sequences of Table 3. As illustrated in FIG. 14 , the XTEN length, in this case, is 36 amino acid residues, but longer lengths are also achieved by this general process. For example, multimerization is performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art that, in this case, result in a construct with 288 amino acid residues. The resulting pool of ligation products is gel-purified and the band with the desired length of XTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene can be cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is then performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, resulting in the gene 500 encoding a CFXTEN fusion protein with a 288 amino acid XTEN linked to the C-terminus of the factor VIII. As would be apparent to one of ordinary skill in the art, the methods are applied to create constructs in alternative configurations and with varying XTEN lengths or in multiple locations.
DNA sequences encoding FVIII are conveniently obtained by standard procedures known in the art from a cDNA library prepared from an appropriate cellular source, from a genomic library, or may be created synthetically (e.g., automated nucleic acid synthesis) using DNA sequences obtained from publicly available databases, patents, or literature references. In the present example, a FVIII B domain deleted (BDD) variant is prepared as described in Example 17. A gene or polynucleotide encoding the FVIII portion of the protein or its complement is then cloned into a construct, such as those described herein, which can be a plasmid or other vector under control of appropriate transcription and translation sequences for high level protein expression in a biological system. A second gene or polynucleotide coding for the XTEN portion or its complement is genetically fused to the nucleotides encoding the terminus of the FVIII gene by cloning it into the construct adjacent and in frame with the gene coding for the CF, through a ligation or multimerization step. In this manner, a chimeric DNA molecule coding for (or complementary to) the CFXTEN fusion protein is generated within the construct. Optionally, a gene encoding for a second XTEN is inserted and ligated in-frame internally to the nucleotides encoding the FVIII-encoding region. The constructs are designed in different configurations to encode various insertion sites of the XTEN in the FVIII sequence, including those of Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9 . Optionally, this chimeric DNA molecule is transferred or cloned into another construct that is a more appropriate expression vector; e.g., a vector appropriate for a mammalian host cell such as CHO, BHK and the like. At this point, a host cell capable of expressing the chimeric DNA molecule is transformed with the chimeric DNA molecule, described more completely, below, or by well-known methods, depending on the type of cellular host, as described supra.
Host cells containing the XTEN-FVIII expression vector are cultured in conventional nutrient media modified as appropriate for activating the promoter. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. After expression of the fusion protein, culture broth is harvested and separated from the cell mass and the resulting crude extract retained for purification of the fusion protein.
Gene expression is measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, gene expression is measured by immunological of fluorescent methods, such as immunohistochemical staining of cells to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against the FVIII sequence polypeptide using a synthetic peptide based on the sequences provided herein or against exogenous sequence fused to FVIII and encoding a specific antibody epitope. Examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (β-gal) or chloramphenicol acetyltransferase (CAT).
The CFXTEN polypeptide product is purified via methods known in the art. Procedures such as gel filtration, affinity purification, salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography or gel electrophoresis are all techniques that may be used in the purification. Specific methods of purification are described in Robert K. Scopes, Protein Purification: Principles and Practice, Charles R. Castor, ed., Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step purification separations are also described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83 (1994).
As illustrated in FIG. 15 , the isolated CFXTEN fusion proteins are characterized for their chemical and activity properties. An isolated fusion protein is characterized, e.g., for sequence, purity, apparent molecular weight, solubility and stability using standard methods known in the art. The fusion protein meeting expected standards is evaluated for activity, which can be measured in vitro or in vivo by measuring one of the factor VIII-associated parameters described herein, using one or more assays disclosed herein, or using the assays of the Examples or Table 49.
In addition, the CFXTEN FVIII fusion protein is administered to one or more animal species to determine standard pharmacokinetic parameters and pharmacodynamic properties, as described in Examples 25 and 26.
By the iterative process of producing, expressing, and recovering CFXTEN constructs, followed by their characterization using methods disclosed herein or others known in the art, the CFXTEN compositions comprising CF and an XTEN are produced and evaluated to confirm the expected properties such as enhanced solubility, enhanced stability, improved pharmacokinetics and reduced immunogenicity, leading to an overall enhanced therapeutic activity compared to the corresponding unfused FVIII. For those fusion proteins not possessing the desired properties, a different sequence or configuration is constructed, expressed, isolated and evaluated by these methods in order to obtain a composition with such properties.
Example 16
Construction of Expression Plasmids for BDD FVIII
I. Construction of B Domain Deleted FVIII (BDD FVIII) Expression Vectors
The expression vector encoding BDD FVIII was created by cloning the BDD FVIII open reading frame into the pcDNA4 vector (Invitrogen, CA) containing a polyA to allow for optimal mammalian expression of the FVIII gene, resulting in a construct designated pBC0100. Several natural sites were identified within this construct for cloning use, including BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, Apa13574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, Agel 4500, PmeI 4527. To facilitate assay development, nucleotides encoding Myc and His tag were introduced into the FVIII open reading frame. pBC0100 was PCR amplified using the following primers: 1) F8-BsiWI-FI: tattccCGTACGgccgccaccATGCAAATAGAGCTCTCCACCT (SEQ ID NO: 1658); 2) F8-nostop-XhoI-R1: GGTGACCTCGAGcgtagaggtcctgtgcctcg (SEQ ID NO: 1659) to introduce BsiWI and XhoI in appropriate locations. The PCR product was digested with BsiWI and XhoI. PcDNA4-Myc-His/C was digested with Acc65I and XhoI, which generated two products of 5003 and 68 bps. The 5003 bps product was ligated with the digested PCR'ed FVIII fragment and used for DHSalpha transformation. The enzymes Acc65I and BsiWI create compatible ends but this ligation destroys the site for future digestion. The resulting construct was designated pBC0102 (pcDNA4-FVIII — 3-Myc-His). To facilitate the design and execution of future cloning strategies, especially ones involving the creation of BDD FVIII expression constructs that contain multiple XTEN insertions, we selected additional unique restriction enzyme sites to incorporate, including BsiWI 908, NheI 1829 and Cla13281. The introduction of these sites was done via the QuikChange method (Agilent, CA) individually. The resulting construct was designated pBC0112 (pcDNA4-FVIII — 4-Myc-His). To avoid problems that may arise from the linker peptides that connects between Myc/His and FVIII/Myc, and to remove restriction enzyme sites that are preferred for future XTEN insertion, we mutated the sequences encoding the peptide sequences from ARGHPF (SEQ ID NO: 1660) to GAGSPGAETA (SEQ ID NO: 178) (between FVIII and Myc), NMHTG (SEQ ID NO: 1661) to SPATG (SEQ ID NO: 1662) (between Myc and His) via the QuikChange method. The construct was designated pBC0114 (pcDNA4-FVIII — 4-GAGSPGAETA-Myc-SPATG-His (‘GAGSPGAETA’ and ‘SPATG’ disclosed as SEQ ID NOS178 and 1662, respectively)) (sequence in Table 21), which was used as the base vector for the design and creation of other expression vectors incorporating XTEN sequences. Expression and FVIII activity data for this construct are presented in
II. Construction of B Domain Deleted FVIII (BDD FVIII) Expression Vectors
The gene encoding BDD FVIII is synthesized by GeneArts (Regensburg, Germany) in the cloning vector pMK (pMK-BDD FVIII). The BDD FVIII proteins contain 1457 amino acids at a total molecular weight of 167539.66. There are 6 domains within the wild-type FVIII protein, the A1, A2, B, A3, C1 and C2 domains. In the BDD FVIII protein, most of the B domain has been deleted as it was shown to be an unstructured domain and the removal of the domain does not alter critical functions of this protein. The pMK vector used by GeneArts contains no promoter, and can not be used as an expression vector. Restriction enzyme sites NheI on the 5′ end and Sfi, SalI and XhoI on the 3′ end are introduced to facilitate subcloning of the DNA sequence encoding BDD FVIII into expression vectors, such as CET1019-HS (Millipore). Several unique restriction enzyme sites are also introduced into the FVIII sequence to allow further manipulation (e.g., insertion, mutagenesis) of the DNA sequences. Unique sites listed with their cut site include, but are not limited to: SacI 391, AfiII 700, Spe1966, PshAI 1417, Acc6512192, KpnI 2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, Apa13893, Bsp1201 3893, SwaI 4265, OliI 4626, XbaI 4644, and BstBI 4673. The HindIII site resides at the very end of the A2 domain and can potentially be used for modification of the B domain. The synthesized pMK-BDD FVIII from GeneArts does not contain a stop codon. The stop codon is introduced by amplifying a 127 bp fragment of FVIII using the following primers: 5′-GTGAACTCTCTAGACCCACCG-3′ (SEQ ID NO: 1663); 5′-CTCCTCGAGGTCGACTCAGTAGAGGTCCTGTGCCTCG-3′ (SEQ ID NO: 1664). The fragment is digested with XbaI and SalI, and ligated to XbaI/SalI digested pMK-BDD FVIII. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The construct named pBC0027 (pMK-BDD FVIII-STOP) contains coding sequences that encode the BDD FVIII protein. The pBC0027 construct is then digested with NheI/SalI, and ligated with NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0025 (CET1019-HS-BDD FVIII-STOP), which encodes the BDD FVIII protein under the control of a human CMV promoter. Introduction of the pBC0025 construct into mammalian cells is expected to allow expression of the BDD FVIII protein with procoagulant activity.
Example 17
Construction of Expression Plasmids for BDD FVIII Containing XTEN
1. B Domain AE42 Insertion
Two PCR reactions were run in parallel to insert XTEN_AE42 into the remaining B domain region of the BDD FVIII constructs. The PCR reactions involved the following primers: cgaaagcgctacgcctgagaGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCTCTGAAACCCCTGCCTC GAGCccaccagtcttgaaacgcc (SEQ ID NO: 1665); TGATATGGTATCATCATAATCGATTTCCTCTTGATCTGACTG (SEQ ID NO: 1666); agcttgaggatccagagttc (SEQ ID NO: 1667); tctcaggcgtagcgctttcgCTTGTCCCCTCTTCTGTTGAGGTGGGGGAGCCAGCAGGAGAACCTGGCG CGCCgttttgagagaagcttcttggt (SEQ ID NO: 1668). The PCR products then served as templates, and a second PCR was performed to introduce the XTEN_AE42 into the FVIII encoding nucleotide sequences flanked by BamHI and ClaI. This PCR product was digested with BamHI and ClaI simultaneously with the digestion of PBC0114 with the same two enzymes. The PCR product was ligated to the digested vector. This construct was designated pBC0135 (pcDNA4-FVIII — 4 XTEN_AE42-GAGSPGAETA-Myc-SPATG-His) (‘GAGSPGAETA’ and ‘SPATG’ disclosed as SEQ ID NOS178 and 1662, respectively), and encodes the BDD FVIII with an AE42 XTEN incorporated within the residual B-domain.
2. AE42 Insertion and R1648A Mutation
The QuikChange method (Agilent, CA) was employed to introduce an R1648A mutation into PBC0135. This construct was designated pBC0149 (pcDNA4-FVIII — 4 XTEN_AE42-GAGSPGAETA-Myc-SPATG-His_R1648A) (‘GAGSPGAETA’ and ‘SPATG’ disclosed as SEQ ID NOS178 and 1662, respectively), eliminating that FVIII processing site.
3. B Domain AE288 Insertion
XTEN_AE288 was PCR amplified using the following primers: tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1669) and tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1670). PBC0075 was used as the template for this PCR reaction. The PCR product was digested with AscI and XhoI, and PBC0135 was digested with the same enzymes. The PCR product was ligated to the PBC0135 fragment. This construct was designated pBC0136 (pcDNA4-FVIII — 4 XTEN_AE288-GAGSPGAETA-Myc-SPATG-His) (‘GAGSPGAETA’ and ‘SPATG’ disclosed as SEQ ID NOS178 and 1662, respectively), and encodes the BDD FVIII with an AE288 XTEN incorporated within the residual B-domain.
4. AE288 Insertion and R1648A Mutation
XTEN_AE288 was PCR amplified using the following primers: tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1671) and tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1672). Construct pBC0075 was used as the template for this PCR reaction. The PCR product was digested with AscI and XhoI, and pBC0149 was digested with the same enzymes. The PCR product was ligated to the pBC0149 fragment. This construct was designated pBC0137 (pcDNA4-FVIII — 4 XTEN_AE288-GAGSPGAETA-Myc-SPATG-His R1648A) (‘GAGSPGAETA’ and ‘SPATG’ disclosed as SEQ ID NOS178 and 1662, respectively) and contains an AE288 XTEN sequence internal to the B domain, with the R1648A mutation eliminating that FVIII processing site.
3. B Domain AE144, AG144, AG288 Insertions with and without R1648A Mutations
Select XTEN fragments were PCR amplified to introduce AscI and XhoI sites to the 5′ and 3′ end respectively. The PCR product was digested with AscI and XhoI, and pBC0135 (for R1648) or pBC0149 (for A1648) were digested with the same enzymes. The PCR product was ligated to the pBC0135 or pBC0149 vector. These constructs were designated pSD0005, 6, 7, 8, 17 and 18.
Construction of Expression Plasmids for BDD FVIII with XTEN Insertion at the C Terminus
1. C Terminal AE288 Insertion
XTEN_AE288 was PCR amplified using the following primers: ggggccgaaacggccggtacctcagagtctgctacc (SEQ ID NO: 1673) and tgttcggccgtttcggcccctggcgcactgccttc (SEQ ID NO: 1674). The construct pBC0075 was used as the template for this PCR reaction. The PCR product was digested with Sfi, and pBC0114 was digested with the same enzyme. The PCR product was ligated to the digested pBC0114 fragment. This construct was designated pBC0145 (pcDNA4-FVIII — 4-XTEN_AE288-GAGSPGAETA-Myc-SPATG-His) (‘GAGSPGAETA’ and ‘SPATG’ disclosed as SEQ ID NOS178 and 1662, respectively), and encodes an AE288 sequence at the C-terminus of the BDD FVIII.
2. C Terminal AG288 Insertion
XTEN_AG288 was designed and synthesized by DNA2.0 (Menlo Park, Calif.). The synthesized gene was PCR amplified using the following primers: ggggccgaaacggccccgggagcgtcacc (SEQ ID NO: 1675) and tgttcggccgtttcggcccctgacccggttgcccc (SEQ ID NO: 1676). The PCR product was digested with Sfi, and PBC0114 based vector was digested with the same enzyme. The PCR product was ligated to the digested PBC0114 fragment. This construct was designated pBC0146 (pcDNA4-FVIII — 4-XTEN_AG288-GAGSPGAETA-Myc-SPATG-His) (‘GAGSPGAETA’ and ‘SPATG’ disclosed as SEQ ID NOS178 and 1662, respectively), and encodes an AG288 sequence at the C-terminus of the BDD FVIII.
3. C Terminal AE/AG144, 288, 864 Insertions
AscI and XhoI sites were introduced into the PBC0114 based vector via QuikChange methods using the primers: 5037-PBC0114-AscI-XhoI-F: CAGGACCTCTACGGCGCgccagcctcgaGCGAACAAAACTCATCTCAGAAGAGG (SEQ ID NO: 1677); 5038-PBC0114-AscI-XhoI-R: CCTCTTCTGAGATGAGTTTTTGTTCGCtcgaggctggcGCGCCGTAGAGGTCCTG (SEQ ID NO: 1678). Various XTEN fragments were PCR amplified with AscI and XhoI introduced into the 5′ and 3′ end respectively. The PCR product was ligated to the digested PBC0114 vector. These constructs were designated pSD0013, pSD0014, pSD0015, pSD0016, pSD0019 and pSD0020.
Construction of Expression Plasmids for BDD FVIII with Inter- and Intra-Domain XTEN Insertions
1. AE7, AE42 and AE144 Insertions
Four distinct strategies are used for insertion of AE42 into the designated sites (e.g., the natural or introduced restriction sites BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, Apa13574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, Agel 4500, PmeI 4527, BsiWI 908, NheI 1829 and Cla13281) within the BDD FVIII encoding sequence, each contributing to the creation of several constructs. By design, these insertions of AE42 create AscI and XhoI sites flanked on either side of the insertion allowing for introduction/substitution of longer XTENs, as well as XTEN with different sequences or incorporated cleavage sequences, as needed. Specifically, the constructs that contain XTEN — 144 insertions are listed in Table 21. These insertions were created by replacing either AE7 or AE42 with a PCRed XTEN — 144 fragment flanked by AscI and XhoI sites.
2. Double PCR-Mediated Method
Two PCR reactions are run in parallel to insert XTEN_AE42 into the designated site. The two PCR reactions introduce XTEN on either the 3′ or the 5′ end via use of a long primer that contains partial XTEN. The PCR products then serve as templates, and a second PCR is performed to introduce the XTEN_AE42 into the FVIII encoding nucleotide sequences flanked by select restriction enzyme sites. This PCR product is digested with the appropriate enzymes simultaneously with the digestion of PBC0114 using the same two enzymes. The PCR product is ligated to the digested vector. Using this method, constructs are created designated pBC0126, pBC0127, pBC0128, and pBC0129, resulting in AE42 insertions at the R3, R3, P130, L216 locations respectively. The sequences are listed in Table 21. Select XTEN — 144 sequences can then be PCRed to introduce AscI and XhoI sites on either end of the fragment, and ligate to digested FVIII-XTEN_AE42 construct. For instance, pSD0053 was created by replacing the AE42 of pBC0129 with XTEN_AE144. Other XTEN — 144 constructs were created via the same strategy and are listed in Table 21.
3. QuikChange Mediated Two Step Cloning Method
The QuikChange method is employed to introduce XTEN_AE7 encoding sequences that are flanked by AscI and XhoI into designated sites. The resulting intermediate construct is then digested with AscI and XhoI. XTEN_AE42 or XTEN_AE144 is PCR amplified to introduce the two sites and digested accordingly. The vector and insert are then ligated to create the final constructs. The sequences are listed in Table 21.
4. Three PCR Type II Restriction Enzyme Mediated Ligation Method
Three PCR reactions are performed to create two pieces of FVIII encoding fragments flanked by one type I restriction enzyme that correlates with a unique site within the FVIII — 4 gene and one type II enzyme (e.g. BsaI, BbsI, BfuAI), the third PCR reaction created the XTEN_AE42 flanked by two type II restriction enzyme sites. The three PCR fragments are digested with appropriate enzymes and ligated into one linear piece that contains the XTEN_AE42 insertion within a fragment of FVIII encoding sequences. This product is then digested with appropriate unique enzymes within the FVIII encoding sequences and ligated to the PBC0114 construct digested with the same enzymes, and result in constructs designated pBC0130 (with XTEN insertion at residue P333), pBC0132 (with XTEN insertion at residue D403), pBC0133 (with XTEN insertion at residue R490). The sequences are listed in Table 21. Select XTEN — 144 sequences can then be PCRed to introduce AscI and XhoI sites on either end of the fragment, and ligate to digested FVIII-XTEN_AE42 construct. For instance, pSD0001 and pSD0003 were created by replacing the AE42 of pBC0132 with XTEN_AE144 and XTEN_AG144 respectively. Other XTEN — 144 constructs listed in Table 21 were created via the same strategy.
5. Custom Gene Synthesis
Custom gene synthesis is performed by GeneArt (Regensburg, Germany). The genes are designed so that they include nucleotides encoding the XTEN_AE42 inserted in the designated site(s) and the genes are flanked by two unique restriction enzyme sites selected within the FVIII — 4 gene. The synthesized genes and PBC0114 are digested with appropriate enzymes and ligated to create the final product with the BDD FVIII incorporating the XTEN_AE42 between the restriction sites. Select XTEN — 144 sequences can then be PCRed to introduce AscI and XhoI sites on either end of the fragment, and ligate to digested FVIII-XTEN_AE42 construct.
Construction of Expression Plasmids with Dual XTEN Insertions in the B Domain and at the C Terminus
The construct pBC0136, which encodes the BDD FVIII with an AE288 XTEN incorporated within the residual B-domain, is digested with BamHI and ClaI, and the resulting 1372 bps fragment from this digestion is the insert. The construct pBC0146 is digested with BamHI and ClaI, and the 9791 bps piece from this digestion is the vector. The vector and insert are ligated together to create pBC0209, containing an AE288 insertion within the B domain and an AG288 on the C terminus. The same strategy is utilized to create constructs containing two AE288 insertions in the B domain and at the C terminus, respectively, using PBC0145 as the vector.
Construction of Expression Plasmids with Multiple XTEN Insertions
The construct pBC0127, which encodes an AE42 XTEN at the R3 position of FVIII, is digested with BsiWI and AflII, and the resulting 468 bps fragment from this digestion is the insert. The construct pBC0209 is digested with BsiWI and Afill, the 10830 bps piece from this digestion is the vector. The vector and insert are ligated together to create a construct designated pBC0210, containing an AE42 insertion in the A1 domain, an extra three ATR amino acid to restore the signal cleavage sequence, an AE288 XTEN insertion within the B domain and an AG288 on the C terminus. The same methodology is used to create constructs encoding multiple XTEN at the natural and introduced restriction sites; e.g., BsiWI 48, Afll 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, Apa13574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, Agel 4500, PmeI 4527, BsiWI 908, NheI 1829 and Cla13281.
Construction of BDD FVIII-Internal-XTEN_AE288 Expression Vectors
Two BsaI restriction enzyme sites are introduced into the PBC0027 pMK-BDD FVIII construct between the base pair 2673 and 2674 using the QuikChange method following manufacturer's protocol (Agilent Technologies, CA). The inserted DNA sequences are gggtctcccgcgccagggtctccc, and the resulting construct is designated pBC0205 (sequence in Table 21). The DNA sequence encoding AE288 (or other variants and lengths of XTEN; e.g. AE42, AG42, AG288, AM288) is then PCR'ed with primers that introduce BsaI sites on both the 5′ and 3′. The pBC0205 vector and the insert (XTEN — 288) are then digested with BsaI and ligated to create pBC0206, which encodes the FVIII gene with an XTEN_AE288 insertion within the B domain (sequence in Table 21). The pBC0206 construct is then digested with NheI/SalI, and ligated with NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0207 (CET1019-HS-BDD FVIII-STOP), which encodes the BDD FVIII protein under the control of a human CMV promoter (sequence in Table 21). Introduction of the pBC0207 construct into mammalian cells is expected to allow expression of the BDD FVIII protein with an internal XTEN_AE288. The same protocol is used to introduce, transform and express constructs containing other variants and lengths of XTEN; e.g. AE42, AG42, AG288, AM288, AE864, AG864, or other XTEN of Table 4.
Construction of BDD FVIII−/−XTEN_AE864 Expression Vectors
The BDD FVIII fragment with NheI and Sfi flanking the 5′ and 3′ end is generated by digesting the pBC0025 construct. This digested fragment is then ligated to a NheI/SfiI digested pSecTag vector (pBC0048 pSecTag-FVIII−/−XTEN_AE864) encoding the FVIII followed by the XTEN_AE864 sequence. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is pBC0060, which encodes the BDD FVIII−/−XTEN_AE864 protein under the control of a human CMV promoter. Introduction of the pBC0060 construct into mammalian cells is expected to express the FVIII protein with a C terminal XTEN fusion (BDD FVIII−/−XTEN_AE864) with procoagulant activity.
Construction of BDD FVIII-/FXI/-XTEN_AE864 Expression Vectors
The BDD FVIII fragment with NheI and Sfi flanking the 5′ and 3′ end is generated by digesting the pBC0025 construct. This digested fragment is then ligated to a NheI/SfiI digested pSecTag vector (pBC0047 pSecTag-FVIII-/FXI/-XTEN_AE864) encoding the FVIII followed by the FXI cleavage sequence (/FXI/) and XTEN_AE864. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is pBC0051, which encodes the BDD FVIII-/FXI/-XTEN_AE864 protein under the control of a human CMV promoter. Introduction of the pBC0051 construct into mammalian cells is expected to express the FVIII protein with a C terminal XTEN fusion (BDD FVIII-/FXI/-XTEN_AE864), which could be subsequently cleaved by FXI, therefore liberating the BDD FVIII protein with procoagulant activity.
Construction of BDD FVIII-/FXI/-XTEN Expression Vectors Comprising AE288 or AG288
The fused AE864 XTEN sequence in pBC0060 is replaced by digesting the XTEN sequences AE288 and AG288 with BsaI and HindIII. A subsequent ligation step using the respective AE288 or AG288 XTEN fragment and BsaI/HindIII digested pBC0051 allows the exchange of the AE288 or AG288 sequences into the BDD FVIII expression vector. The resulting final constructs are pBC0061 for BDD FVII1-AE288 and pBC0062 for BDD FVII1-AG288. Introduction of the pBC0061 construct into mammalian cells is expected to express the FVIII protein with a C-terminal AE288 XTEN fusion (BDD FVIII−/−XTEN_AE288) with procoagulant activity. Introduction of the pBC0062 construct into mammalian cells is expected to express the FVIII protein with a C-terminal AG288 XTEN fusion (BDD FVIII−/−XTEN_AG288) with procoagulant activity.
Construction of BDD FVIII-/FXI/-XTEN Expression Vectors with Alternate XTEN
The fused XTEN sequence in pBC0051 is replaced by digesting DNA encoding other XTEN sequences (e.g. other variants and lengths of XTEN; e.g. AE42, AG42, AG288, AM288) with BsaI and HindIII. A ligation using the XTEN fragment and BsaI/HindIII digested pBC0051 allows the exchange of the various XTEN-encoding sequences into the BDD FVIII expression vector, providing the alternate constructs. Introduction of the alternate constructs into mammalian cells is expected to express the FVIII protein with a C-terminal XTEN (BDD FVIII-/FXI/-XTEN) that can be subsequently cleaved by FXI, releasing the FVIII, resulting in procoagulant FVIII fusion with procoagulant activity.
Example 18
Construction of Expression Plasmids for FVIII Signal Peptide-XTEN-/FXI/-BDD FVIII
Construction of Expression Vectors for FVIII Signal Peptide-XTEN_AE864
The coding sequences for the FVIII signal peptide is generated by annealing the following two oligos: 5′-CTAGCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTG GGTCTCC-3′ (SEQ ID NO: 1679); 5′-ACCTGGAGACCCACTAAAGCAGAATCGCAAAAGGCACAGAAAGAAGCAGGTGGAGAGCTC TATTTGCATG-3′ (SEQ ID NO: 1680). The annealed oligos are flanked by the NheI and BsaI restriction enzyme sites on either end, and is ligated to NheI/BsaI digested pCW0645 vector which encodes the FVII-XTEN_AE864. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants is screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0029, which encodes the signal peptide-XTEN_AE864 protein under the control of a human CMV promoter. This construct is used as an intermediate construct for creating an expression construct with XTEN fused on the N-terminus of the FVIII protein, and can also be used as a master plasmid for creating expression constructs that allow XTEN fusion on the N-terminus of a secreted protein.
Construction of Signal Peptide-XTEN_AE864-/FXI/-BDD FVIII Expression Vectors
An 1800 bp fragment within the FVIII coding region is amplified using primers that introduce NheI-BbsI-/FXI/-AgeI sites on the 5′ and endogenous KpnI restriction enzyme on the 3′ end. The NheI/KpnI digested FVIII fragment is ligated with NheI/KpnI digested pBC0027 vector. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The resulting construct is designated pBC0052, which contains sequences that encode the /FXI/-FVIII protein without the FVIII signal peptide. This construct is used as an intermediate construct for creating an expression construct with XTEN fused on the N-terminus of the FVIII protein.
The pBC0052 vector is digested with BbsI/XhoI enzymes, and is used to ligate with Bbsi/XhoI digested pBC0029. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0053, which encodes the signal peptide-XTEN_AE864-/FXI/-BDD FVIII protein under the control of a human CMV promoter. Introduction of the pBC0053 construct into mammalian cells is expected to express the FVIII protein with an N-terminal XTEN fusion (signal peptide-XTEN_AE864-/FXI/-BDD FVIII), which could be subsequently cleaved by FXI, therefore liberating the BDD FVIII protein.
Construction of Signal Peptide-XTEN-/FXI/-BDD FVIII Expression Vectors
The fused XTEN sequence in pBC0053 can be replaced by digesting other XTEN fragments (e.g. AM, AF, AG) with BsaI and BbsI. A ligation using the XTEN fragment and BsaI/BbsI digested pBC0053 allows the exchange of various XTEN pieces (e.g. AM, AF, AG) into the BDD FVIII expression vector. Various XTEN fusions can increase the half lives of these proteins differently, allowing modification of the properties (e.g. efficacy, potency) of these proteins. Introduction of any of these fusion constructs into mammalian cells is expected to express the FVIII protein with an N-terminal XTEN fusion (signal peptide-XTEN-/FXI/-BDD FVIII), in which the fused XTEN peptide can be subsequently cleaved by FXI, generating the BDD FVIII protein.
Example 19
Construction of BDD FVIII with Interdomain XTEN Insertion
Construction of BDD FVIII Expression Vectors with an XTEN Insertion at the A2-B Domain Boundaries
The pBC0027 construct (pMK-BDD FVIII-STOP) is a cloning vector designed to contain the BDD FVIII protein coding sequences, but not a promoter positioned to initiate the expression of BDD FVIII. This construct is used for manipulation of the coding sequences of BDD FVIII as the vector backbone contains very few restriction enzyme sites. therefore allowing easy cloning strategies. The BDD FVIII proteins contain 1457 amino acids at a total molecular weight of 167539.66. There are 6 domains within the wild-type FVIII protein, the A1, A2, B, A3, C1 and C2 domains. In the BDD FVIII protein, most of the B domain has been deleted as it is believed to be an unstructured domain and the removal of the domain does not alter critical functions of this protein. However, the B domain boundaries seem to be excellent positions for creating XTEN fusions to allow extension of the protein half lives.
Within the pBC0027 construct, there is a unique HindIII restriction enzyme site at the boundary of A2-B junction. The XTEN (e.g., sequences of Tables 4, or 13-17) are amplified using primers that introduce a HindIII and FXI cleavage site on either end of the XTEN coding sequence. The fused XTEN sequence can be altered by amplifying various XTEN fragments. Various XTEN fusions can increase the half lives of these proteins differently, allowing modification of the properties (e.g. efficacy, potency) of these proteins. The HindIII-/FXI/-XTEN-/FXI/-HindIII fragment is digested with HindIII and ligated with HindIII digested pBC0027. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0054, which encodes the BDD FVIII protein with an interdomain XTEN fusion (FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)) but not a promoter to initiate gene expression.
The pBC0054 construct is digested with NheI/SalI, and ligated with NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0055 (CET1019-HS-FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)), which encodes the BDD FVIII protein with an interdomain (inter-A2/B domain) XTEN fusion (FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)) under the control of a human CMV promoter. Introduction of the pBC0055 construct into mammalian cells is expected to express the BDD FVIII protein with an interdomain XTEN fusion (FVIII(A1-A2)-/FXI/-XTEN-/FXI/-FVIII(C1-C2)), which could be subsequently cleaved by FXI, therefore liberating the BDD FVIII protein.
Construction of BDD FVIII Expression Vectors with an XTEN Insertion at the A1-A2 Domain Boundaries
The pBC0027 construct is designed as a template for two PCR reactions using the following four primers:





(Reaction I)

(SEQ ID NO: 1681)

5′-ATGATGGCATGGAAGCCTAT-3′;



(SEQ ID NO: 1682)

5′-ATCCCTCACCTTCGCCAGAACCTTCAGAACCCTCACCTTCAGAACCT



TCACCAGAACCTTCACCATCTTCCGCTTCTTCATTATTTTTCAT-3′.



(Reaction II)

(SEQ ID NO: 1683)

5′-TTCTGGCGAAGGTGAGGGATCTGAAGGCGGTTCTGAAGGTGAAGGTG



GCTCTGAGGGTTCCGAATATGATGATGATCTTACTGATTCTGAAAT-3′;



(SEQ ID NO: 1684)

5′-TATTCTCTGTGAGGTACCAGC-3′.
























The PCR products generated are 150 bps and 800 bps respectively. The 800 bp product is used as the template for the next round of PCR reaction with the 150 bp product as one primer and 5′-TATTCTCTGTGAGGTACCAGC-3′ (SEQ ID NO: 1685) as the other. The product for the second round of PCR is 930 bps and is digested with PshAI and ACC65I restriction enzymes. This PshAI/Acc65I flanked DNA fragment is ligated with PshAI/Acc65I digested pBC0027. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants is screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0058 (pMK-BDD FVII1-D345-XTEN_Y36), which encodes the BDD FVIII protein with an interdomain (inter-A1/A2 domain) XTEN fusion after the D345 residue.
The pBC0058 construct is digested with NheI/SalI, and ligated with NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0059 (CET1019-HS-BDD FVIII D345-XTENY36), which encodes the BDD FVIII protein with an interdomain (inter-A1/A2 domain) XTEN fusion after the D345 residue under the control of a human CMV promoter. Introduction of the pBC0059 construct into mammalian cells is expected to express the BDD FVIII protein with an interdomain XTEN fusion (BDD FVIII D345-XTEN_Y36).
Example 20
Construction of FVIII with Intradomain XTEN Insertion
Construction of BDD FVIII Expression Vectors with an XTEN Insertion after P598 (within the A2 Domain)
The coding sequences for XTEN_Y36 is amplified using PCR techniques with the following primers: 5′-GAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGGTGAAGGTTCTGGT GAAGG-3′ (SEQ ID NO: 1686) 5′-AACTCTGGATCCTCAAGCTGCACTCCAGCTTCGGAACCCTCAGAGCC-3′ (SEQ ID NO: 1687).
The 184 bp PCR product is flanked by the KpnI and BamHI restriction enzyme sites on either end, and is ligated to KpnII/BamHI digested pBC0027 vector which encodes the BDD FVIII gene. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0056, which contains DNA sequences encoding the FVIII protein with an XTEN_Y36 fusion after the P598 residue. This cloning strategy is used to introduce various forms of XTEN into the BDD FVIII protein by altering the template for the PCR reaction and changing the primers accordingly.
The pBC0056 construct is digested with NheI/SalI, and ligated with NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0057 (CET1019-HS-FVIII P598-XTEN_Y32), which encodes the BDD FVIII protein with an intradomain (within A2 domain) XTEN fusion under the control of a human CMV promoter. Introduction of the pBC0057 construct into mammalian cells is expected to express the BDD FVIII protein with an intradomain XTEN fusion (FVIII P598-XTEN_Y32).
Construction of BDD FVIII Expression Vectors with Other Intradomain XTEN Insertions
To introduce various XTEN segments into other intradomain sites within BDD FVIII (e.g., the XTEN of Tables 4, or 13-17), primers are designed that amplify XTEN with an overhang that can anneal with BDD FVIII. The coding sequence of FVIII (pMK-BDD FVIII) is designed with various unique restriction enzyme sites to allow these specific insertions. The unique restriction enzymes are listed below with their cut site: NheI 376, SacI 391, AfiII 700, Spe1966, PshAI 1417, Acc6512192, KpnI 2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, Apa13893, Bsp1201 3893, SwaI 4265, OliI 4626, XbaI 4644, BstBI 4673, SalI4756, and XhoI 4762. The NheI and SalI sites on either end of the coding sequence are used to insert the DNA fragment into a human CMV promoter driven vector, the CET1019-HS (Millipore) for expression in mammalian cells. These constructs express the BDD FVIII protein with an XTEN fusion with sequences listed in Table 21.
Example 21
Construction of FVIII with XTEN Insertions
CFXTEN with Two XTEN:
To obtain CFXTEN with two XTEN insertions in various regions (from N-termini to C-termini: A1-R1, A1-R2, A2-R1, A2-R2, B domain, a3, A3-R1, A3-R2, C-termini), constructs that expressed fusions with single-XTEN insertions that retained FVIII activity were utilized. The coding sequence of FVIII (pBC0114 pcDNA4-FVIII — 4-X10-Myc-SPATG-His extra RE) (‘SPATG’ disclosed as SEQ ID NO: 1662) was designed with various unique restriction enzyme sites to allow these specific combinations. The unique restriction enzymes are listed in Table 18 below with their relative sites between different regions: BsiWI (between N-termini and A1-R1), AflII (between A1-R1 and A1-R2), NheI (between A1-R2 and A2-R1), KpnI (between A2-R1 and A2-R2), BamHI (between A2-R2 and B domain), ClaI (between a3 and A3-R1), PflMI (between A3-R1 and A3-R2), XbaI (between A3-R2 and C-termini), Agel (between FVIII C-termini and stop codon). Building blocks and restriction enzymes for cloning the libraries were chosen, as listed in the table below. The chosen components in each region were mixed at molar ratio of 1:1, and two sets of DNA mixtures were digested with unique restriction enzymes. DNA fragments were separated with 1% agarose gel and purified by Qiagen gel extraction kit. DNA with XTEN insertion in the first desired region was regarded as the insert (the smaller DNA fragment in agarose gel), while DNA with XTEN insertion in the second desired region was regarded as vector (the bigger DNA fragment in agarose gel). The insert and vector were ligated in order to reconstitute the plasmid. The ligated DNA mixture was used to transform DH5a E. coli competent host cells. Transformants were screened by rolling circle amplification (RCA) and Sanger sequencing to cover approximately 3-4 times the potential library size. Unique clones were identified and minipreped. Two distinct restriction digestions were then used to further confirm the integrity of XTEN in each region. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21.
CFXTEN with One or Two XTEN Insertions within the B/a3 Domain and C Terminus:
The B/a3 domain and C-terminus of FVIII are unstructured regions that tolerated XTEN insertions well. The B/a3 domain further mediated interactions with other cofactors, including the von Willibrand Factor. To investigate the optimal XTEN insertions at the B/a3 domain, select deletions and mutations of the region were made via PCR-based mutagenesis methods. Select PCR reactions and the vectors were digested with unique restriction enzymes as listed in Table 18. DNA fragments were separated with 1% agarose gel and purified by Qiagen gel extraction kit. DNA with XTEN insertion in the first desired region was regarded as the insert (the smaller DNA fragment in agarose gel), while DNA with XTEN insertion in the second desired region was regarded as vector (the bigger DNA fragment in agarose gel). The insert and vector were ligated in order to reconstitute the plasmid. The ligated DNA mixture was used to transform DH5a E. coli competent host cells. Transformants were screened by colony PCR and Sanger sequencing to cover approximately 8× the potential library size. Unique clones were identified and minipreped. One three-enzyme restriction digestion was then used to further confirm the integrity of XTEN in each region. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21.





TABLE 18



Cloning design for FVIII libraries with two XTEN insertions






Library

Vector components
Restriction

ID
Insert components (XTEN region)
(XTEN region)
enzymes



LSD0001
pSD0005, pSD0006, pSD0007, pSD0008,
pSD0013 (C-termini)
NheI + ClaI


pSD0017, pSD0018, pBC0136, pBC0137




(B-domain)



LSD0002
pSD0005, pSD0006, pSD0007, pSD0008,
pSD0014 (C-termini)
NheI + ClaI


pSD0017, pSD0018, pBC0136, pBC0137




(B-domain)



LSD0003
pSD0005, pSD0006, pSD0007, pSD0008,
pSD0019 (C-termini)
NheI + ClaI


pSD0017, pSD0018, pBC0136, pBC0137




(B-domain)



LSD0004
pSD0005, pSD0006, pSD0007, pSD0008,
pSD0020 (C-termini)
NheI + ClaI


pSD0017, pSD0018, pBC0136, pBC0137




(B-domain)



LSD0005
pSD0045, pSD0046, pSD0048, pSD0049,
pSD0001 (A2-R1)
BsiWI + AflII


pSD0050, pSD0051, pSD0052 (A1-R1)



LSD0006
pSD0045, pSD0046, pSD0048, pSD0049,
pSD0002 (A2-R1)
BsiWI + AflII


pSD0050, pSD0051, pSD0052 (A1-R1)



LSD0007
pSD0045, pSD0046, pSD0048, pSD0049,
pSD0003 (A2-R1)
BsiWI + AflII


pSD0050, pSD0051, pSD0052 (A1-R1)



LSD0008
pSD0045, pSD0046, pSD0048, pSD0049,
pSD0004 (A2-R1)
BsiWI + AflII


pSD0050, pSD0051, pSD0052 (A1-R1)



LSD0037
pSD0045, pSD0046, pSD0049, pSD0050,
pSD0032 (A2-R1)
BsiWI + AflII


pSD0051, pSD0052 (A1-R1)



LSD0038
pSD0039 (a3)
pSD0045, pSD0046, pSD0049,
BamHI + ClaI



pSD0050, pSD0051, pSD0052 (A1-R1)


LSD0039
pSD0039 (a3)
pSD0032, pSD0001, pSD0003 (A2-R1)
BamHI + ClaI

LSD0040
pSD0040, pSD0010, pSD0041 (A3 -R1)
pSD0045, pSD0046, pSD0049,
ClaI + XbaI



pSD0050, pSD0051, pSD0052 (A1-R1)


LSD0041
pSD0040, pSD0010, pSD0041 (A3-R1)
pSD0032, pSD0001, pSD0003 (A2-R1)
ClaI + XbaI

LSD0042
pSD0062, pSD0063, pSD0043, pSD0044
pSD0045, pSD0046, pSD0049,
ClaI + XbaI


(A3-R2)
pSD0050, pSD0051, pSD0052 (A1-R1)


LSD0043
pSD0062, pSD0063, pSD0043, pSD0044
pSD0032, pSD0001, pSD0003 (A2-R1)
ClaI + XbaI


(A3-R2)



LSD0044
pSD0062, pSD0063, pSD0043, pSD0044
pSD0040, pSD0010, pSD0041 (A3-R1)
PflMI + XbaI


(A3-R2)



LSD0045
pSD0039 (a3)
pSD0040, pSD0010, pSD0041 (A3-R1)
BamHI + ClaI

LSD0046
pSD0039 (a3)
pSD0062, pSD0063, pSD0043,
BamHI + ClaI



pSD0044 (A3-R2)


LSD0047
pSD0046 (A1-R1)
pSD0001, pSD0003 (A2-R1)
BsiWI + AflII

LSD0048
pSD0045, pSD0051 (A1-R1)
pSD0003 (A2-R1)
BsiWI + AflII

pNL0006
PCR product
LSD0003.006 (B Domain and C
BamHI + PflMI



termini)


pNL0007
PCR product
LSD0003.006 (B Domain and C
ClaI + PflMI



termini)


pNL0008
PCR product
LSD0003.009 (B Domain and C
ClaI + PflMI



termini)


pNL0009
PCR product
pSD0039 (a3 Domain)
BamHI + AscI

pNL0010
LSD0003.006 (B Domain and C termini)
pNL0009 (a3 Domain)
XbaI + AgeI




































































Example 22
Construction of BDD FVIII Expression Vectors with 3-5 XTEN Insertions at sites 18/26, 403, 745/1656, 1720, 1900 or 2332
FVIII-fusion constructs with XTEN insertions at sites 18/26, 403, 745/1656, 1720, 1900 or 2332 were chosen to recombine and generate constructs with 3, 4, 5 or 6 XTEN insertions.
Construction of BDD FVIII Expression Vectors with 3-5 XTEN Insertions at Sites 26, 403, 1656, 1720, or 1900
The chosen constructs with single XTEN at the desired sites were: pSD0050, pSD0001, pSD0039, pSD0010, and pSD0062. Constructs with double XTENs at the desired sites included LSD0005.002, LSD0038.001, LSD0040.002, LSD0042.013, LSD0039.010, LSD0041.008, LSD0043.008, LSD0045.002, LSD0046.002, and LSD0044.002. Building blocks and restriction enzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosen components were digested with unique restriction enzymes. DNA of inserts and vectors were separated with 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector were ligated, and then transformed into DH5a E. coli competent host cells. Four colonies for each construct were analyzed by RCA and DNA sequencing. Clones with desired XTEN insertions were minipreped. Restriction digestions were then used to further confirm the integrity of XTEN in each region. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21. The resulting constructs were numbered pSD0077 to pSD0092.
Construction of BDD FVIII Expression Vectors with 4-6 XTEN Insertions at Sites 18, 403, 1656, 1720, 1900 or 2332
Constructs pSD0077 to pSD0092 served as building blocks to generate 4- to 6-XTEN constructs with insertions at 18, 403, 1656, 1720, 1900 and 2332. Building block constructs and restriction enzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosen components were digested with unique restriction enzymes. DNA of inserts and vectors were separated with 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector were ligated, and then transformed into DH5a E. coli competent host cells. Eight colonies for each construct were analyzed by colony PCR and DNA sequencing. Clones with desired XTEN insertions were minipreped. Restriction digestions were then used to further confirm the integrity of XTEN in each region. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21. The resulting constructs were numbered pBC0247 to pBC0257, pNL0022, 23, 24, 25, and 30
Construction of BDD FVIII Expression Vectors with 4-6 XTEN Insertions at Sites 18, 403, 745, 1720, 1900 or 2332
Constructs pBC0247 to pBC0252, pBC0255, pNL0022 to pNL0025 served as building blocks to generate 4- to 6-XTEN constructs with insertions at 18, 403, 745, 1720, 1900 and 2332. Building block constructs and restriction enzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosen components were digested with unique restriction enzymes. DNA of inserts and vectors were separated with 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector were ligated, and then transformed into DH5a E. coli competent host cells. Eight colonies for each construct were analyzed by colony PCR and DNA sequencing. Clones with desired XTEN insertions were minipreped. Restriction digestions were then used to further confirm the integrity of XTEN in each region. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21. The resulting constructs were numbered pBC0258 to pBC0268.





TABLE 19



Cloning design for FVIII libraries with 3-5 XTEN insertions at sites 26, 403, 1656, 1720,

or 1900






Construct

Vector components
Restriction

Name
Insert components (XTEN region)
(XTEN region)
enzymes



pSD0077
pSD0050 (A1-R1)
LSD0039.010 (A2-R1, a3)
BsiWI + AflII

pSD0078
pSD0010 (A3-R1)
LSD0005.002 (A1-R1, A2-R1)
ClaI + XbaI

pSD0079
pSD0062 (A3-R2)
LSD0005.002 (A1-R1, A2-R1)
ClaI + XbaI

pSD0080
pSD0050 (A1-R1)
LSD0045.002 (a3, A3-R1)
BsiWI + AflII

pSD0081
pSD0050 (A1-R1)
LSD0046.002 (a3, A3-R2)
BsiWI + AflII

pSD0082
pSD0050 (A1-R1)
LSD0044.002 (A3-R1, A3-R2)
BsiWI + AflII

pSD0083
pSD0010 (A3-R1)
LSD0039.010 (A2-R1, a3)
ClaI + XbaI

pSD0084
pSD0062 (A3-R2)
LSD0039.010 (A2-R1, a3)
ClaI + XbaI

pSD0085
pSD0062 (A3-R2)
LSD0041.008 (A2-R1, A3-R1)
PflMI + XbaI

pSD0086
pSD0062 (A3-R2)
LSD0045.002 (a3, A3-R1)
PflMI + XbaI

pSD0087
LSD0039.010 (A2-R1, a3)
LSD0040.002 (A1-R1, A3-R1)
NheI + ClaI

pSD0088
LSD0039.010 (A2-R1, a3)
LSD0042.013 (A1-R1, A3-R2)
NheI + ClaI

pSD0089
LSD0044.002 (A3-R1, A3-R2)
LSD0005.002 (A1-R1, A2-R1)
ClaI + XbaI

pSD0090
LSD0044.002 (A3-R1, A3-R2)
LSD0038.001 (A1-R1, a3)
ClaI + XbaI

pSD0091
LSD0044.002 (A3-R1, A3-R2)
LSD0039.010 (A2-R1, a3)
ClaI + XbaI

pSD0092
LSD0044.002 (A3-R1, A3-R2)
pSD0077 (A1-R1, A2-R1, a3)
ClaI + XbaI

pBC0247
pSD0077
LSD0050.003
NheI + BstBI

pBC0248
pSD0078
LSD0050.003
NheI + BstBI

pBC0249
pSD0079
LSD0050.003
NheI + BstBI

pBC0250
pSD0080
LSD0050.003
NheI + BstBI

pBC0251
pSD0082
LSD0050.003
NheI + BstBI

pBC0252
pSD0080
LSD0050.003
NheI + BstBI

pBC0253
pSD0087
LSD0050.003
NheI + BstBI

pBC0254
pSD0088
LSD0050.003
NheI + BstBI

pBC0255
pSD0089
LSD0050.003
NheI + BstBI

pBC0256
pSD0090
LSD0050.003
NheI + BstBI

pBC0257
pSD0092
LSD0050.003
NheI + BstBI

pNL0022
LSD0003.009
pSD0083
XbaI + AgeI

pNL0023
LSD0003.009
pSD0084
XbaI + AgeI

pNL0024
LSD0003.009
pSD0085
XbaI + AgeI

pNL0025
LSD0003.009
pSD0086
XbaI + AgeI

pNL0030
LSD0003.009
pSD0091
XbaI + AgeI

pBC0258
LSD0003.006
pBC0247
BamHI + ClaI

pBC0259
LSD0003.006
pBC0248
BamHI + ClaI

pBC0260
LSD0003.006
pBC0249
BamHI + ClaI

pBC0261
LSD0003.006
pBC0250
BamHI + ClaI

pBC0262
LSD0003.006
pBC0251
BamHI + ClaI

pBC0263
LSD0003.006
pBC0252
BamHI + ClaI

pBC0264
LSD0003.006
pBC0255
BamHI + ClaI

pBC0265
LSD0003.006
pNL0022
BamHI + ClaI

pBC0266
LSD0003.006
pNL0023
BamHI + ClaI

pBC0267
LSD0003.006
pNL0024
BamHI + ClaI

pBC0268
LSD0003.006
pNL0025
BamHI + ClaI

































































Example 23
Construction of CFXTEN Expression Vectors with Three or Four XTENs: The First XTEN in the B Domain, the Second XTEN at the C-Terminus, and the Third or Fourth XTEN Insertion within the A1 or A2 or A3 Domains
Libraries of CFXTEN fusion proteins were constructed with three XTEN insertions by combining coagulation-active clones with XTEN insertions in the A1, A2, or A3 domains and clones with XTEN inserted within the B domain and at the C-terminus. Additional libraries were constructed with a fourth XTEN added in the A1, A2, or A3 domains to select members of the 3 XTEN libraries. The design of the cloning scheme is summarized in the table below. DNA was prepared for the inserts and vectors by restriction enzyme digestion and agarose gel purification. After ligating the inserts with the corresponding vectors, the ligated DNA mixture was used to transform DH5a competent E. coli host cells. Transformants were screened by RCA and sequencing to cover approximately 3-4 times the potential library size. Unique clones were identified and mini-prepped. Three distinct restriction digestions were then used to further confirm the integrity of each XTEN. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21.





TABLE 20



Cloning design for FVIII libraries with 3 XTEN insertions at sites B domain, C-termini,

and A1/A2/A3 domain






Library
Insert component XTEN
Vector components
Restriction

ID
region)
(XTEN region)
enzymes



LSD0049
LSD0003.006 (3 domain and C-
pSD0045, pSD0046, pSD0049, pSD0050,
BamHI + AgeI


termini)
pSD0051, pSD0052 (A1-R1)


LSD0050
LSD0003.009 (B domain and C-
pSD0045, pSD0046, pSD0049, pSD0050,
BamHI + AgeI


termini)
pSD0051, pSD0052 (A1-R1)


LSD0051
LSD0003.006 (B domain and C-
pSD0032, pSD0001, pSD0003 (A2-R1)
BamHI + AgeI


termini)



LSD0052
LSD0003.009 (B domain and C-
pSD0032, pSD0001, pSD0003 (A2-R1)
BamHI + AgeI


termini)



LSD0053
pSD0040, pSD0010, pSD0041
LSD0003.006 (B domain and C-termini)
ClaI + XbaI


(A3-R1)



LSD0054
pSD0040, pSD0010, pSD0041
LSD0003.009 (B domain and C-termini)
ClaI + XbaI


(A3-R1)



LSD0055
pSD0062, pSD0063, pSD0043,
LSD0003.006 (B domain and C-termini)
ClaI + XbaI


pSD0044 (A3-R2)



LSD0056
pSD0062, pSD0063, pSD0043,
LSD0003.009 (B domain and C-termini)
ClaI + XbaI


pSD0044 (A3-R2)



LSD0057
pSD0001 (A2-R1)
LSD0049.021, LSD0049.002, LSD0049.011,
NheI + BamHI



LSD0049.012 (A1-R1, B domain and C-termini)


LSD0058
pSD0003 (A2-R1)
LSD0049.021, LSD0049.002, LSD0049.011,
NheI + BamHI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0059
pNL0005 (A2-R1)
LSD0049.021, LSD0049.002, LSD0049.011,
NheI + BamHI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0060
pBC0246 (A2-R1)
LSD0049.021, LSD0049.002, LSD0049.011,
NheI + BamHI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0061
pSD0009 (A3-R1)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0062
pSD0010 (A3-R1)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0063
pNL0004 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0064
pSD0063 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0065
pNL0002 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0066
pSD0043 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0067
pNL0003 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0068
pSD0044 (A3-R2)
LSD0049.021, LSD0049.002, LSD0049.011,
ClaI + XbaI



LSD0049.012 (A1 -R1, B domain and C-termini)


LSD0069
pSD0009 (A3-R1)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


LSD0070
pSD0010 (A3-R1)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


LSD0071
pNL0004 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


LSD0072
pSD0063 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


LSD0073
pNL0002 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


LSD0074
pSD0043 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


LSD0075
pNL0003 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


LSD0076
pSD0044 (A3-R2)
LSD0051.002, pBC0244, pBC0245 (A2 -R1, B
ClaI + XbaI



domain and C-termini)


pSD0093
pNL0004 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini)
PflMI + XbaI

pSD0094
pSD0063 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini)
PflMI + XbaI

pSD0095
pNL0002 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini)
PflMI + XbaI

pSD0096
pSD0043 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini)
PflMI + XbaI

pSD0097
pNL0003 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini)
PflMI + XbaI

pSD0098
pSD0044 (A3-R2)
LSD0053.022 (A3-R1, B domain and C-termini)
PflMI + XbaI

pCS0001
pBC0168 (A1)
LSD0055.021 (A3-R1, B domain and C-termini)
BsiWI + BamHI

pCS0002
pBC0134 (A2_R2)
LSD0055.021 (A3-R1, B domain and C-termini)
BsiWI + BamHI

pCS0003
pBC0179 (C1)
LSD0055.021 (A3-R1, B domain and C-termini)
ApaI + XbaI

pCS0004
pBC0143 (C1)
LSD0055.021 (A3-R1, B domain and C-termini)
ApaI + XbaI

pCS0005
pBC0182 (C2)
LSD0055.021 (A3-R1, B domain and C-termini)
ApaI + XbaI

pCS0006
pBC0144 (C2)
LSD0055.021 (A3-R1, B domain and C-termini)
ApaI + XbaI

pBC0269
pBC0165 (A1_R1)
LSD0003.006 (B domain and C-termini)
BsiWI + BamHI

pBC0270
pBC0132 (A2_R1)
LSD0003.006 (B domain and C-termini)
BsiWI + BamHI

pBC0271
pBC0138 (A3_R1)
LSD0003.006 (B domain and C-termini)
ClaI + XbaI

pBC0272
pBC0176 (A3_R2)
LSD0003.006 (B domain and C-termini)
ClaI + XbaI

pBC0273
pSD0001 (A2_R1)
LSD0003.006 (B domain and C-termini)
BsiWl + BamHI

pBC0274
pSD0009 (A3_R1)
LSD0003.006 (B domain and C-termini)
ClaI + XbaI

pBC0275
pNL0004 (A3_R2)
LSD0003.006 (B domain and C-termini)
ClaI + XbaI

pBC0276
pBC0280 (A1_R1)
LSD0003.006 (B domain and C-termini)
BsiWI + BamHI

pBC0277
pBC0281 (A2_R1)
LSD0003.006 (B domain and C-termini)
BsiWI + BamHI

pBC0278
pBC0282 (A3_R1)
LSD0003.006 (B domain and C-termini)
ClaI + XbaI

pBC0279
pBC0283 (A3_R2)
LSD0003.006 (B domain and C-termini)
ClaI + XbaI

L09_01
pBC0284 (CT)
pBC0285, 286, 287, 288, 289, 290, 291, 292,
XbaI + AgeI



293 (B domain and A3_R2)


L09_01
pSD0014 (CT)
pBC0285, 286, 287, 288, 289, 290, 291, 292,
XbaI + AgeI



293 (B domain and A3_R2)


L09_01
pSD0020 (CT)
pBC0285, 286, 287, 288, 289, 290, 291, 292,
XbaI + AgeI



293 (B domain and A3_R2)
















































































































TABLE 21



DNA and Amino Acid Sequences of FVIII-XTEN Constructs





Construct
Amino acid sequence disclosed as
DNA sequence disclosed as

Name
SEQ ID NO:
SEQ ID NO:







pBC0114
595
596

pBC0126
597
598

pBC0127
599
600

pBC0165
601
602

pBC0183
603
604

pBC0184
605
606

pBC0166
607
608

pBC0185
609
610

pBC0167
611
612

pBC0128
613
614

pBC0168
615
616

pBC0129
617
618

pBC0169
619
620

pBC0130
621
622

pBC0131
623
624

pBC0132
625
626

pBC0170
627
628

pBC0133
629
630

pBC0171
631
632

pBC0134
633
634

pBC0172
635
636

pBC0135
637
638

pBC0149
639
640

pBC0136
641
642

pBC0137
643
644

pBC0138
645
646

pBC0139
647
648

pBC0140
649
650

pBC0173
651
652

pBC0174
653
654

pBC0175
655
656

pBC0176
657
658

pBC0177
659
660

pBC0178
661
662

pBC0141
663
664

pBC0179
665
666

pBC0180
667
668

pBC0142
669
670

pBC0143
671
672

pBC0181
673
674

pBC0182
675
676

pBC0144
677
678

pBC0145
679
680

pBC0146
681
682

pSD0001
683
684

pSD0002
685
686

pSD0003
687
688

pSD0004
689
690

pSD0005
691
692

pSD0006
693
694

pSD0007
695
696

pSD0008
697
698

pSD0009
699
700

pSD0010
701
702

pSD0011
703
704

pSD0012
705
706

pSD0013
707
708

pSD0014
709
710

pSD0017
711
712

pSD0018
713
714

pSD0019
715
716

pSD0020
717
718

pSD0015
719
720

pSD0016
721
722

pSD0021
723
724

pSD0022
725
726

pSD0023
727
728

pSD0024
729
730

pSD0025
731
732

pSD0026
733
734

pSD0027
735
736

pSD0028
737
738

pSD0029
739
740

pSD0030
741
742

pSD0031
743
744

pSD0032
745
746

pSD0033
747
748

pSD0034
749
750

pSD0035
751
752

pSD0036
753
754

pSD0037
755
756

pSD0038
757
758

pSD0039
759
760

pSD0040
761
762

pSD0041
763
764

pSD0042
765
766

pSD0043
767
768

pSD0044
769
770

pSD0062
771
772

pSD0063
773
774

pSD0045
775
776

pSD0046
777
778

pSD0047
779
780

pSD0048
781
782

pSD0049
783
784

pSD0050
785
786

pSD0051
787
788

pSD0052
789
790

pSD0053
791
792

pSD0054
793
794

pSD0055
795
796

pSD0056
797
798

pSD0057
799
800

pSD0058
801
802

pSD0059
803
804

pSD0060
805
806

pSD0061
807
808

LSD0001.002
809
810

LSD0001.005
811
812

LSD0001.006
813
814

LSD0001.011
815
816

LSD0001.012
817
818

LSD0001.013
819
820

LSD0001.016
821
822

LSD0001.021
823
824

LSD0002.001
825
826

LSD0002.002
827
828

LSD0002.014
829
830

LSD0003.004
831
832

LSD0003.006
833
834

LSD0003.009
835
836

LSD0003.014
837
838

LSD0004.010
839
840

LSD0004.011
841
842

LSD0004.014
843
844

LSD0004.016
845
846

LSD0004.022
847
848

LSD0003.016
849
850

LSD0005.002
851
852

LSD0005.004
853
854

LSD0005.005
855
856

LSD0005.011
857
858

LSD0005.018
859
860

LSD0006.002
861
862

LSD0006.005
863
864

LSD0006.007
865
866

LSD0006.011
867
868

LSD0007.002
869
870

LSD0007.004
871
872

LSD0007.013
873
874

LSD0008.001
875
876

LSD0008.002
877
878

LSD0008.006
879
880

LSD0008.009
881
882

LSD0008.017
883
884

LSD0002.025
885
886

LSD0002.013
887
888

LSD0003.025
889
890

LSD0004.025
891
892

LSD0003.005
893
894

LSD0007.008
895
896

LSD0044.002
897
898

LSD0044.005
899
900

LSD0044.039
901
902

LSD0044.022
903
904

LSD0044.003
905
906

LSD0044.001
907
908

LSD0038.001
909
910

LSD0038.003
911
912

LSD0038.008
913
914

LSD0038.012
915
916

LSD0038.013
917
918

LSD0038.015
919
920

LSD0039.001
921
922

LSD0039.003
923
924

LSD0039.010
925
926

LSD0045.001
927
928

LSD0045.002
929
930

LSD0042.014
931
932

LSD0042.023
933
934

LSD0042.006
935
936

LSD0042.013
937
938

LSD0042.001
939
940

LSD0042.039
941
942

LSD0042.047
943
944

LSD0042.003
945
946

LSD0042.004
947
948

LSD0042.008
949
950

LSD0042.038
951
952

LSD0042.082
953
954

LSD0042.040
955
956

LSD0037.002
957
958

LSD0037.009
959
960

LSD0037.011
961
962

LSD0047.002
963
964

LSD0047.005
965
966

LSD0048.007
967
968

LSD0046.001
969
970

LSD0046.002
971
972

LSD0046.003
973
974

LSD0040.011
975
976

LSD0040.042
977
978

LSD0040.002
979
980

LSD0040.008
981
982

LSD0040.021
983
984

LSD0040.037
985
986

LSD0040.046
987
988

LSD0040.003
989
990

LSD0040.006
991
992

LSD0040.007
993
994

L5D0040.010
995
996

LSD0040.039
997
998

LSD0040.052
999
1000

LSD0041.001
1001
1002

LSD0041.004
1003
1004

LSD0041.006
1005
1006

LSD0041.008
1007
1008

LSD0041.010
1009
1010

LSD0041.014
1011
1012

LSD0041.016
1013
1014

LSD0041.035
1015
1016

LSD0043.001
1017
1018

LSD0043.002
1019
1020

LSD0043.005
1021
1022

LSD0043.006
1023
1024

LSD0043.007
1025
1026

LSD0043.008
1027
1028

LSD0043.015
1029
1030

LSD0043.029
1031
1032

LSD0043.043
1033
1034

pSD0077
1035
1036

pSD0078
1037
1038

pSD0079
1039
1040

pSD0080
1041
1042

pSD0081
1043
1044

pSD0082
1045
1046

pSD0083
1047
1048

pSD0084
1049
1050

pSD0085
1051
1052

pSD0086
1053
1054

pSD0087
1055
1056

pSD0088
1057
1058

pSD0089
1059
1060

pSD0090
1061
1062

pSD0091
1063
1064

pSD0092
1065
1066

LSD0049.002
1067
1068

LSD0049.008
1069
1070

LSD0049.011
1071
1072

LSD0049.012
1073
1074

LSD0049.020
1075
1076

LSD0049.021
1077
1078

LSD0050.002
1079
1080

LSD0050.003
1081
1082

LSD0050.007
1083
1084

LSD0050.010
1085
1086

LSD0050.012
1087
1088

LSD0050.014
1089
1090

LSD0051.002
1091
1092

LSD0051.003
1093
1094

LSD0052.001
1095
1096

LSD0052.003
1097
1098

LSD0053.021
1099
1100

LSD0053.022
1101
1102

LSD0053.024
1103
1104

LSD0054.021
1105
1106

LSD0054.025
1107
1108

LSD0054.026
1109
1110

LSD0055.021
1111
1112

LSD0055.022
1113
1114

LSD0055.026
1115
1116

LSD0056.021
1117
1118

LSD0056.024
1119
1120

LSD0056.025
1121
1122

pNL0001
1123
1124

pNL0002
1125
1126

pNL0003
1127
1128

pNL0004
1129
1130

pNL0005
1131
1132

pNL0006
1133
1134

pNL0007
1135
1136

pNL0008
1137
1138

pNL0009
1139
1140

pNL0010
1141
1142

pBC0244
1143
1144

pBC0245
1145
1146

pBC0246
1147
1148

pBC0247
1149
1150

pBC0248
1151
1152

pBC0249
1153
1154

pBC0250
1155
1156

pBC0251
1157
1158

pBC0252
1159
1160

pBC0253
1161
1162

pBC0254
1163
1164

pBC0255
1165
1166

pBC0256
1167
1168

pBC0257
1169
1170

pBC0259
1171
1172

pBC0260
1173
1174

pBC0262
1175
1176

pBC0263
1177
1178

pBC0264
1179
1180

pBC0266
1181
1182

pBC0267
1183
1184

pBC0268
1185
1186

pNL0016
1187
1188

pNL0017
1189
1190

pNL0018
1191
1192

pNL0022
1193
1194

pNL0023
1195
1196

pNL0024
1197
1198

pNL0025
1199
1200

pNL0030
1201
1202

LSD0057.001
1203
1204

LSD0057.004
1205
1206

LSD0057.005
1207
1208

LSD0057.010
1209
1210

L5D0058.003
1211
1212

LSD0058.005
1213
1214

LSD0058.006
1215
1216

LSD0059.002
1217
1218

LSD0059.003
1219
1220

LSD0059.005
1221
1222

LSD0059.006
1223
1224

LSD0060.001
1225
1226

LSD0060.003
1227
1228

LSD0060.004
1229
1230

LSD0061.002
1231
1232

LSD0061.007
1233
1234

LSD0061.008
1235
1236

LSD0061.012
1237
1238

LSD0062.001
1239
1240

LSD0062.002
1241
1242

LSD0062.006
1243
1244

LSD0062.007
1245
1246

LSD0063.001
1247
1248

LSD0063.003
1249
1250

LSD0063.011
1251
1252

LSD0064.017
1253
1254

LSD0064.018
1255
1256

LSD0064.020
1257
1258

LSD0064.021
1259
1260

LSD0065.001
1261
1262

LSD0065.007
1263
1264

LSD0065.014
1265
1266

LSD0066.001
1267
1268

LSD0066.002
1269
1270

LSD0066.009
1271
1272

LSD0066.011
1273
1274

LSD0067.004
1275
1276

LSD0067.005
1277
1278

LSD0067.006
1279
1280

LSD0067.008
1281
1282

LSD0068.001
1283
1284

LSD0068.002
1285
1286

LSD0068.005
1287
1288

LSD0068.010
1289
1290

LSD0069.004
1291
1292

LSD0069.008
1293
1294

LSD0070.003
1295
1296

LSD0070.004
1297
1298

LSD0070.005
1299
1300

LSD0071.001
1301
1302

LSD0071.002
1303
1304

LSD0071.008
1305
1306

LSD0072.001
1307
1308

LSD0072.002
1309
1310

LSD0072.003
1311
1312

LSD0073.002
1313
1314

LSD0073.004
1315
1316

LSD0073.006
1317
1318

LSD0074.007
1319
1320

LSD0074.010
1321
1322

LSD0074.011
1323
1324

LSD0075.003
1325
1326

LSD0075.004
1327
1328

LSD0075.007
1329
1330

LSD0076.002
1331
1332

LSD0076.003
1333
1334

pSD0093
1335
1336

pSD0094
1337
1338

pSD0095
1339
1340

pSD0096
1341
1342

pSD0097
1343
1344

pSD0098
1345
1346

pSD0099
1347
1348

pSD0100
1349
1350

pSD0101
1351
1352

pSD0102
1353
1354

pSD0103
1355
1356

pSD0104
1357
1358

pCS0001
1359
1360

pCS0002
1361
1362

pCS0003
1363
1364

pCS0004
1365
1366

pCS0005
1367
1368

pCS0006
1369
1370

pBC0269
1371
1372

pBC0270
1373
1374

pBC0271
1375
1376

pBC0272
1377
1378

pBC0273
1379
1380

pBC0274
1381
1382

pBC0275
1383
1384

pBC0276
1385
1386

pBC0277
1387
1388

pBC0278
1389
1390

pBC0279
1391
1392

pBC0280
1393
1394

pBC0281
1395
1396

pBC0282
1397
1398

pBC0283
1399
1400

pBC0284
1401
1402

pBC0285
1403
1404

pBC0286
1405
1406

pBC0287
1407
1408

pBC0288
1409
1410

pBC0289
1411
1412

pBC0290
1413
1414

pBC0291
1415
1416

pBC0292
1417
1418

pBC0293
1419
1420

pBC0294
1421
1422

pBC0295
1423
1424

pBC0296
1425
1426

pBC0297
1427
1428

pBC0298
1429
1430

pBC0299
1431
1432

pBC0300
1433
1434

pBC0301
1435
1436

pBC0302
1437
1438

pBC0303
1439
1440

pBC0304
1441
1442

pBC0305
1443
1444

pBC0306
1445
1446

pBC0307
1447
1448

pBC0308
1449
1450

pBC0309
1451
1452

pBC0310
1453
1454

pBC0311
1455
1456

pBC0312
1457
1458

pBC0313
1459
1460

pBC0314
1461
1462

pBC0315
1463
1464

pBC0316
1465
1466

pBC0317
1467
1468

pBC0318
1469
1470

pBC0319
1471
1472

pBC0320
1473
1474

pBC0321
1475
1476

PBC0322
1477
1478

PBC0323
1479
1480

pNL0040
1481
1482

pNL0041
1483
1484

pNL0042
1485
1486

pNL0043
1487
1488























































































































































































































































































































































































































































































Example 24
Transfection of Mammalian Cells, Expression of FVIII-XTEN and Assessment of FVIII Activity
Mammalian cells, including but not limited to CHO, BHK, COS, and HEK293, are suitable for transformation with the vectors of the Examples, above, in order to express and recover FVIII-XTEN fusion protein. The following are details for methods used to express BDD FVIII and FVIII-XTEN fusion protein constructs pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0146, and pBC0149 by transient transfection, which includes electroporation and chemical (PEI) transfection methods.
Adherent HEK293 cells purchased from ATCC were revived in medium of vendor's recommendation and passaged for a few generations before multiple vials were frozen in the medium with 5% DMSO. One vial was revived and passaged one more time before transfection. The HEK293 cells were plated 1-2 days before transfection at a density of approximately 7×10 5 per ml in one T175 per transfection, using 35 ml medium. On the day of transfection the cells were trypsinized, detached and counted, then rinsed in the medium until an even cell suspension was achieved. The cells were counted and an appropriate volume of cells (based on cell count above) were transferred to 50 mL centrifuge tube, such that there were approximately 4×10 6 cells per transfection. Cells were centrifuged for 5 min at 500 RCF, the supernatant discarded, and the cells resuspended in 10 ml of D-PBS.
Electroporation:
For electroporation, an appropriate volume of resuspension buffer was added using a micropipette (supplied in the Neon™ Transfection System 100 μL Kit), such that 110 μl of buffer was available per transfection. Separate volumes of 110 μl of cell suspension were added to each Eppendorf tube containing 11 μl of plasmid DNA for each of the individual FVIII-XTEN constructs for a total of 6 μg (volume of DNA may be less, qs to 11 μl with sterile H 2 O). A Neon™ Transfection Device was used for transfection. The program was set to electroporate at 1100 v for a pulse width of 20 ms, for a total of two pulses. A Neon™ Tube (supplied in the Neon™ Transfection System 100 μL Kit) was placed into Neon™ Pipette Station. A volume of 3 mL of Electrolytic Buffer E2 (supplied in the Neon™ Transfection System 100 μL Kit) was added to the Neon™ Tube. Neon™ Pipettes and 1001 Neon™ Tips were used to electroporate 100 μl of cell-plasmid DNA mixture using the Neon™ Pipette Station. The electroporation was executed and when complete, the Neon™ Pipette was removed from the Station and the pipette with the transfected cells was used to transfer the cells, with a circular motion, into a 100 mm×20 mm petri plate containing 10 ml of Opti-MEM I Reduced-Serum Medium (1×, Invitrogen), such that transfected cells were evenly distributed on plate. The cells for each transfection were incubated at 37° C. for expression. On day 3 post-transfection, a 10% volume of salt solution of 10 mM Hepes, 5 mM CaCl 2 , and 4M NaCl was added to each cell culture and gently mixed for 30 minutes. Each cell culture was transferred to a 50 ml conical centrifuge tube and was centrifuged at 3000 rpm for 10 minutes at 4° C. The supernatants for each culture were placed into a new 50 ml conical tube and then split into aliquots of 5×1 ml in Eppendorf and 2×15 ml conical tubes for assay or were flash frozen before testing for expression of FVIII-XTEN in ELISA and performance in an FVIII activity assay, as described herein.
Chemical Transfection:
Chemical transfection can be accomplished using standard methods known in the art. In the present Example, PEI is utilized, as described.
Suspension 293 Cells are seeded the day before transfection at 7×10 5 cells/mL in sufficient Freestyle 293 (Invitrogen) medium to provide at least 30 ml working volume, and incubated at 37° C. On the day of transfection, an aliquot of 1.5 ml of the transfection medium is held at room temperature, to which 90 μL of 1 mg/ml PEI is added and vortexed briefly. A volume of 301 of DNA encoding the FVIII-XTEN_AE288 construct (concentration of 1 mg/ml) is added to the PEI solution, which is vortexed for 30 sec. The mixture is held at room temperature for 5-15 min. The DNA/PEI mixture is added to the HEK293 cells and the suspension is incubated at 37° C. using pre-established shake flask conditions. About four hours after the addition of the DNA/PEI mix, a 1× volume of expansion media is added and the cells incubated at 37° C. for 5 days. On the day of harvest, a 10% volume of salt solution of 10 mM Hepes, 5 mM CaCl 2 , and 4M NaCl is added to the cell culture and gently mixed for 30 minutes. The cell culture is transferred to a 50 ml conical centrifuge tube and is centrifuged at 4000 rpm for 10 minutes at 4° C. The supernatant is placed into a new 50 ml conical tube and then split into aliquots of 5×1 ml in Eppendorf and 2×15 ml conical tubes for assay or are flash frozen before testing for expression of FVIII-XTEN in ELISA and/or performance in an FVIII activity assay, as described herein.
Generation of Stable Pools and Cell Lines that Produce FVIII-XTEN
Stable pools are generated by culturing transfected cells for 3-5 weeks in medium containing selection antibiotics such as puromycin, with medium change every 2-3 days. Stable cells can be used for either production or generation of stable clones. For stable cell line selection during primary screening, cells from stable pools either from on-going passaging or revived from frozen vials are seeded in 96-well plates at a target density of 0.5 cell/well. About 1 week after seeding spent medium from wells with single cell cluster as observed under microscope are tested for expression of FVIII by activity assay or antigen measurement.
For additional rounds of screening, normalized numbers of cells are seeded in multi-well plates. Spent medium is harvested and tested for FVIII concentration by ELISA and FVIII activity assay. Cells would also be harvested from the plates and counted using Vi-Cell. Clones are ranked by (1) FVIII titers according to ELISA and activity; (2) ratios of ELISA titer/cell count and activity titer/cell count; and (3) integrity and homogeneity of products produced by the clones as measured by Western blots. A number of clones for each of the constructs are selected from the primary screening for additional rounds of screening.
For the second round of screening, cells in 96-well plates for the top clones selected from primary screening are first expanded in T25 flasks and then seeded in duplicate 24-well plates. Spent medium is collected from the plates for FVIII activity and antigen quantification and cells harvested and counted by Vi-Cell. Clones are ranked and then selected according to titers by ELISA and activity assay, ELISA titer/cell and activity titer/cell count ratios. Frozen vials are prepared for at least 5-10 clones and again these clones were screened and ranked according to titers by ELISA and activity, and ratios of ELISA titer/cell count and activity titer/cell count, and product integrity and homogeneity by Western blot, and 2-3 clones are selected for productivity evaluation in shake flasks. Final clones are selected based on specific productivity and product quality.
Production of FVIII-XTEN Secreted in Cell Culture Medium by Suspension 293 Stable Clones
HEK293 stable cell clones selected by the foregoing methods are seeded in shake flasks at 1-2×10 5 cells/ml in expression medium. Cell count, cell viability, FVIII activity and antigen expression titers are monitored daily. On the day when FVIII activity and antigen titers and product quality are optimal, the culture is harvested by either centrifugation/sterile filtration or depth filtration/sterile filtration. The filtrate is either used immediately for tangential flow filtration (TFF) processing and purification or stored in −80° C. freezer for TFF processing and purification later.
Example 25
Purification and Characterization of CFXTEN Constructs
Exemplary methods for the purification and characterization of CFXTEN constructs with one or more XTEN follow.
Purification of FVII-XTEN AE864 by FVIII Affinity Chromatography
CFXTEN containing supernatant is filtered using a Cuno ZetaPlus Biocap filter and a Cuno BioAssure capsule and subsequently concentrated by tangential flow filtration using a Millipore Pellicon 2 Mini cartridge with a 30,000 Da MWCO. Using the same tangential flow filtration cartridge the sample is diafiltered into 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0. FVIIISelect resin (GE 17-5450-01) selectively binds FVIII or B domain deleted FVIII using a 13 kDa recombinant protein ligand coupled to a chromatography resin. The resin is equilibrated with 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0 and the supernatant loaded. The column is washed with 20 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0, then is washed with 20 mM histidine, 20 mM calcium chloride, 1.0 M sodium chloride, and 0.02% Tween 80 at pH 7.0, and eluted with 20 mM histidine, 20 mM calcium chloride, 1.5 M sodium chloride, and 0.02% Tween 80 dissolved in 50% ethylene glycol at pH 7.0.
Concentration and Buffer Exchange by Tangential Flow Filtration and Diafiltration
Supernatant batches totaling at least 10 L in volume, from stable CHO cells lines expressing CFXTEN are filtered using a Cuno ZetaPlus Biocap filter and a Cuno BioAssure capsule. They are subsequently concentrated approximately 20-fold by tangential flow filtration using a Millipore Pellicon 2 Mini cartridge with a 30,000 Da MWCO. Using the same tangential flow filtration cartridge the sample is diafiltered with 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0 10 mM tris pH 7.5, 1 mM EDTA with 5 volumes worth of buffer exchange. Samples are divided into 50 ml aliquots and frozen at −80° C.
Purification of CFXTEN by Anion Exchange Chromatography
Using an Akta FPLC system the sample is purified using a SuperQ-650M column. The column is equilibrated into buffer A (0.02 mol/L imidazole, 0.02 mol/L glycine ethylester hydrochloride, 0.1 5 mol/L, NaCl, 2.5% glycerol, pH 6.9) and the sample loaded. The sample is eluted using buffer B (5 mmol/L histidine HCl (His/HCl), 1.15 mol/L NaCl, pH 7.0). The 215 nm chromatogram is used to monitor the elution profile. The eluted fractions are assayed for FVIII by ELISA, SDS-PAGE or activity assay. Peak fractions are pooled and stored or subjected to thrombin activation immediately (O'Brien et al., Blood (1990) 75:1664-1672). Fractions are assayed for FVIII activity using an aPTT based factor assay. A Bradford assay is performed to determine the total amount of protein in the load and elution fractions.
Purification of CFXTEN by Hydrophobic Interaction Chromatography
CFXTEN samples in Buffer A (50 mmol/l histidine, 1 mmol/l CaCl 2, 1 M NaCl, and 0.2 g/l Tween 80®, pH 7.0) are loaded onto a toyopearl ether 650M resin equilibrated in Buffer A. The column is washed with 10 column volumes of Buffer A to remove DNA, incorrectly folded forms and FVIII, and other contaminant proteins. The CFXTEN is eluted with Buffer B (25 mmol/l histidine, 0.5 mmol/l CaCl 2 and 0.4 mol/l NaCl, pH 7.0) as a single step elution (U.S. Pat. No. 6,005,082). Fractions are assayed for FVIII activity using an aPTT based factor assay. A Bradford assay is performed to determine the total amount of protein in the load and elution fractions.
Removal of Aggregated Protein from Monomeric CFXTEN with Anion Exchange Chromatography
Using an Akta FPLC system the sample is purified using a macrocap Q column. The column is equilibrated into buffer A (20 mM MES, 1 mM CaCl 2 , pH 7.0) and the sample is loaded. The sample is eluted using a linear gradient of 30% to 80% buffer B (20 mM MES, 1 mM CaCl 2 , pH 7.0+500 mM NaCl) over 20 column volumes. The 215 nm chromatogram is used to monitor the elution profile. The fractions corresponding to the early portion of the elution contain primarily monomeric protein, while the late portion of the elution contains primarily the aggregated species. Fractions from the macrocapQ column is analyzed via size exclusion chromatography with 60 cm BioSep G4000 column to determine which to pool to create an aggregate free sample.
Activation of FVIII by Thrombin
Purified FVIII in 5 mmol/L histidine HCl (His/HCl), 1.15 mol/L NaCl, pH 7.0 is treated with thrombin at a 1:4 ratio of units of human thrombin to units FVIII, and the sample is incubated at 37° C. for up to 2 hours. To monitor the activation process, aliquots of this sample are then withdrawn, and acetone precipitated by the addition of 4.5 vol ice-cold acetone. The sample is incubated on ice for 10 minutes, and the precipitate is collected by centrifugation at 13,000 g in a microfuge for 3 minutes. The acetone is removed, and the precipitate is resuspended in 30 μL SDS-PAGE reducing sample buffer and boiled for 2 minutes. Samples are then assayed by SDS-PAGE or western blot. The conversion of FVIII to FVIIIa is examined by looking for the conversion of the heavy chain into 40 and 50 kDa fragments and the conversion of the light chain into a 70 kDa fragment (O'Brien et al., Blood (1990) 75:1664-1672).
SEC Analysis of CFXTEN
FVII-XTEN purified by affinity and anion exchange chromatography is analyzed by size exclusion chromatography with 60 cm BioSep G4000 column. A monodispersed population with a hydrodynamic radius of ˜10 nm/apparent MW of ˜1.7 MDa (XTEN-288 fusion) or ˜12 nm/an apparent MW of 5.3 MDa (XTEN-864 fusion) is indicative of an aggregation-free sample. CFXTEN is expected to have an apparent molecular weight factor up to or about 8 (for an XTEN-288 fusion with FVIII) or up to or about −15 (for an XTEN-864 fusion with FVIII).
ELISA Based Concentration Determination of CFXTEN
The quantitative determination of factor VIII/CFXTEN antigen concentrations using the double antibody enzyme linked immuno-sorbent assay (ELISA) is performed using proven antibody pairings (VisuLize™ FVIII Antigen kit, Affinity Biologicals, Ontario Canada). Strip wells are pre-coated with sheep polyclonal antibody to human FVIII. Plasma samples are diluted and applied to the wells. The FVIII antigen that is present binds to the coated antibody. After washing away unbound material, peroxidase-labeled sheep detecting antibody is applied and allowed to bind to the captured FVIII. The wells are again washed and a solution of TMB (the peroxidase substrate tetramethylbenzidine) is applied and allowed to react for a fixed period of time. A blue color develops which changes to yellow upon quenching the reaction with acid. The color formed is measured spectrophotometrically in a microplate reader at 450 nm. The absorbance at 450 nm is directly proportional to the quantity of FVIII antigen captured onto the well. The assay is calibrated using either the calibrator plasma provided in the kit or by substituting a CFXTEN standard in an appropriate matrix.
Assessment of CFXTEN Activity Via a FXa Coupled Chromogenic Substrate Assay
Using the Chromogenix Coamatic Factor VIII (Chromogenix, cat#82258563) the activity of FVIII or CFXTEN comprising FVIII is assessed as follows. In the presence of calcium ions and phospholipids, factor X is activated to factor Xa by factor IXa. This activation is greatly stimulated by factor VIII which acts as a cofactor in this reaction. By using optimal amounts of Ca 2+ , phospholipid and factor IXa, and an excess of factor X, the rate of activation of factor X is linearly related to the amount of factor VIII. Factor Xa hydrolyses the chromogenic substrate S-2765 thus liberating the chromophoric group, pNA. The color is then read spectrophotometrically at 405 nm. The generated factor Xa and thus the intensity of color is proportional to the factor VIII activity in the sample. Hydrolysis of S-2765 by thrombin formed is prevented by the addition of the synthetic thrombin inhibitor 1-2581 together with the substrate. The activity of an unknown sample is determined by comparing final A405 of that sample to those from a standard curve constructed from known FVIII amounts. By also determining the amount of FVIII antigen present in the samples (via A280 or ELISA), a specific activity of a sample is determine to understand the relative potency of a particular preparation of FVIII. This enables the relative efficiency of different isolation strategies or construct designs for CFXTEN fusions to be assessed for activity and ranked.
aPTT Based Assays for CFXTEN Activity Determination
CFXTEN acts to replace FVIII in the intrinsic or contact activated coagulation pathway. The activity of this coagulation pathway is assessed using an activated partial thromboplastin time assay (aPTT). FVIII activity specifically is measured as follows: a standard curve is prepared by diluting normal control plasma (Pacific Hemostasis cat#100595) two-fold with FVIII deficient plasma (cat#100800) and then conducting 6.4-fold serial dilutions again with factor VIII deficient plasma. This creates a standard curve with points at 500, 130, 31, 7.8, 2.0, 0.5 and 0.1 IU/ml of activity, where one unit of activity is defined as the amount of FVIIIC activity in 1 ml of normal human plasma. A FVIII-deficient plasma also is included to determine the background level of activity in the null plasma. The sample is prepared by adding CFXTEN to FVIII deficient plasma at a ratio of 1:10 by volume. The samples is tested using an aPTT assay as follows. The samples are incubated at 37 C in a molecular devices plate reader spectrophotometer for 2 minutes at which point an equal volume of aPTT reagent (Pacific Hemostasis cat#100402) is added and an additional 3 minute 37 C incubation performed. After the incubation the assay is activated by adding one volume of calcium chloride (Pacific Hemostasis cat#100304). The turbidity is monitored at 450 nm for 5 minutes to create reaction profiles. The aPTT time, or time to onset of clotting activity, is defined as the first time where OD405 nm increased by 0.06 over baseline. A log—linear standard curve is created with the log of activity relating linearly to the aPTT time. From this the activity of the sample in the plate well is determined and then the activity in the sample is determined by multiplying by 11 to account for the dilution into the FVIII deficient plasma. By also determining the amount of FVIII antigen present in the samples (via A280 or ELISA), a specific activity of a sample can be determine to understand the relative potency of a particular preparation of FVIII. This enables the relative efficiency of different isolation strategies or construct designs for CFXTEN fusions to be ranked.
Western Blot Analysis of FVIII/FVIII-XTEN Expressed Proteins
Samples were run on a 8% homogeneous SDS gel and subsequently transferred to PVDF membrane. The samples in lanes 1-15 were: MW Standards, FVIII(42.5 ng), pBC0100B, pBC0114A, pBC0100, pBC0114, pBC0126, pBC0127 (8/5/11; #9), pBC0128, pBC0135, pBC0136, pBC0137, pBC0145, pBC0149, and pBC0146, respectively. The membrane was initially blocked with 5% milk then probed with anti-FVIII monoclonal antibody, GMA-012, specific to the A2 domain of the heavy chain (Ansong C, Miles S M, Fay P J. J Thromb Haemost. 2006 April; 4(4):842-7). Insertion of XTEN288 in the B-domain was observed for pBC0136 (lane 8, FIG. 22 ) and pBC0137 (lane 9, FIG. 22 ), whereas XTEN288 insertion at the C-terminus was observed for pBC0146 (lane 12, FIG. 22 ). All of the assayed FVIII-XTEN proteins revealed the presence of single chain protein with molecular weight of at least 21 kDa higher than that of pBC0114 base construct or FVIII standard. In addition, AE42 insertion was observed for pBC0135 (lane 7, FIG. 22 ) and pBC0149 (lane 11, FIG. 22 ) with the single chain running ˜5 kDa higher than that of pBC0114 base protein and heavy chain running at −5 kDa higher than 90 kDa band of the base protein.
Assay of Expressed FVIII by ELISA
To verify and quantitate the expression of FVIII-XTEN fusion proteins of the constructs by cell culture, an ELISA assay was established. Capture antibodies, either SAF8C-AP (Affinity Biologicals), or GMA-8002 (Green Mountain Antibodies), or GMA011 antibodies (Green Mountain Antibodies) for FVIII-LC ELISA) or by GMA016 antibodies were immobilized onto wells of an ELISA plate. The wells were then incubated with blocking buffer (1×PBS/3% BSA) to prevent non-specific binding of other proteins to the anti-FVIII antibody. FVIII standard dilutions (˜50 ng-0.024 ng range), quality controls, and cell culture media samples were then incubated for 1.5 h in the wells to allow binding of the expressed FVIII protein to the coated antibody. Wells were then washed extensively, and bound protein is incubated with anti-FVIII detection antibody, SAF8C-Biotinylated (Affinity Biologicals). Then streptavidin-HRP, which binds the biotin conjugated to the FVIII detection antibody, is added to the well and incubated for 1 h. Finally, OPD substrate is added to the wells and its hydrolysis by HRP enzyme is monitored with a plate reader at 490 nm wavelength. Concentrations of FVIII-containing samples were then calculated by comparing the colorimetric response at each culture dilution to a standard curve. The results, in Table 22, below, show that FVIII-XTEN of the various constructs are expressed at 0.4-1 μg/ml in the cell culture media. The results obtained by ELISA and the activity data indicate that FVIII-XTEN fusion proteins were very well expressed using the described transfection methods. Furthermore, under the experimental conditions, the results demonstrate that the specific activity values of the FVIII-XTEN proteins were similar or greater than that of pBC0114 base construct (expressing BDD FVIII) and support that XTEN insertion into the C-terminus or B-domain of FVIII results in preservation of FVIII protein function.
Chromogenic Activity Assay for CFXTEN Fusion Protein
BDD FVIII and CFXTEN fusion protein constructs pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0146, and pBC0149, in various configurations, including XTEN AE288 and AG288 inserted at the C-terminus of the FVIII BDD sequence and FVIII-XTEN fusion proteins with AE42 and AE288 inserted after residue 745 (or residue 743) and before residue 1640 (or residue 1638) of the B-domain (including constructs with the P1648 processing site mutated to alanine), were expressed in transiently transfected Freestyle 293 cells, as described above, and tested for procoagulant activity. The procoagulant activity of each of the FVIII-XTEN proteins present in cell culture medium was assessed using a Chromogenix Coamatic® Factor VIII assay, an assay in which the activation of factor X was linearly related to the amount of factor VIII in the sample. The assay was performed according to manufacturer's instructions using the end-point method, which was measured spectrophotometrically at OD405 nm. A standard curve was created using purified FVIII protein at concentrations of 250, 200, 150, 100, 75, 50, 37.5, 25, 12.5, 6.25, 3.125 and 1.56 mU/ml. Dilutions of factor VIII standard, quality controls, and samples were prepared with assay buffer and PEI culture medium to account for the effect of the medium in the assay performance. Positive controls consist of purified factor VIII protein at 20, 40, and 80 mU/ml concentrations and cell culture medium of pBC0114 FVIII base construct, lacking the XTEN insertions. Negative controls consisted of assay buffer or PEI culture medium alone. The cell culture media of the FVIII-XTEN constructs were obtained as described, above, and were tested in replicates at 1:50, 1:150, and 1:450 dilutions and the activity of each was calculated in U/ml. Each FVIII-XTEN construct exhibited procoagulant activity that was at least comparable, and in some cases greater than that of the base construct positive control, and support that under the conditions of the experiments, the linkage of XTEN, including AE288 or AG288, at the C-terminus of FVIII or insertion of XTEN, including AE42 or AE288 within the B-domain resulted in retention or even enhancement of FVIII procoagulant activity.





TABLE 22



Results of ELISA and Chromogenic FVIII activity assays







FVIII-


Specific


XTEN
Activity
Concentration
Activity


Construct
(IU/ml)
(μg/ml)
(IU/mg)
Description of Construct









pBC0114
3.0
0.6
5000
BDD FVIII base construct used for





XTEN insertions

pBC0146
7.4
0.6
12759
FVIII construct with XTEN AG288





inserted at the C-terminus of FVIII

pBC0145
3.1
0.6
4844
FVIII construct with XTEN AE288





inserted at the C-terminus of FVIII

pBC0135
4.0
1.0
4124
FVIII construct with XTEN AE42





inserted between residue 745 and 1640

pBC0149
4.9
0.9
5581
FVIII construct with XTEN AE42





inserted between residue 745 and 1640





and with Arg1648 to Ala mutation

pBC0136
2.7
0.4
7670
FVIII construct with XTEN AE288





inserted between residue 745 and 1640

pBC0137
1.9
0.3
6013
FVIII construct with XTEN AE288





inserted between residue 745 and 1640





and with Arg1648 to Ala mutation









































Coatest Assay for Cell Culture Sample Activity Assay Containing CFXTEN Fusion Protein
Using the Coatest assay, the activity of FVIII or CFXTEN comprising FVIII is assessed as follows.
Assay Matrix:
All wells in the same plate were adjusted to the same percentage of media to control for matrix effects. The test samples were diluted such that the OD405 reading would fall within the linear range of the standard. The range of concentrations for the FVIII standard was 100 mU/mL to 0.78 mU/mL, prepared by four-fold serial dilutions of the FVIII standard in 1× Coatest buffer (DiaPharma) plus the pre-determined percentage of culture media.
The Coatest SP FVIII (DiaPharma) reagent package includes the 10× Coatest buffer stock solution, factor IXa+factor X, phospholipid, CaCl 2 and substrate. The 1× Coatest solution was prepared by adding 9× volume of cold ddH 2 O to 1× volume of the stock. The cell culture media was then added to the prepared 1× solution at a pre-determined ratio to normalize the percentage of matrix in all test wells. Factor IXa+factor X, phospholipid, and substrate were reconstituted according to manufacturer's recommendations.
Coatest Assay Procedure:
Assay reagents were prepared and kept on ice until needed. 25 μl of the diluted test samples and standards were added to a 96 well plate in duplicate. 50 μl of phospholipid/factor IXa/factor X was added to each well and mixed by gently tapping the side of the plate. Plates were incubated at 37° C. for 5 min on a 37° C. plate heater. 25 μl of CaCl 2 was added to each well and mixed. The plates were incubated at 37° C. for 5 min on a plate heater. 50 μl of substrate was then added to each well, mixed, and the plates incubated at 37° C. for an additional 5-10 min until the top standard developed an OD405 reading of about 1.5. 25 μl of 20% acetic acid was added to each well with mixing to stop the reaction and wells were read at OD405 using a SpectraMAX® plus (Molecular Devices) spectrophotometer. Data analysis was performed using the SoftMax program (verion 5.2). The LLOQ varied per assay, but was generally 0.0039 IU/ml.
Results:
The data are presented in Tables 23-26. Table 23 presents results from CFXTEN fusion proteins with XTEN inserted in single sites chosed on the basis of criteria described herein, including Example 34. The pBC00114 FVIII positive control showed good expression and FVIII activity. Of the 106 single-XTEN fusion proteins assayed, 68% retained measurable FVIII activity, with 30% exhibiting 3+ to 4+activity in the coagulation assay. Thirty-one percent of the fusion proteins assayed had results below the limits of quantitation (which may be due to poor expression, reflected in the corresponding expression ELISA results). All four B-domain insertion constructs exhibited good activity, as did the C-terminal linked constructs, indicating that these are likely favorable insertion sites
The results of the single insertion site data guided the creation of XTEN constructs with 2 XTEN insertions, the results of which are presented in Table 24. Overall, the positivity rate was 67%, with 31% of fusion proteins exhibiting 3+ to 4+ activity in the coagulation assay.
The results of the foregoing data guided the creation of XTEN constructs with 3 XTEN insertions, the results of which are presented in Table 25. Overall, 92% of the samples had measurable FVIII activity, with fully 79% exhibiting 3+ to 4+ activity in the coagulation assay.
A limited number of constructs with 4 XTEN inserted in the A1, A2 and A3 domains were created and assayed, with 4 of 5 exhibiting FVIII activity (Table 26), suggesting that insertion of multiple XTEN does not compromise the ability of the resulting fusion proteins to retain FVIII activity.
Conclusions:
Under the conditions of the experiments, the results support that the criteria used to select XTEN insertion sites are valid, that insertion of one or more XTEN into the selected sites of FVIII is more likely than not to result in retention of procoagulant activity of the resulting CFXTEN molecule, and that insertion of three XTEN appears to result in a greater proportion of fusion proteins retaining high levels of FVIII procoagulant activity compared to single or double XTEN insertion constructs.





TABLE 23



Results of Coagulation Activity Assays for

CFXTEN comprising one XTEN







Insertion



Expression

Site
Domain
Construct
Activity
ELISA









pBC0114


+++
+++

3
A1
pBC0126
LLOQ*
LLOQ

3
A1
pBC0127
+
+

18
A1
pBC0165
++
++

22
A1
pBC0183
+++
++

26
A1
pBC0184
++
++

40
A1
pBC0166
++
++

60
A1
pBC0185
LLOQ
LLOQ

116
A1
pBC0167
LLOQ
LLOQ

130
A1
pBC0128
LLOQ
LLOQ

188
A1
pBC0168
++
++

216
A1
pBC0129
++
++

230
A1
pBC0169
LLOQ
LLOQ

333
A1
pBC0130
++
++

375
A2
pBC0131
LLOQ
+++

403
A2
pBC0132
++
++

442
A2
pBC0170
++
++

490
A2
pBC0133
+
++

518
A2
pBC0171
LLOQ
+

599
A2
pBC0134
++
++

713
A2
pBC0172
+
+++

745
B
pBC0135
+++
+++

745
B
pBC0149
+++
+++

745
B
pBC0136
++
++

745
B
pBC0137
+++
+++

1720
A3
pBC0138
+++
+++

1796
A3
pBC0139
+
++

1802
A3
pBC0140
+
++

1827
A3
pBC0173
LLOQ
LLOQ

1861
A3
pBC0174
LLOQ
LLOQ

1896
A3
pBC0175
LLOQ
LLOQ

1900
A3
pBC0176
+++
+++

1904
A3
pBC0177
+
+

1937
A3
pBC0178
LLOQ
LLOQ

2019
A3
pBC0141
LLOQ
+

2068
C1
pBC0179
++
++

2111
C1
pBC0180
LLOQ
LLOQ

2120
C1
pBC0142
LLOQ
+

2171
C2
pBC0143
++
+++

2188
C2
pBC0181
LLOQ
LLOQ

2227
C2
pBC0182
++
+++

2277
C2
pBC0144
++
++

2332
CT
pBC0145
+++
+++

2332
CT
pBC0146
+++
+++

403
A2
pSD0001
+++
+++

599
A2
pSD0002
+
+

403
A2
pSD0003
+++
+++

599
A2
pSD0004
+
+

745
B
pSD0005
+++
++

745
B
pSD0006
+++
+++

745
B
pSD0007
+++
++

745
B
pSD0008
+++
+++

1720
A3
pSD0009
+
+

1720
A3
pSD0010
++
++

2171
C2
pSD0011
+
++

2171
C2
pSD0012
+
++

2332
CT
pSD0013
+++
++

2332
CT
pSD0014
+++
+++

745
B
pSD0017
+++
+++

745
B
pSD0018
+++
+++

2332
CT
pSD0019
+++
+++

2332
CT
pSD0020
+++
+++

2332
CT
pSD0015
++
++

2332
CT
pSD0016
+++
+++

0
N-term
pSD0021
+
+

32
A1
pSD0022
+++
+++

65
A1
pSD0023
LLOQ
LLOQ

81
A1
pSD0024
LLOQ
LLOQ

119
A1
pSD0025
LLOQ
LLOQ

211
A1
pSD0026
+
+

220
A1
pSD0027
+
+

224
A1
pSD0028
+
+

336
A1
pSD0029
++
+++

339
A1
pSD0030
++
+++

378
A2
pSD0031
LLOQ
++

399
A2
pSD0032
++
++

409
A2
pSD0033
++
++

416
A2
pSD0034
+
+

487
A2
pSD0035
LLOQ
+

494
A2
pSD0036
LLOQ
+

500
A2
pSD0037
LLOQ
+

603
A2
pSD0038
+
+

1656
A3
pSD0039
+++
+++

1656
A3
pNL009**
++++
ND

1711
A3
pSD0040
++
+

1725
A3
pSD0041
LLOQ
++

1749
A3
pSD0042
LLOQ
LLOQ

1905
A3
pSD0043
++
++

1910
A3
pSD0044
+
+

1900
A3
pSD0062
++
++

1900
A3
pSD0063
+++
++

18
A1
pSD0045
+++
+++

18
A1
pSD0046
+++
+++

22
A1
pSD0047
LLOQ
LLOQ

22
A1
pSD0048
LLOQ
LLOQ

26
A1
pSD0049
+++
+++

26
A1
pSD0050
+++
+++

40
A1
pSD0051
+++
+++

40
A1
pSD0052
+++
+++

216
A1
pSD0053
LLOQ
LLOQ

216
A1
pSD0054
LLOQ
LLOQ

375
A2
pSD0055
LLOQ
+

442
A2
pSD0056
LLOQ
LLOQ

442
A2
pSD0057
LLOQ
LLOQ

1796
A3
pSD0058
LLOQ
LLOQ

1796
A3
pSD0059
+
+

1802
A3
pSD0060
+
+

1802
A3
pSD0061
LLOQ
LLOQ



*LLOQ: below the limits of quantitation

**pNL009 includes a deletion of 745-1656










































































































































TABLE 24



Results of Coagulation Activity Assays for

CFXTEN comprising two XTEN





Insertion 1
Insertion 2








Insertion

Insertion










Site
Domain
Site
Domain
Construct
Activity










745
B
2332
CT
LSD0001.002
+++

745
B
2332
CT
LSD0001.005
+++

745
B
2332
CT
LSD0001.006
+++

745
B
2332
CT
LSD0001.011
+++

745
B
2332
CT
LSD0001.012
+++

745
B
2332
CT
LSD0001.013
+++

745
B
2332
CT
LSD0001.016
+++

745
B
2332
CT
LSD0001.021
+++

745
B
2332
CT
LSD0002.001
+++

745
B
2332
CT
LSD0002.002
+++

745
B
2332
CT
LSD0002.014
+++

745
B
2332
CT
LSD0003.004
+++

745
B
2332
CT
LSD0003.006
+++

745
B
2332
CT
LSD0003.009
+++

745
B
2332
CT
LSD0003.014
+

745
B
2332
CT
LSD0004.010
+++

745
B
2332
CT
LSD0004.011
LLOQ

745
B
2332
CT
LSD0004.014
+++

745
B
2332
CT
LSD0004.016
+++

745
B
2332
CT
LSD0004.022
+++

745
B
2332
CT
LSD0003.016
+++

0745
B
2332
CT
pNL006
+++

0745
B
2332
CT
pNL007
+++

0745
B
2332
CT
pNL008
++

1656
a3
2332
CT
pNL010
+++

26
A1
403
A2
LSD0005.002
++

26
A1
403
A2
LSD0005.004
++

40
A1
403
A2
LSD0005.005
++

40
A1
403
A2
LSD0005.011
++

18
A1
403
A2
LSD0005.018
++

26
A1
599
A2
LSD0006.002
+

40
A1
599
A2
LSD0006.005
++

40
A1
599
A2
LSD0006.007
++

40
A1
599
A2
LSD0006.011
+++

40
A1
403
A2
LSD0007.002
+

40
A1
403
A2
LSD0007.004
+

26
A1
403
A2
LSD0007.013
++

26
A1
599
A2
LSD0008.001
++

40
A1
599
A2
LSD0008.002
++

26
A1
599
A2
LSD0008.006
+

18
A1
599
A2
LSD0008.009
++

40
A1
599
A2
LSD0008.017
+

745
B
2332
CT
LSD0002.025
+++

745
B
2332
CT
LSD0002.013
+++

745
B
2332
CT
LSD0003.025
+++

745
B
2332
CT
LSD0004.025
+++

745
B
2332
CT
LSD0003.005
++

26
A1
403
A2
LSD0007.008
++

1720
A3
1900
A3
LSD0044.002
LLOQ

1725
A3
1900
A3
LSD0044.005
LLOQ

1720
A3
1900
A3
LSD0044.039
LLOQ

1711
A3
1905
A3
LSD0044.022
LLOQ

1720
A3
1905
A3
LSD0044.003
LLOQ

1725
A3
1905
A3
LSD0044.001
LLOQ

1656
A3
26
A1
LSD0038.001
++

1656
A3
18
A1
LSD0038.003
++

1656
A3
18
A1
LSD0038.008
+++

1656
A3
40
A1
LSD0038.012
++

1656
A3
40
A1
LSD0038.013
++

1656
A3
26
A1
LSD0038.015
++

1656
A3
399
A2
LSD0039.001
+

1656
A3
403
A2
LSD0039.003
++

1656
A3
403
A2
LSD0039.010
++

1656
A3
1725
A3
LSD0045.001
+

1656
A3
1720
A3
LSD0045.002
++

1900
A3
18
A1
LSD0042.014
+

1900
A3
18
A1
LSD0042.023
+

1900
A3
26
A1
LSD0042.006
+

1900
A3
26
A1
LSD0042.013
++

1900
A3
40
A1
LSD0042.001
+

1900
A3
40
A1
LSD0042.039
+

1900
A3
26
A1
LSD0042.047
+

1905
A3
18
A1
LSD0042.003
+

1905
A3
40
A1
LSD0042.004
LLOQ

1905
A3
26
A1
LSD0042.008
LLOQ

1905
A3
26
A1
LSD0042.038
LLOQ

1905
A3
40
A1
LSD0042.082
LLOQ

1910
A3
26
A1
LSD0042.040
LLOQ

18
A1
399
A2
LSD0037.002
++

26
A1
399
A2
LSD0037.009
+

40
A1
399
A2
LSD0037.011
++

18
A1
403
A2
LSD0047.002
++

18
A1
403
A2
LSD0047.005
+

18
A1
403
A2
LSD0048.007
+

1656
A3
1900
A3
LSD0046.001
++

1656
A3
1900
A3
LSD0046.002
+

1656
A3
1905
A3
LSD0046.003
+

1711
A3
40
A1
LSD0040.011
LLOQ

1711
A3
26
A1
LSD0040.042
LLOQ

1720
A3
26
A1
LSD0040.002
+

1720
A3
40
A1
LSD0040.008
+

1720
A3
18
A1
LSD0040.021
+

1720
A3
26
A1
LSD0040.037
LLOQ

1720
A3
18
A1
LSD0040.046
+

1725
A3
26
A1
LSD0040.003
LLOQ

1725
A3
40
A1
LSD0040.006
LLOQ

1725
A3
26
A1
LSD0040.007
LLOQ

1725
A3
18
A1
LSD0040.010
LLOQ

1725
A3
40
A1
LSD0040.039
LLOQ

1725
A3
18
A1
LSD0040.052
+

1720
A3
403
A2
LSD0041.001
+

1720
A3
399
A2
LSD0041.004
LLOQ

1711
A3
403
A2
LSD0041.006
LLOQ

1720
A3
403
A2
LSD0041.008
LLOQ

1725
A3
403
A2
LSD0041.010
LLOQ

1725
A3
403
A2
LSD0041.014
LLOQ

1725
A3
399
A2
LSD0041.016
LLOQ

1711
A3
403
A2
LSD0041.035
LLOQ

1900
A3
399
A2
LSD0043.001
LLOQ

1900
A3
403
A2
LSD0043.002
LLOQ

1905
A3
403
A2
LSD0043.005
LLOQ

1900
A3
399
A2
LSD0043.006
LLOQ

1900
A3
403
A2
LSD0043.007
LLOQ

1900
A3
403
A2
LSD0043.008
LLOQ

1905
A3
399
A2
LSD0043.015
LLOQ

1905
A3
403
A2
LSD0043.029
LLOQ

1910
A3
403
A2
LSD0043.043
LLOQ


























































































































































TABLE 25



Results of Coagulation Activity Assays for CFXTEN comprising three XTEN







Insertion 1
Insertion 2
Insertion 3












Insertion Site
Domain
Insertion Site
Domain
Insertion Site
Domain
Construct
Activity












26
A1
403
A2
1656
A3
pSD0077
+++

26
A1
403
A2
1720
A3
pSD0078
++

26
A1
403
A2
1900
A3
pSD0079
++

26
A1
1656
A3
1720
A3
pSD0080
+++

26
A1
1656
A3
1900
A3
pSD0081
LLOQ

26
A1
1720
A3
1900
A3
pSD0082
+

403
A2
1656
A3
1720
A3
pSD0083
+++

403
A2
1656
A3
1900
A3
pSD0084
+++

403
A2
1720
A3
1900
A3
pSD0085
+

1656
A3
1720
A3
1900
A3
pSD0086
+++

18
A1
745
B
2332
CT
LSD0049.002
+++

26
A1
745
B
2332
CT
LSD0049.008
+++

26
A1
745
B
2332
CT
LSD0049.011
+++

40
A1
745
B
2332
CT
LSD0049.012
+++

40
A1
745
B
2332
CT
LSD0049.020
+++

18
A1
745
B
2332
CT
LSD0049.021
+++

40
A1
745
B
2332
CT
LSD0050.002
+++

18
A1
745
B
2332
CT
LSD0050.003
+++

26
A1
745
B
2332
CT
LSD0050.007
LLOQ

18
A1
745
B
2332
CT
LSD0050.010
+++

26
A1
745
B
2332
CT
LSD0050.012
+++

40
A1
745
B
2332
CT
LSD0050.014
+++

403
A2
745
B
2332
CT
LSD0051.002
+++

399
A2
745
B
2332
CT
LSD0051.003
+++

403
A2
745
B
2332
CT
LSD0052.001
+++

399
A2
745
B
2332
CT
LSD0052.003
+++

1725
A3
745
B
2332
CT
LSD0053.021
LLOQ

1720
A3
745
B
2332
CT
LSD0053.022
+++

1711
A3
745
B
2332
CT
LSD0053.024
+++

1720
A3
745
B
2332
CT
LSD0054.021
+++

1711
A3
745
B
2332
CT
LSD0054.025
+++

1725
A3
745
B
2332
CT
LSD0054.026
+++

1900
A3
745
B
2332
CT
LSD0055.021
+++

1905
A3
745
B
2332
CT
LSD0055.022
+++

1900
A3
745
B
2332
CT
LSD0055.026
+++

1900
A3
745
B
2332
CT
LSD0056.021
+++

1900
A3
745
B
2332
CT
LSD0056.024
+++

1910
A3
745
B
2332
CT
LSD0056.025
+++

0745
B
1900
A3
2332
CT
pBC0294*


0745
B
1900
A3
2332
CT
pBC0295*


0745
B
1900
A3
2332
CT
pBC0296*


0745
B
1900
A3
2332
CT
pBC0297*


0745
B
1900
A3
2332
CT
pBC0298*


0745
B
1900
A3
2332
CT
pBC0299*


0745
B
1900
A3
2332
CT
pBC0300*


0745
B
1900
A3
2332
CT
pBC0301*


0745
B
1900
A3
2332
CT
pBC0302*


0745
B
1900
A3
2332
CT
pBC0303*


0745
B
1900
A3
2332
CT
pBC0304*


0745
B
1900
A3
2332
CT
pBC0305*


0745
B
1900
A3
2332
CT
pBC0306*


0745
B
1900
A3
2332
CT
pBC0307*


0745
B
1900
A3
2332
CT
pBC0308*


0745
B
1900
A3
2332
CT
pBC0309*


0745
B
1900
A3
2332
CT
pBC0310*


0745
B
1900
A3
2332
CT
pBC0311*


0745
B
1900
A3
2332
CT
pBC0312*


0745
B
1900
A3
2332
CT
pBC0313*


0745
B
1900
A3
2332
CT
pBC0314*


0745
B
1900
A3
2332
CT
pBC0315*


0745
B
1900
A3
2332
CT
pBC0316*


0745
B
1900
A3
2332
CT
pBC0317*


0745
B
1900
A3
2332
CT
pBC0318*


0745
B
1900
A3
2332
CT
pBC0319*


0745
B
1900
A3
2332
CT
pBC0320*


0018
A1
0745
B
2332
CT
pBC0269*


0403
A2
0745
B
2332
CT
pBC0270*


1720
A3
0745
B
2332
CT
pBC0271*


1900
A3
0745
B
2332
CT
pBC0272*


0403
A2
0745
B
2332
CT
pBC0273*


1720
A3
0745
B
2332
CT
pBC0274*


1900
A3
0745
B
2332
CT
pBC0275*


0018
A1
0745
B
2332
CT
pBC0276*


0403
A2
0745
B
2332
CT
pBC0277*


1720
A3
0745
B
2332
CT
pBC0278*


1900
A3
0745
B
2332
CT
pBC0279*



*Construct with R1648A mutation











































































































TABLE 26



Results of Coagulation Activity Assays

for CFXTEN comprising four XTEN










XTEN Insert 1
XTEN Insert 2
XTEN Insert 3
XTEN Insert 4
XTEN Insert 5
XTEN Insert 6
Construct ID
Activity












26
403
1656
1720
—
—
pSD0087
+++

26
403
1656
1900
—
—
pSD0088
+++

26
403
1720
1900
—
—
pSD0089
LLOQ

26
1656
1720
1900
—
—
pSD0090
++

403
1656
1720
1900
—
—
pSD0091
++

0040
0403
745
2332
—
—
LSD0058.006*
++

0018
0409
745
2332
—
—
LSD0059.002*
+

0040
0409
745
2332
—
—
LSD0059.006*
+

0040
0409
745
2332
—
—
LSD0060.001*
+

0018
0409
745
2332
—
—
LSD0060.003*
+

0040
1720
745
2332
—
—
LSD0061.002*
+

0026
1720
745
2332
—
—
LSD0061.007*
++

0018
1720
745
2332
—
—
LSD0061.008*
++

0018
1720
745
2332
—
—
LSD0061.012*
++

0018
1720
745
2332
—
—
LSD0062.001*
++

0026
1720
745
2332
—
—
LSD0062.002*
++

0018
1720
745
2332
—
—
LSD0062.006*
++

0018
1900
745
2332
—
—
LSD0063.001*
++

0018
1900
745
2332
—
—
LSD0064.017*
++

0026
1900
745
2332
—
—
LSD0064.020*
++

0040
1900
745
2332
—
—
LSD0064.021*
++

0040
1905
745
2332
—
—
LSD0065.001*
+

0018
1905
745
2332
—
—
LSD0065.014*
+

0040
1905
745
2332
—
—
LSD0066.001*
+

0026
1905
745
2332
—
—
LSD0066.002*
+

0018
1905
745
2332
—
—
LSD0066.009*
++

0018
1905
745
2332
—
—
LSD0066.011*
++

0018
1910
745
2332
—
—
LSD0067.004*
++

0018
1910
745
2332
—
—
LSD0067.005*
+

0040
1910
745
2332
—
—
LSD0067.006*
+

0026
1910
745
2332
—
—
LSD0067.008*
+

0018
1910
745
2332
—
—
LSD0068.001*
+

0026
1910
745
2332
—
—
LSD0068.002*
+

0040
1910
745
2332
—
—
LSD0068.005*
+

0018
1910
745
2332
—
—
LSD0068.010*
++

0409
1720
745
2332
—
—
LSD0069.004*
+

0403
1720
745
2332
—
—
LSD0069.008*
+

0409
1720
745
2332
—
—
LSD0070.003*
+

0403
1720
745
2332
—
—
LSD0070.004*
++

0403
1720
745
2332
—
—
LSD0070.005*
++

0403
1900
745
2332
—
—
LSD0071.001*
++

0403
1900
745
2332
—
—
LSD0071.002*
+

0409
1900
745
2332
—
—
LSD0071.008*
++

0403
1900
745
2332
—
—
LSD0072.001*
++

0403
1900
745
2332
—
—
LSD0072.002*
+

0409
1900
745
2332
—
—
LSD0072.003*
+

0409
1905
745
2332
—
—
LSD0073.002*
+

0403
1905
745
2332
—
—
LSD0073.004*
+

0403
1905
745
2332
—
—
LSD0073.006*
+

0403
1905
745
2332
—
—
LSD0074.007*
++

0409
1905
745
2332
—
—
LSD0074.010*
+

0403
1905
745
2332
—
—
LSD0074.011*
+

0409
1910
745
2332
—
—
LSD0075.004*
+

0403
1910
745
2332
—
—
LSD0075.007*
+

0403
1910
745
2332
—
—
LSD0076.002*
+

0403
1910
745
2332
—
—
LSD0076.003*
+

0403
1910
745
2332
—
—
pSD0093*
+

1720
1900
745
2332
—
—
pSD0094*
++

1720
1905
745
2332
—
—
pSD0095*
+

1720
1910
745
2332
—
—
pSD0097*
+

1720
1910
745
2332
—
—
pSD0098*
+

0403
1656
1720
2332
—
—
pNL0022
+

0403
1656
1900
2332
—
—
pNL0023
+

0403
1720
1900
2332
—
—
pNL0024
LLOQ

1656
1720
1900
2332
—
—
pNL0025
+

0018
0403
1656
2332
—
—
pBC0247
++

0018
0403
1720
2332
—
—
pBC0248
+

0018
0403
1900
2332
—
—
pBC0249
+

0018
1656
1720
2332
—
—
pBC0250
+

0018
1656
1900
2332
—
—
pBC0251
++

0018
1720
1900
2332
—
—
pBC0252
LLOQ

0018
0403
0745
2332
—
—
LSD57.005
++

0018
0745
1720
2332
—
—
LSD62.001
++

0018
0745
1900
2332
—
—
pBC0262
++

0403
0745
1720
2332
—
—
LSD70.004
+

0403
0745
1900
2332
—
—
pBC0266
+

0745
1720
1900
2332
—
—
pBC0268
+

0188
1900
0745
2332
—
—
pCS0001*
ND

0599
1900
0745
2332
—
—
pCS0002*
ND

2068
1900
0745
2332
—
—
pCS0003*
ND

2171
1900
0745
2332
—
—
pCS0004*
ND

2227
1900
0745
2332
—
—
pCS0005*
ND

2277
1900
0745
2332
—
—
pCS0006*
ND

0403
1656
1720
1900
2332
—
pNL0030
LLOQ

0018
0403
1656
1720
2332
—
pBC0253
+

0018
0403
1656
1900
2332
—
pBC0254
+

0018
0403
1720
1900
2332
—
pBC0255
LLOQ

0018
1656
1720
1900
2332
—
pBC0256
+

0018
0403
0745
1720
2332
—
pBC0259*
+

0018
0403
0745
1900
2332
—
pBC0260*
+

0018
0745
1720
1900
2332
—
pBC0263
+

0403
0745
1720
1900
2332
—
pBC0267
LLOQ

0018
0403
1656
1720
1900
2332
pBC0257
LLOQ

0018
0403
0745
1720
1900
2332
pBC0264
LLOQ



*Construct with R1648A mutation





















































































































Example 26
Determination of XTEN Radii and Related Parameters
In order to quantify the hydrodynamic radii of the XTEN components of CFXTEN fusion proteins and how the value of multiple XTEN versus single XTEN varies, a series of formulae were created based on empirically-derived data from size exclusion chromatography assays of various fusion proteins comprising one or more XTEN. It is believed that the incorporation of multiple XTEN into a CFXTEN provides a higher total hydrodynamic radius of the XTEN component compared to CFXTEN with fewer XTEN yet having approximately the same total of XTEN amino acids. The maximum radius of a single XTEN polypeptide is calculated (hereinafter “XTEN Radius”) according to the formula given by Equation II:
XTEN Radius=(√XTEN length 0.2037)+3.4627 II

The sum of the maximum of the XTEN Radii for all XTEN segments in a CFXTEN is calculated (hereinafter “Sum XTEN Radii”) according to the formula given by Equation III:








Sum





XTEN





Radii

=


∑

i
=
1

m



XTEN






Radius
i






III








wherein: n=the number of XTEN segments
and i is an iterator




The ratio of the SUM XTEN Radii of a CFXTEN comprising multiple XTEN to that of an XTEN Radius for a single XTEN of an equivalent length (in total amino acid residues to that of the CFXTEN) is calculated (hereinafter “Ratio XTEN Radii”) according to the formula given by Equation IV:








Ratio





XTEN





Radii

=



∑

i
=
1

n



XTEN






Radius
i





(




∑

i
=
1

n



XTEN






Length
i




*
0.2037

)

+
3.4627





IV








wherein: n=the number of XTEN segments
and i is an iterator




Results:
Equation II was applied to XTEN of lengths 144, 288, 576 and 864. The results are presented in Table 27. Equation IV was applied to various CFXTEN fusion proteins described herein with two, three, or four XTEN. The Ratio of XTEN Radii has a value of 1 for all CFXTEN that contain a single XTEN. The Ratio XTEN Radii are presented in Table 28. The Ratio of XTEN Radii for pSD0092, which contains 5 XTEN insertions, has a value of 3.31. Collectively, the results indicate that the inclusion of multiple XTEN increases the Ratio XTEN Radii to values greater than 2, with four insertions resulting in higher values than three insertions.





TABLE 27



Results of Radii Calculations for

CFXTEN comprising XTEN






XEN Length
XTEN Radius









42
4.8


144
5.9


288
6.9


576
8.4


864
9.5



































TABLE 28



Results of Radii Calculations for CFXTEN comprising XTEN








Insertion 1
Insertion 2
Insertion 3
Insertion 4

Ratio












Insert

Insert

Insert

Insert


XTEN

Site
Domain
Site
Domain
Site
Domain
Site
Domain
Construct
Radii














40
A1






pBC0166
1.00

745
B
2332
CT




LSD0001.002
1.67

745
B
2332
CT




LSD0001.005
1.71

745
B
2332
CT




LSD0001.006
1.71

745
B
2332
CT




LSD0001.011
1.71

745
B
2332
CT




LSD0001.012
1.71

745
B
2332
CT




LSD0001.013
1.67

745
B
2332
CT




LSD0001.016
1.67

745
B
2332
CT




LSD0001.021
1.67

745
B
2332
CT




LSD0002.001
1.67

745
B
2332
CT




LSD0002.002
1.67

745
B
2332
CT




LSD0002.004
1.71

745
B
2332
CT




LSD0002.008
1.67

745
B
2332
CT




LSD0002.014
1.67

745
B
2332
CT




LSD0003.001
1.67

745
B
2332
CT




LSD0003.004
1.66

745
B
2332
CT




LSD0003.006
1.67

745
B
2332
CT




LSD0003.009
1.67

745
B
2332
CT




LSD0003.014
1.66

745
B
2332
CT




LSD0003.018
1.67

745
B
2332
CT




LSD0004.010
1.66

745
B
2332
CT




LSD0004.011
1.67

745
B
2332
CT




LSD0004.014
1.66

745
B
2332
CT




LSD0004.016
1.66

745
B
2332
CT




LSD0004.022
1.66

745
B
2332
CT




LSD0003.016
1.67

26
A1
403
A2




LSD0005.002
1.71

26
A1
403
A2




LSD0005.004
1.71

40
A1
403
A2




LSD0005.005
1.71

40
A1
403
A2




LSD0005.011
1.71

18
A1
403
A2




LSD0005.018
1.71

26
A1
599
A2




LSD0006.002
1.71

40
A1
599
A2




LSD0006.005
1.71

40
A1
599
A2




LSD0006.007
1.71

40
A1
599
A2




LSD0006.011
1.71

40
A1
403
A2




LSD0007.002
1.71

40
A1
403
A2




LSD0007.004
1.71

26
A1
403
A2




LSD0007.013
1.71

26
A1
599
A2




LSD0008.001
1.71

40
A1
599
A2




LSD0008.002
1.71

26
A1
599
A2




LSD0008.006
1.71

18
A1
599
A2




LSD0008.009
1.71

40
A1
599
A2




LSD0008.017
1.71

745
B
2332
CT




LSD0002.025
1.71

745
B
2332
CT




LSD0002.013
1.67

745
B
2332
CT




LSD0003.025
1.67

745
B
2332
CT




LSD0004.025
1.67

745
B
2332
CT




LSD0003.005
1.66

26
A1
403
A2




LSD0007.008
1.71

1720
A3
1900
A3




LSD0044.002
1.71

1725
A3
1900
A3




LSD0044.005
1.71

1720
A3
1900
A3




LSD0044.039
1.71

1711
A3
1905
A3




LSD0044.022
1.71

1720
A3
1905
A3




LSD0044.003
1.71

1725
A3
1905
A3




LSD0044.001
1.71

1656
A3
26
A1




LSD0038.001
1.71

1656
A3
18
A1




LSD0038.003
1.71

1656
A3
18
A1




LSD0038.008
1.71

1656
A3
40
A1




LSD0038.012
1.71

1656
A3
40
A1




LSD0038.013
1.71

1656
A3
26
A1




LSD0038.015
1.71

1656
A3
399
A2




LSD0039.001
1.71

1656
A3
403
A2




LSD0039.003
1.71

1656
A3
403
A2




LSD0039.010
1.71

1656
A3
1725
A3




LSD0045.001
1.71

1656
A3
1720
A3




LSD0045.002
1.71

1900
A3
18
A1




LSD0042.014
1.71

1900
A3
18
A1




LSD0042.023
1.71

1900
A3
26
A1




LSD0042.006
1.71

1900
A3
26
A1




LSD0042.013
1.71

1900
A3
40
A1




LSD0042.001
1.71

1900
A3
40
A1




LSD0042.039
1.71

1900
A3
26
A1




LSD0042.047
1.71

1905
A3
18
A1




LSD0042.003
1.71

1905
A3
40
A1




LSD0042.004
1.71

1905
A3
26
A1




LSD0042.008
1.71

1905
A3
26
A1




LSD0042.038
1.71

1905
A3
40
A1




LSD0042.082
1.71

1910
A3
26
A1




LSD0042.040
1.71

18
A1
399
A2




LSD0037.002
1.71

26
A1
399
A2




LSD0037.009
1.71

40
A1
399
A2




LSD0037.011
1.71

18
A1
403
A2




LSD0047.002
1.71

18
A1
403
A2




LSD0047.005
1.71

18
A1
403
A2




LSD0048.007
1.71

1656
A3
1900
A3




LSD0046.001
1.71

1656
A3
1900
A3




LSD0046.002
1.71

1656
A3
1905
A3




LSD0046.003
1.71

1711
A3
40
A1




LSD0040.011
1.71

1711
A3
26
A1




LSD0040.042
1.71

1720
A3
26
A1




LSD0040.002
1.71

1720
A3
40
A1




LSD0040.008
1.71

1720
A3
18
A1




LSD0040.021
1.71

1720
A3
26
A1




LSD0040.037
1.71

1720
A3
18
A1




LSD0040.046
1.71

1725
A3
26
A1




LSD0040.003
1.71

1725
A3
40
A1




LSD0040.006
1.71

1725
A3
26
A1




LSD0040.007
1.71

1725
A3
18
A1




LSD0040.010
1.71

1725
A3
40
A1




LSD0040.039
1.71

1725
A3
18
A1




LSD0040.052
1.71

1720
A3
403
A2




LSD0041.001
1.71

1720
A3
399
A2




LSD0041.004
1.71

1711
A3
403
A2




LSD0041.006
1.71

1720
A3
403
A2




LSD0041.008
1.71

1725
A3
403
A2




LSD0041.010
1.71

1725
A3
403
A2




LSD0041.014
1.71

1725
A3
399
A2




LSD0041.016
1.71

1711
A3
403
A2




LSD0041.035
1.71

1900
A3
399
A2




LSD0043.001
1.71

1900
A3
403
A2




LSD0043.002
1.71

1905
A3
403
A2




LSD0043.005
1.71

1900
A3
399
A2




LSD0043.006
1.71

1900
A3
403
A2




LSD0043.007
1.71

1900
A3
403
A2




LSD0043.008
1.71

1905
A3
399
A2




LSD0043.015
1.71

1905
A3
403
A2




LSD0043.029
1.71

1910
A3
403
A2




LSD0043.043
1.71

26
A1
403
A2
1656
A3


pS0077
2.30

26
A1
403
A2
1720
A3


pS0078
2.30

26
A1
403
A2
1900
A3


pS0079
2.30

26
A1
1656
A3
1720
A3


pS0080
2.30

26
A1
1656
A3
1900
A3


pS0081
2.30

26
A1
1720
A3
1900
A3


pS0082
2.30

403
A2
1656
A3
1720
A3


pS0083
2.30

403
A2
1656
A3
1900
A3


pS0084
2.30

403
A2
1720
A3
1900
A3


pS0085
2.30

1656
A3
1720
A3
1900
A3


pS0086
2.30

26
A1
403
A2
1656
A3
1720
A3
pS0087
2.83

26
A1
403
A2
1656
A3
1900
A3
pS0088
2.83

26
A1
403
A2
1720
A3
1900
A3
pS0089
2.83

26
A1
1656
A3
1720
A3
1900
A3
pS0090
2.83

403
A2
1656
A3
1720
A3
1900
A3
pS0091
2.83

26
A1
403
A2
1656
A3
1720
A3
pS0092
2.83

18
A1
745
B
2332
CT


LSD0049.002
2.24

26
A1
745
B
2332
CT


LSD0049.008
2.24

26
A1
745
B
2332
CT


LSD0049.011
2.24

40
A1
745
B
2332
CT


LSD0049.012
2.24

40
A1
745
B
2332
CT


LSD0049.020
2.24

18
A1
745
B
2332
CT


LSD0049.021
2.24

40
A1
745
B
2332
CT


LSD0050.002
2.24

18
A1
745
B
2332
CT


LSD0050.003
2.24

26
A1
745
B
2332
CT


LSD0050.007
2.24

18
A1
745
B
2332
CT


LSD0050.010
2.24

26
A1
745
B
2332
CT


LSD0050.012
2.24

40
A1
745
B
2332
CT


LSD0050.014
2.24

403
A2
745
B
2332
CT


LSD0050.002
2.24

399
A2
745
B
2332
CT


LSD0050.003
2.24

403
A2
745
B
2332
CT


LSD0050.001
2.24

399
A2
745
B
2332
CT


LSD0050.003
2.24

1725
A3
745
B
2332
CT


LSD0050.021
2.24

1720
A3
745
B
2332
CT


LSD0050.022
2.24

1711
A3
745
B
2332
CT


LSD0051.024
2.24

1720
A3
745
B
2332
CT


LSD0051.021
2.24

1711
A3
745
B
2332
CT


LSD0052.025
2.24

1725
A3
745
B
2332
CT


LSD0052.026
2.24
























































































































































































Example 27
Binding Interference of FVIII-XTEN to Anti-FVIII Antibody
The ability of XTEN inserted into different locations of CFXTEN fusion proteins to affect the binding of anti-FVIII antibodies was determined by sandwich ELISA assays. Two anti-FVIII antibodies; i.e. GMA-8021 (Green Mountain Antibodies, Burlington, Vt.) and ESH8 (American Diagnostica Inc., Stamford, Conn.), that bind to the A2 and C2 domains, respectively were utilized as capture antibodies. A non-XTEN containing FVIII-His-Myc protein was used as a calibration standard and positive control for all ELISAs. Ten CFXTEN fusion proteins with single XTEN insertions in either the A1, A2 or A3 domains were created that additionally contained His and Myc affinity tags. The protein concentrations of each test sample was normalized to 100% based on an anti-His capture-anti-Myc detection ELISA run concurrently on the same plate as the anti-FVIII antibody capture-anti-Myc detection ELISA.
Briefly, appropriate wells on a 96-well plate were coated with GMA-8021, ESH8 or anti-His antibody overnight at 4° C., then were washed and blocked with BSA. Equal volumes of the respective control or fusion proteins were introduced into duplicate wells and allowed to interact with coated GMA-8021, ESH8 or anti-His antibody for 2 h at room temperature. After incubation, unbound material was washed away and a rabbit anti-Myc detection antibody was added and incubated for an additional h at room temperature. The plate was then washed and a peroxidase-conjugated donkey anti-rabbit secondary antibody was introduced and incubated for 1 h at room temperature. The plate was washed again, followed by the addition of TMB substrate and the reaction was allowed to proceed for 5-20 min. H2SO4 was introduced to stop the reaction and absorbance was read by spectrophotometer at 450 nm.
Results:
The results are presented in Table 29. Collectively, the results demonstrate that the two antibodies against the CFXTEN fusion proteins with XTEN inserted into the A2 domain exhibited reduced binding of FVIII compared to CFXTEN with XTEN inserted into the A1 or A3 domain when the anti-FVIII capture antibody was GMA-8021 (with binding affinity to the A2 domain). In contrast, there was no discernible pattern of inhibition or enhancement of binding by any of the CFXTEN when the anti-FVIII capture antibody was ESH8, with binding affinity to the C2 domain.





TABLE 29



Binding Interference of FVIII-XTEN to anti-FVIII Antibody






XTEN insertion
Concentration on aFVIII/MYc ÷ concentration on aHis/MYc








(Domain, site,

GMA-8021/Myc
ESH8/Myc

Sample Tested
XTEN)
His/Myc
(A2 domain)
(C2 domain)









FVIII-His-Myc
None
100%
92%
104%

FVIII-XTEN-
A2, 403, AE144
100%
103% ± 1%
141% ± 24%

His-Myc
A2, 403, AG144
100%
104% ± 6%
129% ± 12%


A2, 399, AE144
100%
100% ± 8%
140% ± 18%


A3, 1656, AG144
100%
153%
158%


A1, 18, AE144
100%
129%
130%


A1, 18, AG144
100%
150%
131%


A1, 26, AE144
100%
155%
87%


A1, 26, AG144
100%
157%
147%


A1, 40, AE144
100%
137%
147%


A1, 40, AG144
100%
164% ± 0%
153% ± 18%



aFVIII/Myc = GMA-8021/Myc or ESH8/Myc antibody condition;

aHis/Myc = anti-His/Myc antibody condition







































Example 28
Activity Assay of CFXTEN Fusion Proteins in the Presence of FVIII Inhibitors
Inhibitor Testing Titration Procedure
Select antibodies inhibiting FVIII procoagulant activity were purchased from commercial sources. The antibodies target select domains of FVIII (e.g. A2, A3, C1, C2) and inhibit FVIII-dependent procoagulant activity. In order to establish the optimal concentration of FVIII inhibitors to utilize in the assay, an initial titration experiment was performed using varying amounts of each inhibitory antibody incubated at 37° C. for 2 hrs with the base vector expressing wild-type FVIII with a His/Myc double tag, and a second sample with antibody and at least one CFXTEN fusion protein. The samples were then utilized in a coagulation assay to determine the FVIII activity. The activity was measured by the Coatest assay procedure described herein. The concentration that resulted in optimal inhibition of FVIII activity was determined for each antibody individually.
Inhibitor Testing Procedure:
The FVIII inhibitor antibodies were then used at their optimal concentration for assay of test samples. CFXTEN and positive control samples were individually incubated with each antibody at 37° C. for 2 hrs and the samples were then collected and utilized in the Coatest activity assay, along with untreated aliquots of the CFXTEN and positive control. In some cases, CFXTEN constructs with a R1648A mutation were tested to determine the effect, if any, of this mutation on resistance to inhibitors as measured by the retention of FVIII activity.
Results:
The results of the titration experiment are shown in FIG. 26 . The data indicate a right-shift of approximately 0.7 order of magnitude in the amount of antibody required to inhibit the procoagulant activity of the CFXTEN LSD0049.002 to the 50% level, compared to FVIII positive control, indicating that the CFXTEN with three XTEN insertions (at insertion points corresponding to amino acid residue 18, 745 and 2332 of the BDD-FVIII) had lower binding with the antibody compared to FVIII, reflected in the retention of coagulation activity.
The results of the Coatest assays are presented in Tables 30 and 31, for the FVIII inhibitor antibodies GMA8008 and GMA8021, respectively. All of the untreated CFXTEN fusion protein constructs tested exhibited procoagulant activity, as did the pBC00114 FVIII positive control. The positive control sample pre-incubated with FVIII inhibitor antibodies resulted in a sharp decrease in the measured coagulation activity to 0.05-0.15 (5-15%) relative to the untreated sample, as did the majority of the CFXTEN constructs treated with the GMA8008 antibody to the C2 domain. However, three CFXTEN fusion proteins retained at least twice the relative remaining activity compared to the FVIII control; LSD0049.020, LSD0053.024, and LSD0056.025, each with three XTEN inserts.
The CFXTEN samples showed a lower degree of inhibition with the GMA8021 antibody to the A2 domain compared to untreated samples that was further reduced by either the additional numbers of XTEN inserts (tabular data shown in Table 30). FIG. 29 shows the graph of median values of the ratio to control of retained activity showing a linear relationship between numbers of XTEN inserted and reduced inhibition to the GMA8021 antibody relative to the inhibition of the FVIII control. Similarly, the means±S.E. for the ratio to control values were 2.26+0.12 for 1 XTEN, 3.48+0.26 for 2 XTEN and 5.70+0.29 for 3 XTEN insertions. CFTXEN with at least three XTEN inserts treated with the GMA8021 antibody had at least 4.5 to 9.2-fold greater retention of FVIII activity compared to FVIII control. In addition, in those CFXTEN with three XTEN insertions, constructs with a higher degree of separation (in numbers of amino acid residues) between any two insertions appeared to result in a higher degree of procoagulant activity and, hence, less binding by the FVIII inhibitor antibody, compared to insertions clustered more closely; e.g. on the C-terminal side of the B-domain. The assay results of constructs with the R1648A mutation appeared to be comparable to those without the mutation.
Conclusions:
The results support that, under the conditions of the experiments, insertion of XTEN into FVIII resulted in protection against binding by FVIII inhibitors, with retention of procoagulant activity, and that inclusion of multiple XTEN inserts increased resistance to, in particular, the A2 domain inhibitor antibody. Lastly, there appears to be an effect by having spatial separation between the XTEN inserts.
TABLE 30 Results of Coagulation Assay with CFXTEN treated with antibody GMA8008 to C2 Domain Relative Ratio Construct Remaining to XTEN XTEN XTEN Name Activity Control Insertion 1 Insertion 2 Insertion 3 Mutations pBC0114 CT 0.05-0.15 1 pBC0149 0.1 0.8 0745_AE42_1 pSD0045 0.3 1.1 0018_AE144_5A pSD0046 0.3 1.0 0018_AG144_F pSD0050 0.2 0.9 0026_AG144_F pSD0051 0.3 1.3 0040_AE144_5A pSD0052 0.2 1.0 0040_AG144_F pSD0001 0.2 0.9 0403_AE144_2A pBC0136 0.2 1.2 0745_AE288_1 pBC0137 0.2 1.1 0745_AE288_1 pSD0013 0.1 0.9 2332_AE144_6B pSD0014 0.1 0.8 2332_AE144_1 pBC0145 0.1 0.6 2332_AE288_1 pSD0019 0.1 0.5 2332_AE288_1 pBC0146 0.1 0.7 2332_AE288_1 pSD0015 0.1 0.8 2332_AE864 LSD0038.008 0.1 0.9 0018_AG144_F 1656_AG144_C LSD0038.013 0.1 0.6 0040_AG144_F 1656_AG144_C LSD003.09 0.1 0.9 0745_AE144_3B 2332_AE288_1 LSD003.06 0.0 0.8 0745_AE144_3B 2332_AE288_1 R1648A LSD0046.001 0.0 0.6 1656_AG144_C 1900_AG144_C PSD077 0.1 1.0 0026_AG144_F 0403_AE144_2A 1656_AG144_C PSD080 0.1 1.0 0026_AG144_F 1656_AG144_C 1720_AG144_C PSD083 0.1 0.8 0403_AE144_2A 1656_AG144_C 1720_AG144_C PSD084 0.1 0.9 0403_AE144_2A 1656_AG144_C 1900_AE144_4A LSD0050.010 0.1 0.7 0018_AE144_5A 0745_AE144_3B 2332_AE288_1 LSD0049.021 0.0 0.6 0018_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.002 0.1 0.9 0018_AE144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.008 0.1 0.9 0026_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.011 0.1 0.9 0026_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.020 0.2 2.6 0040_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0050.002 0.0 0.2 0040_AG144_F 0745_AE144_3B 2332_AE288_1 LSD0053.024 0.2 2.5 1711_AE144_4A 0745_AE144_3B 2332_AE288_1 LSD0054.021 0.2 1.5 1720_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0055.021 0.2 1.6 1900_AE144_4A 0745_AE144_3B 2332_AE288_1 R1648A LSD0056.021 0.2 1.6 1900_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0056.025 0.3 2.0 1910_AG144_C 0745_AE144_3B 2332_AE288_1
proportion of activity remaining relative to corresponding untreated sample The ratio of the relative remaining activity (relative to its own control) compared to FVIII pBC0114 positive control

TABLE 31 Results of Coagulation Assay with CFXTEN treated with antibody GMA8021 to A2 Domain Re1ative Ratio Construct Remaining to XTEN XTEN XTEN Name Activity Contro1 Insertion 1 Insertion 2 Insertion 3 Mutation pBC0114 0.05-0.15 1 pBC0149 0.2 1.3 0745_AE42_1 pSD0045 0.3 2.7 0018_AE144_5A pSD0046 0.2 2.1 0018_AG144_F pSD0050 0.2 2.4 0026_AG144_F pSD0051 0.3 3.1 0040_AE144_5A pSD0052 0.3 2.7 0040_AG144_F pSD0001 0.2 1.6 0403_AE144_2A pBC0136 0.3 2.4 0745_AE288_1 pBC0137 0.3 2.4 0745_AE288_1 R1648A pSD0013 0.2 1.8 2332_AE144_6B pSD0014 0.2 2.1 2332_AG144_1 pBC0145 0.3 2.1 2332_AE288_1 pSD0019 0.3 2.3 2332_AE288_1 pBC0146 0.3 2.1 2332_AG288_1 pSD0015 0.3 2.8 2332_AE864 LSD0038.008 0.4 3.0 0018_AG144_F 1656_AG144_C LSD0038.013 0.4 3.0 0040_AG144_F 1656_AG144_C LSD003.09 0.3 3.6 0745_AE144_3B 2332_AE288_1 LSD003.06 0.3 3.4 0745_AE144_3B 2332_AE288_1 R1648A LSD0046.001 0.2 4.4 1656_AG144_C 1900_AG144_C PSD077 0.4 5.8 0026_AG144_F 0403_AE144_2A 1656_AG144_C PSD080 0.4 5.7 0026_AG144_F 1656_AG144_C 1720_AG144_C PSD083 0.3 5.0 0403_AE144_2A 1656_AG144_C 1720_AG144_C PSD084 0.3 4.5 0403_AE144_2A 1656_AG144_C 1900_AE144_4A LSD0050.010 0.4 6.7 0018_AE144_5A 0745_AE144_3B 2332_AE288_1 LSD0049.021 0.4 6.7 0018_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.002 0.5 9.2 0018_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.008 0.4 5.9 0026_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.011 0.4 5.6 0026_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.020 0.3 5.0 0040_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0050.002 0.3 6.2 0040_AG144_F 0745_AE144_3B 2332_AE288_1 LSD0053.024 0.3 4.5 1711_AE144_4A 0745_AE144_3B 2332_AE288_1 LSD0054.021 0.5 5.2 1720_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0055.021 0.5 5.4 1900_AE144_4A 0745_AE144_3B 2332_AE288_1 R1648A LSD0056.021 0.5 5.1 1900_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0056.025 0.5 4.8 1910_AG144_C 0745_AE144_3B 2332_AE288_1
proportion of activity remaining relative to corresponding untreated sample The ratio of the relative remaining activity (relative to its own control) compared to FVIII pBC0114 positive control

Example 29
Protein Purification of CFXTEN Fusion Proteins pBC0145 and pBC0146
Two CFXTEN constructs with C-terminal XTEN were utilized to establish a purification method. For both pBC0145 with a C-terminal XTEN of 288 amino acids of the AE family (see sequence in Table 21) and pBC0146 with a C-terminal XTEN of 288 amino acids of the AG family (see sequence in Table 21), a tangential flow filtration (TFF) step was used to buffer exchange the clarified conditioned media from cell culture. Products were then captured using a strong anion exchange chromatography resin, and then further purified using VIIISelect affinity chromatography (GE Healthcare). An additional size exclusion chromatography (GE Healthcare) was applied to FVIII-pBC0146 as a third polish step to remove high molecule weight species. The purity of both fusion proteins was deemed acceptable by HPLC-SEC and was further confirmed by SDS-PAGE analysis of the two CFXTEN constructs showing CFXTEN products at expected sizes. The specific activity of both molecules was comparable to B-domain deleted FVIII, as measured by aPTT coagulation assay and ELISA determination of FVIII concentration.
Example 30
Pharmacokinetics of CFXTEN Fusion Proteins pBC0145 and pBC0146 in HemA and FVIII/VWF DKO Mice
Male FVIII knock-out (HemA) mice or FVIII/VWF double knock-out (DKO) mice, 8-12 weeks old, were treated with a single intravenous administration of either recombinant BDD-FVIII, the CFXTEN pBC0145 or pBC0146 fusion purified proteins (from Example 23) at 200 IU/kg dose (n=4/time point). At select time points, blood samples were collected via vena cava sampling. In HemA mice, blood samples were collected at 5 min, 1 4, 8, 16, 20, 24, 32, and 48 hrs post-dosing for rBDD-FVIII, and at 5 min, 8, 16, 24, 32, 48, 55 and 72 hrs post-dosing for pBC0145 and pBC0146 fusion proteins. In the FVIII/VWF DKO mice, blood samples were collected at 5 min, 30 min and 1 hr post-dosing for rBDD-FVIII, and at 5 min, 4, 8, 16 and 24 hr post-dosing for the pBC0145 and pBC0146 fusion proteins. Plasma FVIII activity was measured by FVIII chromogenic assay and the PK profile was analyzed by the WinNonlin program.
Results:
As show in Table 32 and FIG. 24 , CFXTEN with the AE C-terminus XTEN insertion (pBC0145) exhibited 1.6-fold and 14.1-fold FVIII half-life (T1/2) extension compared to rBDD FVIII in HemA mice and FVIII/VWF DKO mice, respectively. The CFXTEN with the AG C-terminus XTEN insertion (pBC0146) had 1.4-fold and 14.4-fold extended half-life compared to rBDD-FVIII in the HemA mice and FVIII/VWF DKO mice, respectively. The magnitude of the FVIII half-life extension conferred by XTEN insertion was much more pronounced in the FVIII/VWF DKO mice compared to the HemA mice, demonstrated by the 14-fold longer FVIII half-life from both FVIII-AE-XTEN and FVIII-AG-XTEN compared to rBDD-FVIII. In addition, in comparison to rBDD-FVIII, FVIII with C-terminal AE or AG-XTEN insertion also had significantly improved FVIII recovery at the 5 min interval, reduced clearance and volume of distribution, and increased AUC in the DKO mice. Under the conditions of the experiment, CFXTEN with C-terminus XTEN insertions demonstrated great potential on FVIII half-life extension, and, when combined with other FVIII intra-domain insertions could potentially further extend FVIII half-life.
TABLE 32 Pharmacokinetic parameters of CFXTEN in HemA and FVIII/VWF DKO mice 5 min Cl Vss AUC_D T 1/2 Mouse Treat- Recovery T 1/2 MRT (mL/ (mL/ (hrkgmIU/m Fold Mouse Strain ment (%) (hr) (hr) hr/kg) kg) L/mIU) Increase Strain HemA pBC0145 73 11.88 16.47 3.81 62.74 0.26 1.6 HemA pBC0146 64 10.54 13.31 5.66 75.34 0.18 1.4 rBDD- 89 7.58 11.02 4.33 47.68 0.23 FVIII FVIII/ pBC0145 74 3.38 3.76 13.06 63.68 0.0765 13.9 FVIII/ VWF VWF DKO DKO pBCF0146 61 3.45 3.61 17.40 86.63 0.0575 14.2 rBDD- 23 0.24 0.24 460.62 161.51 0.0022 FVIII
Compared to rBDD-FVIII

Example 31
Cell Culture and Concentration of Cell Culture Media for CFXTEN Fusion Proteins pSD0050 and pSD0062
CFXTEN construct variants pSD0050 with an intradomain AG XTEN of 144 amino acids inserted after amino acid residue 26 of BDD FVIII, pSD0062 with an intradomain AE XTEN of 144 amino acids inserted after residue 1900 of BDD FVIII (Note: amino acid numbering based full length FVIII), as well as a construct encoding rBDD-FVIII, were transfected into HEK293F cells (Invitrogen, Carlsbad, Calif.) using polyethyleneimine (PEI, Polysciences Inc. Warrington, Pa.). The transiently transfected cells were grown in 293 Free Style medium media (Invitrogen, Carlsbad, Calif.) for 4 days and 50-100 ml cell culture media were then concentrated 10- to 20-fold by Centricon Spin Column (100 kDa MW cut-off) to reach 10-30 IU/ml FVIII activity. The concentrated materials were then flash-frozen and stored at −80° C. for future in vitro analysis and in vivo pharmacokinetic studies.
Example 32
Pharmacokinetics of CFXTEN Fusion Proteins pSD0050 and pSD0062 in HemA and FVIII/VWF DKO Mice
Male HemA or FVIII/VWF double knock-out (DKO) mice, 8-12 weeks old, were treated with a single intravenous administration of cell culture concentrates from Example 31 containing either recombinant BDD-FVIII, the CFXTEN pBD0050 or pBD062 at 100-300 IU/kg (n=3/group). At select time points, blood samples were collected via retro orbital bleeds from the same set of mice. In HemA mice, blood samples were collected at 5 min, 24 hr and 48 hr post-dosing, while in FVIII/VWF DKO mice blood samples were collected at 5 min, 8 hr and 16 hr. The FVIII activity of plasma samples and cell culture concentrates were analyzed by a FVIII chromogenic assay, and the PK profile of rBDD FVIII and FVIII-XTEN variants were analyzed using the WinNonlin program.
Results:
The PK profiles of the two CFXTEN intradomain insertion variants pSD0050 and pSD0062 and rBDD-FVIII in HemA mice and FVIII/VWF DKO mice are shown in FIG. 25 and Table 33. In HemA mice, a comparable initial recovery at the 5 min interval was observed for the three test FVIII molecules. Both CFXTEN fusion proteins demonstrated two-fold longer half-life compared to wild-type BDD-FVIII. In FVIII-VWF DKO mice, because of the loss of VWF protection, rBDD-FVIII had only a 15 min plasma half-life. In the case of the two CFXTEN, however, half-life were extended to 3.15 hr and 3.83 hr, respectively; values that are comparable to the CFXTEN with 288 C-terminus XTEN insertions (Example 24), suggesting that further extension of the XTEN length at a given insertion point may not be necessary. Under the experimental conditions, the study results clearly demonstrate that intradomain insertion of an XTEN with 144 amino acid residues not only preserved FVIII activity, but also provided similar FVIII half-life benefit as the C-terminus 288 amino acid XTEN insertion variants, suggesting that the combination of the FVIII intradomain and C-terminus insertions may allow further extension of FVIII half-life.
TABLE 33 Pharmacokinetic parameters of CFXTEN in HemA and FVIII/VWF DKO mice 5 min Mouse Recovery T 1/2 MRT Cl Vss AUC_D T 1/2 Fold Strain Treatment (%) (hr) (hr) (mL/hr/kg) (mL/kg) (hrkgmIU/mL/mIU) Increase HemA pSD0050 40 14.12 14.25 5.27 75.03 0.19 2.3 pSD0062 43 12.96 14.79 4.24 62.67 0.24 2.1 rBDD- 47 6.19 2.62 6.35 16.62 0.16 FVIII FVIII/ pSD0050 34 3.15 2.59 21.73 56.28 0.05 ~12 VWF pSD0062 35 3.83 3.71 18.51 68.69 0.05 ~15 DKO rBDD- 23 ~0.25 FVIII
Compared to rBDD-FVIII

Example 33
Pharmacokinetic Analysis of CFXTEN Fusion Polypeptides in Rats
The pharmacokinetics of various CFXTEN fusion proteins, compared to FVIII alone, are tested in Sprague-Dawley rats. CFXTEN and FVIII are administered to female Sprague-Dawley rats (n=3) IV through a jugular vein catheter at 3-10 g/rat. Blood samples (0.2 mL) are collected into pre-chilled heparinized tubes at predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 hour time points, and processed into plasma. Quantitation of the test articles is performed by ELISA assay using an anti-FVIII antibody for both capture and detection. A non-compartmental analysis is performed in WinNonLin with all time points included in the fit to determine the PK parameters. Results are expected to show increased terminal half-life and area under the curve, and a reduced volume of distribution for the CFXEN compared to FVIII alone, and the results are used in conjunction with results from coagulation and pharmacodynamic assays to select those fusion protein configurations with desired properties.
Example 34
Analysis of FVIII for XTEN Insertion Sites
The selection of XTEN insertion sites within the factor VIII molecule was performed by predicting the locations of permissive sites within loop structures or otherwise flexible surface exposed structural elements. For these analyses, the atomic coordinates of two independently determined X-ray crystallographic structures of FVIII were use (Shen B W, et al. The tertiary structure and domain organization of coagulation factor VIII. Blood. (2008) February 1; 111(3):1240-1247; Ngo J C, et al. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex. Structure (2008) 16 (4):597-606), as well as those of factor VIII and factor VIIIa derived from molecular dynamic simulation (MDS) (Venkateswarlu, D. Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study. BMC Struct Biol. (2010) 10:7). Atomic coordinates in Protein Data Bank (PDB) format were analyzed to identify regions of the FVIII/FVIIIa predicted to have a high degree solvent accessible surface area using the algorithms ASAView (Ahmad S, et al. ASAView: database and tool for solvent accessibility representation in proteins. BMC Bioinformatics (2004) 5:51) and GetArea (Rychkov G, Petukhov M. Joint neighbors approximation of macromolecular solvent accessible surface area. J Comput Chem (2007) 28(12):1974-1989). The resulting set of sites was then further prioritized on the basis of high predicted atomic positional fluctuation based on the basis of the published results of the MDS study. Sites within the acidic peptide regions flanking the A1, A2, and A3 domains, as well as those that appeared by visual inspection to be in areas other than surface exposed loops were deprioritized. The resulting set of potential sites was evaluated on the basis of interspecies sequence conservation, with those sites in regions of high sequence conservation among 20 vertebrate species being ranked more favorably. Additionally, putative clearance receptor binding sites, FVIII interaction sites with other molecules (such as vWF, FIX), domain and exon boundaries were also considered in fusion site selection. Finally, sites within close proximity to mutations implicated in hemophilia A listed in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database were eliminated (Kemball-Cook G, et al. The factor VIII Structure and Mutation Resource Site: HAMSTeRS version 4. Nucleic Acids Res. (1998) 26(1):216-219). Based on these criteria, the construction of 42 FVIII-XTEN variants was proposed for XTEN insertions. Of these, three represent XTEN insertions within the residual B domain sequence, two represent extensions to the C-terminus of the factor VIII molecule, and 37 represent XTEN insertions within structurally defined inter- and intradomain structural elements; i.e., residues 3, 18, 22, 26, 40, 60, 116, 130, 188, 216, 230, 333, 375, 403, 442, 490, 518, 599, 713, 745, 1720, 1796, 1802, 1827, 1861, 1896, 1900, 1904, 1937, 2019, 2068, 2111, 2120, 2171, 2188, 2227, 2277, and 2332.
Example 35
Functional Analysis of FVIII-XTEN Constructs
Two FVIII-XTEN fusion proteins, FVII1-AE288 (F8X-40) and FVII1-AG288 (F8X-41), contain an AE288 — 1 XTEN or an AG288 — 1 XTEN, respectively, fused at the C-terminus of FVIII C2 domain. To determine if FVIII activity was retained after XTEN fusion, HEK293 cells were transfected separately with these two FVIII-XTEN fusion constructs by using polyethylenimine (PEI) in serum-free medium. At 3 or 5 days post-transfection, the cell culture supernatant was tested for FVIII activity by a two-stage chromogenic assay. Purified recombinant FVIII, calibrated against WHO international standard, was used to establish the standard curve in the chromogeinic assay. The fusion protein products of both F8X-40 and F8X-41 constructs were expressed at levels comparable to those of wild-type BDD-FVIII constructs. (Table 34).





TABLE 34



FVIII Titer of FVIII-XTEN fusion proteins

in transient transfection cell culture







FVIII Molecules
FVIII 066 a
pBC 0114 a
F8X-40
F8X-41










FVIII activity
Sample A
6.42
6.68
7.47
3.32 b

(IU/ml)
Sample B
7.13
7.61
8.25
Not done



a Both FVIII 066 and pBC 0114 contain B-domain deleted FVIII without XTEN fusion.

b The F8X-41sample was from a 3-day transfection while other samples were from a 5-day transient transfection.


























Example 36
Functional Analysis of FVIII-XTEN Constructs: FVIII Activity and PK Properties
The half-life extension potential of the F8X-40 and F8X-41 constructs was evaluated in FVIII and von Willebrand factor double knock-out mice by hydrodynamic plasmid DNA injection, with a FVIIIFc DNA construct serving as a positive control. Mice were randomly divided into 3 groups with 4 mice per group. Plasmid DNA encoding BDD FVIIIFc fusion protein, F8X-40 or F8X-41, all sharing the same DNA vector backbone, was administered to mice in the respective groups. Approximately 100 micrograms of the appropriate plasmid DNA was injected into each mouse via hydrodynamic injection, and blood plasma samples were collected at 24 hours and 48 hours post-injection. The plasma FVIII activity was measured by a two-stage chromogenic assay using calibrated recombinant FVIII as a standard. As shown in FIG. 23 , samples from the F8X-40 and F8X-41 groups showed higher plasma FVIII titers than did those from the BDD FVIIIFc, suggesting FVIII fusion with XTEN prolongs the half-life of FVIII in vivo. Taken together, these data support the conclusion that FVIII-XTEN fusion proteins retained FVIII activity in transient transfection and exhibited prolonged circulating half-life in an animal model.
Example 37
Pharmacodynamic Evaluation of CFXTEN in Animal Models
The in vivo pharmacologic activity of CFXTEN fusion proteins are assessed using a variety of preclinical models of bleeding including but not limited to those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding and hydrodynamic injection. These models are developed in multiple species including mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FVIII approaches. CFXTEN compositions are provided in an aqueous buffer compatible with in vivo administration (for example: phosphate-buffered saline or Tris-buffered saline). The compositions are administered at appropriate doses, dosing frequency, dosing schedule and route of administration as optimized for the particular model. Efficacy determinations include measurement of FVIII activity, one-stage clotting assay, FVIII chromogenic assay, activated partial prothrombin time (aPTT), bleeding time, whole blood clotting time (WBCT), thrombelastography (TEG or ROTEM), among others.
In one example of a PD model, CFXTEN and FVIII are administered to genetically-deficient or experimentally-induced HemA mice. At various time points post-administration, levels of FVIII and CFXTEN are measured by ELISA, activity of FVIII and CFXTEN is measured by commercially-available FVIII activity kits and clotting time is measured by aPTT assay. Overall, the results can indicate that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII with less frequent or more convenient dosing intervals.
In a mouse bleeding challenge PD model CFXTEN and FVIII are administered to genetically-deficient or experimentally-induced HemA mice and effect on hemostatic challenge is measured. Hemostatic challenge can include tail transaction challenge, hemarthropthy challenge, joint bleeding or saphenous vein challenge among others. At various time points post-administration levels of FVIII and CFXTEN are measured by ELISA, activity of FVIII and CFXTEN are measured by commercially available FVIII activity kit, bleeding time is measured and clotting time is measured by aPTT assay. Overall the results are expected to indicate that the CFXTEN constructs are more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII with less frequent or more convenient dosing intervals, and the results are used in conjunction with results from coagulation and other assays to select those fusion protein configurations with desired properties.
In a dog PD model, CFXTEN and FVIII are administered to genetically-deficient hemophiliac dogs. At various time points post administration, levels of FVIII and CFXTEN are measured by ELISA, activity of FVIII and CFXTEN are measured by commercially available FVIII activity kit and clotting time is measured by aPTT assay. Overall the results indicates that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII with less frequent or more convenient dosing, and the results are used in conjunction with results from coagulation and other assays to select those fusion protein configurations with desired properties.
In a dog bleeding challenge PD model CFXTEN and FVIII are administered to genetically deficient hemophiliac dogs and effect on hemostatic challenge is measured. Hemostatic challenge includes cuticle bleeding time among others. At various time points post-administration levels of FVIII and CFXTEN are measured by ELISA, activity of FVIII and CFXTEN are measured by commercially available FVIII activity kit, bleeding time is measured and clotting time are measured by aPTT assay. Overall the results indicate that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII with less frequent or more convenient dosing intervals, and the results are used in conjunction with results from coagulation and other assays to select those fusion protein configurations with desired properties.
Additional preclinical models of bleeding include but are not limited to those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin-induced bleeding and hydrodynamic injection. These models can developed in multiple species including mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FVIII approaches. Overall the results indicate that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII with less frequent or more convenient dosing intervals, and the results are used in conjunction with results from coagulation and other assays to select those fusion protein configurations with desired properties.
Example 38
CFXTEN with Cleavage Sequences
C-terminal XTEN releasable by FXIa
A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in FIG. 12 . Exemplary sequences are provided in Table 51. In this case, the release site cleavage sequence is incorporated into the CFXTEN that contains an amino acid sequence that is recognized and cleaved by the FXIa protease (EC 3.4.21.27, Uniprot P03951). Specifically the amino acid sequence KLTRAET (SEQ ID NO: 1688) is cut after the arginine of the sequence by FXIa protease. FXI is the procoagulant protease located immediately before FVIII in the intrinsic or contact activated coagulation pathway. Active FXIa is produced from FXI by proteolytic cleavage of the zymogen by FXIIa. Production of FXIa is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the KLTRAET cleavage sequence (SEQ ID NO: 1688), the XTEN domain is only be removed from FVIII concurrent with activation of the intrinsic coagulation pathway and when coagulation is required physiologically. This creates a situation where the CFXTEN fusion protein is processed in one additional manner during the activation of the intrinsic pathway.
C-Terminal XTEN Releasable by FIIa (Thrombin)
A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in FIG. 12 . In this case, the release site contains an amino acid sequence that is recognized and cleaved by the FIIa protease (EC 3.4.21.5, Uniprot P00734). Specifically the sequence LTPRSLLV (SEQ ID NO: 1618) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after the arginine at position 4 in the sequence. Active FIIa is produced by cleavage of FII by FXa in the presence of phospholipids and calcium and is down stream from factor IX in the coagulation pathway. Once activated its natural role in coagulation is to cleave fibrinogen ( FIG. 2 ), which then in turn, begins clot formation. FIIa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the LTPRSLLV sequence (SEQ ID NO: 1618), the XTEN domain is only removed from FVIII concurrent with activation of either the extrinsic or intrinsic coagulation pathways, and when coagulation is required physiologically. This creates a situation where CFXTEN fusion is processed in one additional manner during the activation of coagulation.
C-Terminal XTEN Releasable by Elastase-2
A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in FIG. 12 . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the elastase-2 protease (EC 3.4.21.37, Uniprot P08246). Specifically the sequence LGPVSGVP (SEQ ID NO: 1689) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 in the sequence. Elastase is constitutively expressed by neutrophils and is present at all times in the circulation. Its activity is tightly controlled by serpins and is therefore minimally active most of the time. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.
C-Terminal XTEN Releasable by MMP-12
A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in FIG. 12 . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-12 protease (EC 3.4.24.65, Uniprot P39900). Specifically the sequence GPAGLGGA (SEQ ID NO: 1690) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 of the sequence. MMP-12 is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.
C-Terminal XTEN Releasable by MMP-13
A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in FIG. 12 . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-13 protease (EC 3.4.24.-, Uniprot P45452). Specifically the sequence GPAGLRGA (SEQ ID NO: 1691) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4. MMP-13 is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.
C-Terminal XTEN Releasable by MMP-17
A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in FIG. 12 . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot Q9ULZ9). Specifically the sequence APLGLRLR (SEQ ID NO: 1692) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 in the sequence. MMP-17 is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.
C-Terminal XTEN Releasable by MMP-20
A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in FIG. 12 . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-20 protease (EC.3.4.24.-, Uniprot 060882). Specifically the sequence PALPLVAQ (SEQ ID NO: 1693) [Rawlings N. D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 (depicted by the arrow). MMP-20 is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.
Optimization of the Release Rate of XTEN
Variants of the foregoing Examples can be created in which the release rate of XTEN incorporated at the C-terminus, the N-terminus, or internal XTEN is altered. As the rate of XTEN release by an XTEN release protease is dependent on the sequence of the XTEN release site, by varying the amino acid sequence in the XTEN release site one can control the rate of XTEN release. The sequence specificity of many proteases is well known in the art, and is documented in several data bases. In this case, the amino acid specificity of proteases is mapped using combinatorial libraries of substrates [Harris, J. L., et al. (2000) Proc Natl Acad Sci USA, 97: 7754] or by following the cleavage of substrate mixtures as illustrated in [Schellenberger, V., et al. (1993) Biochemistry, 32: 4344]. An alternative is the identification of optimal protease cleavage sequences by phage display [Matthews, D., et al. (1993) Science, 260: 1113]. Constructs are made with variant sequences and assayed for XTEN release using standard assays for detection of the XTEN polypeptides.
Example 39
Human Clinical Trial Designs for Evaluating CFXTEN Comprising FVIII
Kogenate® FS is recombinant human coagulation factor VIII, intended for promoting hemostasis in hemophilia A subjects. Due to its short half-life, Kogenate is dosed intravenously every other day for prophylaxis and 8 to every 12 h in treatment of bleeds until hemostasis is achieved. It is believed that fusion of one or more XTEN to FVIII improves the half-life of the protein, enabling a reduced dosing frequency using such CFXTEN-containing fusion protein compositions.
Clinical trials are designed such that the efficacy and advantages of CFXTEN, relative to Kogenate or other commercially available FVIII preparations, can be verified in humans. Such studies comprises three phases. First, a Phase I safety and pharmacokinetics study in adult patients is conducted to determine the maximum tolerated dose and pharmacokinetics and pharmacodynamics in humans (either normal subjects or patients with hemophilia), as well as to define potential toxicities and adverse events to be tracked in future studies. The Phase I studies are conducted in which single rising doses of CFXTEN compositions are administered by the route (e.g., subcutaneous, intramuscular, or intravenously) and biochemical, PK, and clinical parameters are measured at defined intervals. This permits the determination of the minimum effective dose and the maximum tolerated dose and establishes the threshold and maximum concentrations in dosage and circulating drug that constitute the therapeutic window for the respective components, as well as bioavailability when administered by the intramuscular or subcutaneous routes. From this information, the dose and dose schedule that permits less frequent administration of the CFXTEN compositions, yet retains the pharmacologic response, is obtained. Thereafter, clinical trials are conducted in patients with the condition, verifying the effectiveness of the CFXTEN compositions under the dose conditions, which can be conducted in comparison to a positive control such as Kogenate to establish the enhanced properties of the CFXTEN compositions.
Phase II and III clinical trials are conducted in patients suffering from any disease in which factor VIII may be expected to provide clinical benefit. For example, the CFXTEN is used in clinical trials for treatment of indications approved for use of factor VIII; such indications include bleeding episodes in hemophilia A, patients with inhibitors to factor VIII, prevention of bleeding in surgical interventions or invasive procedures in hemophilia A patients with inhibitors to factor VIII, treatment of bleeding episodes in patients with congenital factor VIII deficiency, and prevention of bleeding in surgical interventions or invasive procedures in patients with congenital factor VIII deficiency. CFXTEN may also be indicated for use in additional patient populations. A phase II dosing study is conducted in hemophilia A patients where pharmacodynamic, coagulation, bleeding and other physiologic, PK, safety and clinical parameters and clinical endpoints appropriate for trials are measured as a function of the dosing of the fusion proteins compositions, yielding dose-ranging information on doses that is appropriate for a subsequent Phase III trial, in addition to collecting safety data related to adverse events. The PK parameters are correlated to the physiologic, clinical and safety parameter data to establish the therapeutic window and the therapeutic dose regimen for the CFXTEN composition, permitting the clinician to establish the appropriate dose ranges for the composition. In one trial, hemophilia A patients with factor VIII inhibitors would be evaluated to establish doses and dose regimen of CFXTEN pharmaceutical compositions that result in achieving and maintaining hemostasis and preventing or attenuating bleeding episodes. Finally, a phase III efficacy study is conducted wherein patients are administered the CFXTEN pharmaceutical composition and a positive control (such as a commercially-available Kogenate) are administered using a dosing schedule deemed appropriate given the pharmacokinetic and pharmacodynamic properties of the respective compositions derived from the Phase II findings, with all agents administered for an appropriately extended period of time to achieve the study endpoints. Parameters that are monitored include aPTT assay, one- or two-stage clotting assays, control of bleeding episodes, or the occurrence of spontaneous bleeding episodes; parameters that are tracked relative to the placebo or positive control groups. Efficacy outcomes are determined using standard statistical methods. Toxicity and adverse event markers are also be followed in this study to verify that the compound is safe when used in the manner described. In another phase III trial, hemophilia A patients with factor VIII inhibitors would be evaluated to establish the effectiveness of CFXTEN pharmaceutical compositions in achieving and maintaining hemostasis and preventing or attenuating bleeding episodes.
Example 40
Analytical Size Exclusion Chromatography of XTEN Fusion Proteins with Diverse Payloads
Size exclusion chromatography analyses were performed on fusion proteins containing various therapeutic proteins and unstructured recombinant proteins of increasing length. An exemplary assay used a TSKGel-G4000 SWXL (7.8 mm×30 cm) column in which 40 μg of purified glucagon fusion protein at a concentration of 1 mg/ml was separated at a flow rate of 0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram profiles were monitored using OD214 nm and OD280 nm. Column calibration for all assays were performed using a size exclusion calibration standard from BioRad; the markers include thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). Representative chromatographic profiles of Glucagon-Y288, Glucagon-Y144, Glucagon-Y72, Glucagon-Y36 are shown as an overlay in FIG. 21 . The data show that the apparent molecular weight of each compound is proportional to the length of the attached XTEN sequence. However, the data also show that the apparent molecular weight of each construct is significantly larger than that expected for a globular protein (as shown by comparison to the standard proteins run in the same assay). Based on the SEC analyses for all constructs evaluated, including a CFXTEN composition, the apparent molecular weights, the apparent molecular weight factor (expressed as the ratio of apparent molecular weight to the calculated molecular weight) and the hydrodynamic radius (R H in nm) are shown in Table 35. The results indicate that incorporation of different XTENs of 576 amino acids or greater confers an apparent molecular weight for the fusion protein of approximately 339 kDa to 760, and that XTEN of 864 amino acids or greater confers an apparent molecular weight greater than approximately 800 kDA. The results of proportional increases in apparent molecular weight to actual molecular weight were consistent for fusion proteins created with XTEN from several different motif families; i.e., AD, AE, AF, AG, and AM, with increases of at least four-fold and ratios as high as about 17-fold. Additionally, the incorporation of XTEN fusion partners with 576 amino acids or more into fusion proteins with the various payloads (and 288 residues in the case of glucagon fused to Y288) resulted with a hydrodynamic radius of 7 nm or greater, well beyond the glomerular pore size of approximately 3-5 nm. Accordingly, it is expected that fusion proteins comprising growth and XTEN have reduced renal clearance, contributing to increased terminal half-life and improving the therapeutic or biologic effect relative to a corresponding un-fused biologic payload protein.





TABLE 35



SEC analysis of various polypeptides










XTEN



Apparent



or
Thera-
Actual
Apparent
Molecular


Construct
fusion
peutic
MW
MW
Weight
R H

Name
partner
Protein
(kDa)
(kDa)
Factor
(nm)











AC14
Y288
Glucagon
28.7
370
12.9
7.0

AC28
Y144
Glucagon
16.1
117
7.3
5.0

AC34
Y72
Glucagon
9.9
58.6
5.9
3.8

AC33
Y36
Glucagon
6.8
29.4
4.3
2.6

AC89
AF120
Glucagon
14.1
76.4
5.4
4.3

AC88
AF108
Glucagon
13.1
61.2
4.7
3.9

AC73
AF144
Glucagon
16.3
95.2
5.8
4.7

AC53
AG576
GFP
74.9
339
4.5
7.0

AC39
AD576
GFP
76.4
546
7.1
7.7

AC41
AE576
GFP
80.4
760
9.5
8.3

AC52
AF576
GFP
78.3
526
6.7
7.6

AC398
AE288
FVII
76.3
650
8.5
8.2

AC404
AE864
FVII
129
1900
14.7
10.1

AC85
AE864
Exendin-4
83.6
938
11.2
8.9

AC114
AM875
Exendin-4
82.4
1344
16.3
9.4

AC143
AM875
hGH
100.6
846
8.4
8.7

AC227
AM875
IL-1ra
95.4
1103
11.6
9.2

AC228
AM1318
IL-1ra
134.8
2286
17.0
10.5












































Example 41
Pharmacokinetics of Extended Polypeptides Fused to GFP in Cynomolgus Monkeys
The pharmacokinetics of GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-XTEN_Y576 and XTEN_AD836-GFP were tested in cynomolgus monkeys to determine the effect of composition and length of the unstructured polypeptides on PK parameters. Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are summarized in FIG. 19 . They show a surprising increase of half-life with increasing length of the XTEN sequence. For example, a half-life of 10 h was determined for GFP-XTEN_L288 (with 288 amino acid residues in the XTEN). Doubling the length of the unstructured polypeptide fusion partner to 576 amino acids increased the half-life to 20-22 h for multiple fusion protein constructs; i.e., GFP-XTEN_L576, GFP-XTEN_AF576, GFP-XTEN_Y576. A further increase of the unstructured polypeptide fusion partner length to 836 residues resulted in a half-life of 72-75 h for XTEN_AD836-GFP. Thus, increasing the polymer length by 288 residues from 288 to 576 residues increased in vivo half-life by about 10 h. However, increasing the polypeptide length by 260 residues from 576 residues to 836 residues increased half-life by more than 50 h. These results show that there is a surprising threshold of unstructured polypeptide length that results in a greater than proportional gain in in vivo half-life. Thus, fusion proteins comprising extended, unstructured polypeptides are expected to have the property of enhanced pharmacokinetics compared to polypeptides of shorter lengths.
Example 42
Serum Stability of XTEN
A fusion protein containing XTEN_AE864 fused to the N-terminus of GFP was incubated in monkey plasma and rat kidney lysate for up to 7 days at 37° C. Samples were withdrawn at time 0, Day 1 and Day 7 and analyzed by SDS PAGE followed by detection using Western analysis and detection with antibodies against GFP as shown in FIG. 20 . The sequence of XTEN_AE864 showed negligible signs of degradation over 7 days in plasma. However, XTEN_AE864 was rapidly degraded in rat kidney lysate over 3 days. The in vivo stability of the fusion protein was tested in plasma samples wherein the GFP_AE864 was immunoprecipitated and analyzed by SDS PAGE as described above. Samples that were withdrawn up to 7 days after injection showed very few signs of degradation. The results demonstrate the resistance of CFXTEN to degradation due to serum proteases; a factor in the enhancement of pharmacokinetic properties of the CFXTEN fusion proteins.
Example 43
Increasing Solubility and Stability of a Peptide Payload by Linking to XTEN
In order to evaluate the ability of XTEN to enhance the physicochemical properties of solubility and stability, fusion proteins of glucagon plus shorter-length XTEN were prepared and evaluated. The test articles were prepared in Tris-buffered saline at neutral pH and characterization of the Gcg-XTEN solution was by reverse-phase HPLC and size exclusion chromatography to affirm that the protein was homogeneous and non-aggregated in solution. The data are presented in Table 36. For comparative purposes, the solubility limit of unmodified glucagon in the same buffer was measured at 60 μM (0.2 mg/mL), and the result demonstrate that for all lengths of XTEN added, a substantial increase in solubility was attained. Importantly, in most cases the glucagon-XTEN fusion proteins were prepared to achieve target concentrations and were not evaluated to determine the maximum solubility limits for the given construct. However, in the case of glucagon linked to the AF-144 XTEN, the limit of solubility was determined, with the result that a 60-fold increase in solubility was achieved, compared to glucagon not linked to XTEN. In addition, the glucagon-AF144 CFXTEN was evaluated for stability, and was found to be stable in liquid formulation for at least 6 months under refrigerated conditions and for approximately one month at 37° C. (data not shown).
The data support the conclusion that the linking of short-length XTEN polypeptides to a biologically active protein such as glucagon can markedly enhance the solubility properties of the protein by the resulting fusion protein, as well as confer stability at the higher protein concentrations.





TABLE 36



Solubility of Glucagon-XTEN constructs






Test Article
Solubility










Glucagon
60
μM


Glucagon-Y36
>370
μM


Glucagon-Y72
>293
μM


Glucagon-AF108
>145
μM


Glucagon-AF120
>160
μM


Glucagon-Y144
>497
μM


Glucagon-AE144
>467
μM


Glucagon-AF144
>3600
μM


Glucagon-Y288
>163
μM

































Example 44
Analysis of Sequences for Secondary Structure by Prediction Algorithms
Amino acid sequences can be assessed for secondary structure via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe-Robson, or “GOR” method (Garnier J, Gibrat J F, Robson B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553). For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as total and/or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation.
Several representative sequences from XTEN “families” have been assessed using two algorithm tools for the Chou-Fasman and GOR methods to assess the degree of secondary structure in these sequences. The Chou-Fasman tool was provided by William R. Pearson and the University of Virginia, at the “Biosupport” internet site, URL located on the World Wide Web at .fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=miscl as it existed on Jun. 19, 2009. The GOR tool was provided by Pole Informatique Lyonnais at the Network Protein Sequence Analysis internet site, URL located on the World Wide Web at .npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl as it existed on Jun. 19, 2008.
As a first step in the analyses, a single XTEN sequence was analyzed by the two algorithms. The AE864 composition is an XTEN with 864 amino acid residues created from multiple copies of four 12 amino acid sequence motifs consisting of the amino acids G, S, T, E, P, and A. The sequence motifs are characterized by the fact that there is limited repetitiveness within the motifs and within the overall sequence in that the sequence of any two consecutive amino acids is not repeated more than twice in any one 12 amino acid motif, and that no three contiguous amino acids of full-length the XTEN are identical. Successively longer portions of the AF 864 sequence from the N-terminus were analyzed by the Chou-Fasman and GOR algorithms (the latter requires a minimum length of 17 amino acids). The sequences were analyzed by entering the FASTA format sequences into the prediction tools and running the analysis. The results from the analyses are presented in Table 37.
The results indicate that, by the Chou-Fasman calculations, short XTEN of the AE and AG families, up to at least 288 amino acid residues, have no alpha-helices or beta-sheets, but amounts of predicted percentage of random coil by the GOR algorithm vary from 78-99%. With increasing XTEN lengths of 504 residues to greater than 1300, the XTEN analyzed by the Chou-Fasman algorithm had predicted percentages of alpha-helices or beta-sheets of 0 to about 2%, while the calculated percentages of random coil increased to from 94-99%. Those XTEN with alpha-helices or beta-sheets were those sequences with one or more instances of three contiguous serine residues, which resulted in predicted beta-sheet formation. However, even these sequences still had approximately 99% random coil formation.
The data provided herein suggests that 1) XTEN created from multiple sequence motifs of G, S, T, E, P, and A that have limited repetitiveness as to contiguous amino acids are predicted to have very low amounts of alpha-helices and beta-sheets; 2) that increasing the length of the XTEN does not appreciably increase the probability of alpha-helix or beta-sheet formation; and 3) that progressively increasing the length of the XTEN sequence by addition of non-repetitive 12-mers consisting of the amino acids G, S, T, E, P, and A results in increased percentage of random coil formation. Results further indicate that XTEN sequences defined herein (including e.g., XTEN created from sequence motifs of G, S, T, E, P, and A) have limited repetitiveness (including those with no more than two identical contiguous amino acids in any one motif) are expected to have very limited secondary structure. Any order or combination of sequence motifs from Table 3 can be used to create an XTEN polypeptide that will result in an XTEN sequence that is substantially devoid of secondary structure, though three contiguous serines are not preferred. The unfavorable property of three contiguous series however, can be ameliorated by increasing the length of the XTEN. Such sequences are expected to have the characteristics described in the CFXTEN embodiments of the invention disclosed herein.





TABLE 37



CHOU-FASMAN and GOR prediction calculations

of polypeptide sequences











GOR


SEQ ID
No.
Chou-Fasman
Calcu-

SEQ NAME
NO:
Residues
Calculation
lation









AE36:
1489
36
Residue totals: H: 0 E: 0
94.44%

LCW0402_002


percent: H: 0.0 E: 0.0


AE36:
1490
36
Residue totals: H: 0 E: 0
94.44%

LCW0402_003


percent: H: 0.0 E: 0.0


AG36:
1491
36
Residue totals: H: 0 E: 0
77.78%

LCW0404_001


percent: H: 0.0 E: 0.0


AG36:
1492
36
Residue totals: H: 0 E: 0
83.33 %

LCW0404_003


percent: H: 0.0 E: 0.0


AE42_1
1493
42
Residue totals: H: 0 E: 0
90.48%




percent: H: 0.0 E: 0.0


AE42_1
1494
42
Residue totals: H: 0 E: 0
90.48%




percent: H: 0.0 E: 0.0


AG42_1
1495
42
Residue totals: H: 0 E: 0
88.10%




percent: H: 0.0 E: 0.0


AG42_2
1496
42
Residue totals: H: 0 E: 0
88.10%




percent: H: 0.0 E: 0.0


AE144
1497
144
Residue totals: H: 0 E: 0
98.61%




percent: H: 0.0 E: 0.0


AG144_1
1498
144
Residue totals: H: 0 E: 0
91.67%




percent: H: 0.0 E: 0.0


AE288
1499
288
Residue totals: H: 0 E: 0
99.31%




percent: H: 0.0 E: 0.0


AG288_2
1500
288
Residue totals: H: 0 E: 0
92.71




percent: H: 0.0 E: 0.0


AF504
1501
504
Residue totals: H: 0 E: 0
94.44%




percent: H: 0.0 E: 0.0


AD 576
1502
576
Residue totals: H: 7 E: 0
99.65%




percent: H: 1.2 E: 0.0


AE576
1503
576
Residue totals: H: 2 E: 0
99.65%




percent: H: 0.4 E: 0.0


AG576
1504
576
Residue totals: H: 0 E: 3
99.31%




percent: H: 0.4 E: 0.5


AF540
1505
540
Residue totals: H: 2 E: 0
99.65




percent: H: 0.4 E: 0.0


AD836
1506
836
Residue totals: H: 0 E: 0
98.44%




percent: H: 0.0 E: 0.0


AE864
1507
864
Residue totals: H: 2 E: 3
99.77%




percent: H: 0.2 E: 0.4


AF864
1508
875
Residue totals: H: 2 E: 0
95.20%




percent: H: 0.2 E: 0.0


AG864
1509
864
Residue totals: H: 0 E: 0
94.91%




percent: H: 0.0 E: 0.0


AM875
1510
875
Residue totals: H: 7 E: 3
98.63%




percent: H: 0.8 E: 0.3


AM1318
1511
1318
Residue totals: H: 7 E: 0
99.17%




percent: H: 0.7 E: 0.0


AM923
1512
924
Residue totals: H: 4 E: 3
98.70%




percent: H: 0.4 E: 0.3


AE912
1513
913
Residue totals: H: 8 E: 3
99.45%




percent: H: 0.9 E: 0.3


BC 864
1514

Residue totals: H: 0 E: 0
99.77%




percent: H: 0 E: 0




H: alpha-helix

E: beta-sheet














































































Example 45
Analysis of Polypeptide Sequences for Repetitiveness
In this Example, different polypeptides, including several XTEN sequences, were assessed for repetitiveness in the amino acid sequence. Polypeptide amino acid sequences can be assessed for repetitiveness by quantifying the number of times a shorter subsequence appears within the overall polypeptide. For example, a polypeptide of 200 amino acid residues length has a total of 165 overlapping 36-amino acid “blocks” (or “36-mers”) and 198 3-mer “subsequences”, but the number of unique 3-mer subsequences will depend on the amount of repetitiveness within the sequence. For the analyses, different polypeptide sequences were assessed for repetitiveness by determining the subsequence score obtained by application of the following equation:








Subsequence





score





=







∑

i
=
1

m







Count
i


m




I








wherein: m=(amino acid length of polypeptide)-(amino acid length of subsequence)+1; and Count i =cumulative number of occurrences of each unique subsequence within sequence,

In the analyses of the present Example, the subsequence score for the polypeptides of Table 38 were determined using the foregoing equation in a computer program using the algorithm depicted in FIG. 27 , wherein the subsequence length was set at 3 amino acids. The resulting subsequence score is a reflection of the degree of repetitiveness within the polypeptide.





The results, shown in Table 38, indicate that the unstructured polypeptides consisting of 2 or 3 amino acid types have high subsequence scores, while those of consisting of the 12 amino acid motifs of the six amino acids G, S, T, E, P, and A with a low degree of internal repetitiveness, have subsequence scores of less than 10, and in some cases, less than 5. For example, the L288 sequence has two amino acid types and has short, highly repetitive sequences, resulting in a subsequence score of 50.0. The polypeptide J288 has three amino acid types but also has short, repetitive sequences, resulting in a subsequence score of 33.3. Y576 also has three amino acid types, but is not made of internal repeats, reflected in the subsequence score of 15.7 over the first 200 amino acids. W576 consists of four types of amino acids, but has a higher degree of internal repetitiveness, e.g., “GGSG” (SEQ ID NO: 1694),”, resulting in a subsequence score of 23.4. The AD576 consists of four types of 12 amino acid motifs, each consisting of four types of amino acids. Because of the low degree of internal repetitiveness of the individual motifs, the overall subsequence score over the first 200 amino acids is 13.6. In contrast, XTEN's consisting of four motifs contains six types of amino acids, each with a low degree of internal repetitiveness have lower subsequence scores; i.e., AE864 (6.1), AF864 (7.5), and AM875 (4.5), while XTEN consisting of four motifs containing five types of amino acids were intermediate; i.e., AE864, with a score of 7.2.
Conclusions:
The results indicate that the combination of 12 amino acid subsequence motifs, each consisting of four to six amino acid types that are non-repetitive, into a longer XTEN polypeptide results in an overall sequence that is substantially non-repetitive, as indicated by overall subsequence scores less than 10 and, in many cases, less than 5. This is despite the fact that each subsequence motif may be used multiple times across the sequence. In contrast, polymers created from smaller numbers of amino acid types resulted in higher subsequence scores, with polypeptides consisting of two amino acid type having higher scores that those consisting of three amino acid types.





TABLE 38



Subsequence score calculations

of polypeptide sequences







Seq Name
SEQ ID NO:
Score










J288
1515
33.3


K288
1516
46.9


L288
1517
50.0


Y288
1518
26.8


Q576
1519
18.5


U576
1520
18.1


W576
1521
23.4


Y576
1522
15.7


AE288
1523
6.0


AG288_1
1524
6.9


AD576
1525
13.6


AE576
1526
6.1


AF540
1527
8.8


AF504
1528
7.0


AE864
1529
6.1


AF864
1530
7.5


AG864
1531
7.2


AG868
1532
7.5


AM875
1533
4.5


AE912
1534
4.5


AM923
1535
4.5


AM1296
1536
4.5















































Example 46
Calculation of TEPITOPE Scores
TEPITOPE scores of 9mer peptide sequence can be calculated by adding pocket potentials as described by Sturniolo [Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555]. In the present Example, separate Tepitope scores were calculated for individual HLA alleles. Table 39 shows as an example the pocket potentials for HLA0101B, which occurs in high frequency in the Caucasian population. To calculate the TEPITOPE score of a peptide with sequence P1-P2-P3-P4-P5-P6-P7-P8-P9, the corresponding individual pocket potentials in Table 39 were added. The HLA0101B score of a 9mer peptide with the sequence FDKLPRTSG (SEQ ID NO: 1695) is the sum of 0, −1.3, 0, 0.9, 0, −1.8, 0.09, 0, 0.
To evaluate the TEPITOPE scores for long peptides one can repeat the process for all 9mer subsequences of the sequences. This process can be repeated for the proteins encoded by other HLA alleles. Tables 40-43 give pocket potentials for the protein products of HLA alleles that occur with high frequency in the Caucasian population.
TEPITOPE scores calculated by this method range from approximately −10 to +10. However, 9mer peptides that lack a hydrophobic amino acid (FKLMVWY (SEQ ID NO: 1696)) in P1 position have calculated TEPITOPE scores in the range of −1009 to −989. This value is biologically meaningless and reflects the fact that a hydrophobic amino acid serves as an anchor residue for HLA binding and peptides lacking a hydrophobic residue in P1 are considered non binders to HLA. Because most XTEN sequences lack hydrophobic residues, all combinations of 9mer subsequences will have TEPITOPEs in the range in the range of −1009 to −989. This method confirms that XTEN polypeptides may have few or no predicted T-cell epitopes.





TABLE 39



Pocket potential for HLA0101B allele.












Amino










Acid
P1
P2
P3
P4
P5
P6
P7
P8
P9














A
−999
0
0
0
—
0
0
—
0

C
−999
0
0
0
—
0
0
—
0

D
−999
−1.3
−1.3
−2.4
—
−2.7
−2
—
−1.9

E
−999
0.1
−1.2
−0.4
—
−2.4
−0.6
—
−1.9

F
0
0
0.8
0.08
—
−2.1
0.3
—
−0.4

G
−999
0.5
0.2
−0.7
—
−0.3
−1.1
—
−0.8

H
−999
0.8
0.2
−0.7
—
−2.2
0.1
—
−1.1

I
−1
1.1
1.5
0.5
—
−1.9
0.6
—
0.7

K
−999
1.1
0
−2.1
—
−2
−0.2
—
−1.7

L
−1
1
1
0.9
—
−2
0.3
—
0.5

M
−1
1.1
1.4
0.8
—
−1.8
0.09
—
0.08

N
−999
0.8
0.5
0.04
—
−1.1
0.1
—
−1.2

P
−999
−0.5
0.3
−1.9
—
−0.2
0.07
—
−1.1

Q
−999
1.2
0
0.1
—
−1.8
0.2
—
−1.6

R
−999
2.2
0.7
−2.1
—
−1.8
0.09
—
−1

S
−999
−0.3
0.2
−0.7
—
−0.6
−0.2
—
−0.3

T
−999
0
0
−1
—
−1.2
0.09
—
−0.2

V
−1
2.1
0.5
−0.1
—
−1.1
0.7
—
0.3

W
0
−0.1
0
−1.8
—
−2.4
−0.1
—
−1.4

Y
0
0.9
0.8
−1.1
—
−2
0.5
—
−0.9

















































TABLE 40



Pocket potential for HLA0301B allele.












Amino










acid
P1
P2
P3
P4
P5
P6
P7
P8
P9














A
−999
0
0
0
—
0
0
—
0

C
−999
0
0
0
—
0
0
—
0

D
−999
−1.3
−1.3
2.3
—
−2.4
−0.6
—
−0.6

E
−999
0.1
−1.2
−1
—
−1.4
−0.2
—
−0.3

F
−1
0.8
0.8
−1
—
−1.4
0.5
—
0.9

G
−999
0.5
0.2
0.5
—
−0.7
0.1
—
0.4

H
−999
0.8
0.2
0
—
−0.1
−0.8
—
−0.5

I
0
1.1
1.5
0.5
—
0.7
0.4
—
0.6

K
−999
1.1
0
−1
—
1.3
−0.9
—
−0.2

L
0
1
1
0
—
0.2
0.2
—
−0

M
0
1.1
1.4
0
—
−0.9
1.1
—
1.1

N
−999
0.8
0.5
0.2
—
−0.6
−0.1
—
−0.6

P
−999
−0.5
0.3
−1
—
0.5
0.7
—
−0.3

Q
−999
1.2
0
0
—
−0.3
−0.1
—
−0.2

R
−999
2.2
0.7
−1
—
1
−0.9
—
0.5

S
−999
−0.3
0.2
0.7
—
−0.1
0.07
—
1.1

T
−999
0
0
−1
—
0.8
−0.1
—
−0.5

V
0
2.1
0.5
0
—
1.2
0.2
—
0.3

W
−1
−0.1
0
−1
—
−1.4
−0.6
—
−1

Y
−1
0.9
0.8
−1
—
−1.4
−0.1
—
0.3

















































TABLE 41



Pocket potential for HLA0301B allele.












Amino










acid
P1
P2
P3
P4
P5
P6
P7
P8
P9














A
−999
0
0
0
—
0
0
—
0

C
−999
0
0
0
—
0
0
—
0

D
−999
−1.3
−1.3
1.4
—
−1.1
−0.3
—
−1.7

E
−999
0.1
−1.2
1.5
—
−2.4
0.2
—
−1.7

F
0
0.8
0.8
−0.9
—
−1.1
−1
—
−1

G
−999
0.5
0.2
−1.6
—
−1.5
−1.3
—
−1

H
−999
0.8
0.2
1.1
—
−1.4
0
—
0.08

I
−1
1.1
1.5
0.8
—
−0.1
0.08
—
−0.3

K
−999
1.1
0
−1.7
—
−2.4
−0.3
—
−0.3

L
−1
1
1
0.8
—
−1.1
0.7
—
−1

M
−1
1.1
1.4
0.9
—
−1.1
0.8
—
−0.4

N
−999
0.8
0.5
0.9
—
1.3
0.6
—
−1.4

P
−999
−0.5
0.3
−1.6
—
0
−0.7
—
−1.3

Q
−999
1.2
0
0.8
—
−1.5
0
—
0.5

R
−999
2.2
0.7
−1.9
—
−2.4
−1.2
—
−1

S
−999
−0.3
0.2
0.8
—
1
−0.2
—
0.7

T
−999
0
0
0.7
—
1.9
−0.1
—
−1.2

V
−1
2.1
0.5
−0.9
—
0.9
0.08
—
−0.7

W
0
−0.1
0
−1.2
—
−1
−1.4
—
−1

Y
0
0.9
0.8
−1.6
—
−1.5
−1.2
—
−1

















































TABLE 42



Pocket potential for HLA0301B allele.












Amino










acid
P1
P2
P3
P4
P5
P6
P7
P8
P9














A
−999
0
0
0
—
0
0
—
0

C
−999
0
0
0
—
0
0
—
0

D
−999
−1.3
−1.3
−1.6
—
−2.5
−1.3
—
−1.2

E
−999
0.1
−1.2
−1.4
—
−2.5
0.9
—
−0.3

F
0
0.8
0.8
0.2
—
−0.8
2.1
—
2.1

G
−999
0.5
0.2
−1.1
—
−0.6
0
—
−0.6

H
−999
0.8
0.2
0.1
—
−0.8
0.9
—
−0.2

I
−1
1.1
1.5
1.1
—
−0.5
2.4
—
3.4

K
−999
1.1
0
−1.3
—
−1.1
0.5
—
−1.1

L
−1
1
1
−0.8
—
−0.9
2.2
—
3.4

M
−1
1.1
1.4
−0.4
—
−0.8
1.8
—
2

N
−999
0.8
0.5
−1.1
—
−0.6
1.4
—
−0.5

P
−999
−0.5
0.3
−1.2
—
−0.5
−0.2
—
−0.6

Q
−999
1.2
0
−1.5
—
−1.1
1.1
—
−0.9

R
−999
2.2
0.7
−1.1
—
−1.1
0.7
—
−0.8

S
−999
−0.3
0.2
1.5
—
0.6
0.4
—
−0.3

T
−999
0
0
1.4
—
−0.1
0.9
—
0.4

V
−1
2.1
0.5
0.9
—
0.1
1.6
—
2

W
0
−0.1
0
−1.1
—
−0.9
1.4
—
0.8

Y
0
0.9
0.8
−0.9
—
−1
1.7
—
1.1

















































TABLE 43



Pocket potential for HLA0301B allele.












Amino










acid
P1
P2
P3
P4
P5
P6
P7
P8
P9



A
−999
0
0
0
—
0
0
—
0

C
−999
0
0
0
—
0
0
—
0

D
−999
−1.3
−1.3
−0.4
—
−0.4
−0.7
—
−1.9

E
−999
0.1
−1.2
−0.6
—
−1
−0.7
—
−1.9

F
−1
0.8
0.8
2.4
—
−0.3
1.4
—
−0.4

G
−999
0.5
0.2
0
—
−0.5
0
—
−0.8

H
−999
0.8
0.2
1.1
—
−0.5
0.6
—
−1.1

I
0
1.1
1.5
0.6
—
−0.05
1.5
—
0.7

K
−999
1.1
0
−0.7
—
−0.3
−0.3
—
−1.7

L
0
1
1
0.5
—
−0.2
1.9
—
0.5

M
0
1.1
1.4
1
—
−0.1
1.7
—
0.08

N
−999
0.8
0.5
−0.2
—
−0.7
0.7
—
−1.2

P
−999
−0.5
0.3
−0.3
—
−0.2
0.3
—
−1.1

Q
−999
1.2
0
−0.8
—
−0.8
−0.3
—
−1.6

R
−999
2.2
0.7
0.2
—
1
−0.5
—
−1

S
−999
−0.3
0.2
−0.3
—
−0.6
0.3
—
−0.3

T
−999
0
0
−0.3
—
−0
0.2
—
−0.2

V
0
2.1
0.5
0.2
—
−0.3
0.3
—
0.3

W
−1
−0.1
0
0.4
—
−0.4
0.6
—
−1.4

Y
−1
0.9
0.8
2.5
—
−0.4
0.7
—
−0.9









































Example 46
Assessment of Insertion of XTEN into Permissive Loops
XTEN AE42-4 Insertion
The construction and expression of FVIII with XTEN AE42 insertions were described in Example 17 and 24. Thus, where residue X designates the site of insertion and residue Z designates the next residue in the native FVIII polypeptide sequence, the polypeptide resulting from insertion of XTEN AE42 would contain the sequence:







(SEQ ID NO: 1697)


X-GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS-Z









16 different sites in the FVIII sequence were selected for XTEN AE42 insertion, and these were designed Batch 1. An additional 21 sites selected for XTEN AE42 insertion were designed Batch 2. Collectively, the Batch 1 and Batch 2 sites represent 12 sites in the A1 domain, 7 sites in the A2 domain, 10 sites in the A3 domain, 4 sites in the C1 domain, and 3 sites in the C2 domain. Locations of Batch 1 and 2 sites in the 3-D structure of FVIII are depicted in FIG. 32 .
The location of these Batch 1 and Batch 2 insertion sites results in 37 constructs designated pSD0001-pSD0004, pSD0009-pSD0012, pSD0023-pSD0032, pSD0034-pSD0063 [the foregoing ranges include all intermediate numbers, as well], the sequences of which are set forth in Table 21 and the insertions sites of which are set forth in Table 23.
In Vitro Assays
To assess FVIII tolerability to XTEN AE42-4 insertion, the FVIII activity in culture media samples from FVIII-XTEN cell cultures was analyzed using a FVIII chromogenic assay. Antigen expression levels were analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain) ELISA.
FVIII Activity Measurement by Chromogenic Assay
The FVIII activity was measured using the COATEST® SP FVIII kit from DiaPharma (lot# N089019) and all incubations were performed on a 37° C. plate heater with shaking. Cell culture harvests from transient transfection media of FVIII-XTEN AE42-4 variants from 6 well plates were diluted to the desired FVIII activity range using 1×FVIII COATEST R buffer. FVIII standards were prepared in 1×FVIII COATEST R buffer containing mock transfection media with matching culture media concentration as the testing sample. The range of recombinant Factor VIII (rFVIII) standard was from 100 mIU/mL to 0.78 mIU/mL. The standards, diluted cell culture samples, and a pooled normal human plasma assay control were added to Immulon® 2HB 96-well plates in duplicates (25 μL/well).
Freshly prepared IXa/FX/Phospholipid mix (50 μL), 25 μL of 25 mM CaCl 2 , and 50 μL of FXa substrate were added sequentially into each well, with 5 minutes incubation between each addition. After incubating with the substrate, 25 μL of 20% acetic acid was added to terminate the color reaction, and the absorbance at 405 nm was measured with a SpectraMAX R plus (Molecular Devices) instrument.
Data analysis was performed using SoftMax Pro software (version 5.2). The Lowest Level of Quantification (LLOQ) was 39 mIU/mL. Results are presented in Table 22.
Expression Measurement by FVIII-HC and FVIII-LC ELISA
Expression of variants was quantified using ELISA. The FVIII antigen expression levels of DNA constructs corresponding to XTEN insertions in the A1 and A2 domains of FVIII were analyzed by FVIII-LC ELISA. The FVIII antigen expression levels of DNA constructs corresponding to XTEN insertions in the A3, C1 and C2 domains of FVIII were analyzed by FVIII-HC ELISA. Results are presented in Table 22.
FVIII-XTEN antigens in cell culture media after harvest were captured by GMA011 antibodies (Green Mountain Antibodies) for FVIII-LC ELISA) or by GMA016 antibodies (Green Mountain Antibodies) for FVIII-HC ELISA. Immulon® 2HB 96-well plates were coated with 100 μl/well of anti-FVIII antibody (2 μg/ml) by overnight incubation at 4° C. Plates were then washed four times with Phosphate Buffer saline with Tween-20 (PBST) and blocked with blocking buffer (PBST with 10% heat inactivated horse serum) for 1 hour at room temperature.
Cell culture harvests from transient transfection media of FVIII-XTEN variants from a 6-well plate were diluted to the desired FVIII antigen range using 1× blocking buffer. FVIII standards were prepared in 1×FVIII blocking buffer containing mock transfection media with matching media concentration as the testing samples. The range of rFVIII standard was from 50 ng/mL to 0.39 ng/mL.
Standards, diluted cell culture samples, and a pooled normal human plasma assay control were added into Immulon® 2HB 96-well plates in duplicates (100 μL/well) and incubated at 37° C. for 2 hours. Following four times washing with PBST, 100 μl of HRP-sheep anti-hFVIII antibody (Affinity Biologicals, F8C-EIC-D) were added into each well and plates were incubated for 1 hour at 37° C. After another four washes with PBST, 100 μl of TMB Super Sensitive Substrate (BioFX) were added to each well, followed by 5-10 min color development. To terminate the color reaction, 50 μL of H2SO4 were added to each well, and the absorbance of at 450 nm was measured with a SpectraMAX plus (Molecular Devices) instrument.
Data analysis was performed using SoftMax Pro software (version 5.4). The Lowest Level of Quantification (LLOQ) was 0.0039 μg/mL. Results are presented in Table 22.
Permissive sites into which XTEN sequences were inserted without eliminating procoagulant activity of the recombinant protein, or the ability of the recombinant proteins to be expressed in the host cell were clustered within loops in each of the three A domains of FVIII. FIG. 36 shows the location of insertion sites in the recombinant FVIII proteins that showed FVIII activity on domains A1, A2 and A3. FIG. 33 shows a structural representation depicting the location of insertion sites in the recombinant FVIII proteins that showed FVIII activity.
The permissive sites clustered in solvent exposed, highly flexible surface loops (XTEN permissive loops). The A1 domain loops were located in a region corresponding approximately to amino acid positions 15 to 45, and 201 to 232, respectively, in the sequence of mature human FVIII ( FIG. 30 ). The A2 domain loops were located in a region corresponding approximately to amino acid positions 395 to 421, and 577 to 635, respectively, in the sequence of mature human FVIII ( FIG. 30 ). The A3 domain loops were located in a region corresponding approximately to amino acid positions 1705 to 1732, and 1884 to 1917, respectively, in the sequence of mature human FVIII ( FIG. 30 ). FIGS. 37A and 37B show the location of the XTEN permissive loops relative to secondary structure elements in the tridimensional structure of FVIII.
Example 47
CFXTEN with Insertions of XTEN Having 144 Amino Acids
Analysis of the preliminary data presented above (Example 46) suggested the existence of defined regions within the linear polypeptide sequences and 3-D structures of the FVIII A domains that can accommodate the insertion of XTEN sequences. To test this hypothesis and further define the boundaries of putative regions that can accommodate the insertion of XTEN sequences without loss of FVIII activity, 23 additional insertion sites not present in either Batch 1 or 2 were chosen and designated Batch 3.
Batch 3 constructs were generated by the insertion of a 144 residue XTEN AE polypeptide, comprising amino acid residues Gly (G), Ala (A), Pro (P), Ser (S), Thr (T), and Glu (E), or a 144 residue XTEN AG polypeptide, comprising amino acid residues Gly (G), Ala (A), Pro (P), Ser (S), and Thr (T). Five different version of the 144 residue AE polypeptide were generated and designated XTEN-AE144-2A, XTEN-AE144-3B, XTEN-AE144-4A, XTEN-AE144-5A, XTEN-AE144-6B. The amino acid sequences are as set forth in Table 4. Five different versions of the 144 residue polypeptide were generated and designated XTEN-AG144-1, XTEN-AG144-A, XTEN-AG144-B, XTEN-AG144-C, and XTEN-AG144-F. The amino acid sequences are as set forth in Table 4.
The 144 residue XTEN encoding DNA sequence was introduced by the chemical synthesis of DNA segments (DNA 2.0, Redwood City, Calif.) spanning the nearest unique restriction sites within the base vector on either side of the site of insertion.
The DNA sequences corresponding to the XTEN 144 peptides were inserted such that the resulting DNA construct would encode a FVIII protein in which the XTEN 144 protein sequence is inserted immediately after the residue indicated in the site selection, and flanked by AscI and XhoI sites.
In addition to these sites, those sites from Batch 1 and 2 at which insertion of the XTEN AE42 polypeptide did not abolish FVIII procoagulant acitivity were modified by excision of the AE42 polypeptide encoding DNA segment with restriction enzymes AscI and XhoI, and introduction of XTEN AE144 and XTEN AG144 coding sequences at the same sites. The location of these Batch 1, Batch 2 and Batch insertion sites is summarized in Table III. FIG. 34 presents a structural representation of FVIII showing the location of the XTEN 144 insertion sites.
A total of 48 constructs with 144 XTEN inserts were created. The constructs are pSD0001-pSD0004, pSD0009-pSD0012, pSD0023-63 [the foregoing ranges include all intermediate numbers, as well], the sequences of which are set forth in Table 21 and the insertion sites of which are detailed in Table 22.
Expression of FVIII-XTEN 144 Variants
FVIII variants with XTEN 144 insertions were tranfected into HEK293F cells (Invitrogen, Carlsbad, Calif.) using polyethyleneimine (PEI, Polysciences Inc. Warrington, Pa.) or Lipofectamine transfection reagent (Invitrogen, Carlsbad, Calif.). The transiently transfected cells were grown in 293 Free Style medium or a mixture of 293 Free Style and CD Opti CHO media (Invitrogen, Carlsbad, Calif.). The cell culture medium was harvested 3-5 days after transfection and analyzed for FVIII expression by chromogenic FVIII activity assay and FVIII ELISA conducted as described herein.
Cell culture media from transient transfection were concentrated 10-fold in Centricon® spin columns (100 kd cut-off). Concentrated material was then flash frozen and stored at −80° C. for future in vitro analysis and in vivo PK studies.
In Vitro Assays
To assess FVIII tolerability to insertions, the FVIII activity in culture media samples from cell cultures was analyzed using a FVIII chromogenic assay. Antigen expression levels were analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain) ELISA.
FVIII Activity Measurement by Chromogenic Assay and Expression Measurement by FVIII-HC and FVIII-LC ELISA
Chromogenic and ELISA assay methods were conducted as described. The results obtained are summarized in Table 23.
Permissive sites into which XTEN sequences were inserted without eliminating procoagulant activity of the recombinant protein, or the ability of the recombinant proteins to be expressed in the host cell clustered within loops in each of the three A domains of FVIII. The same XTEN permissive loop regions tolerating the shorter XTEN sequences inserted were found to tolerate the insertion of the longer XTEN sequences. FIG. 38 shows the location of XTEN 144 insertion sites in the recombinant FVIII proteins that showed FVIII activity on domains A1, A2 and A3. FIG. 35 shows a structural representation depicting the location of insertion sites in the recombinant FVIII proteins that showed FVIII activity.
These observation indicate that two regions within each of the A domains of FVIII are able to accommodate insertion of XTEN sequences without loss of FVIII cofactor activity. A structural depiction of these so-called XTEN permissive loops ( FIGS. 40 and 41 ) demonstrate that they occupy structurally analogous positions in each of the A domains and project from one face of the FVIII molecule. The identified XTEN permissive loops correspond to highly flexible loops located between beta strands in the three-dimensional structures of the A1, A2, and A3 domains, as shown in FIGS. 37A and 37B .
The in vivo evaluation of XTEN 144 insertions on FVIII Half-life Extension, as determined by pharmacokinetics, is described in Example 32.
Example 48
Rescue or Enhancement of FVIII Expression by Insertion of an XTEN Sequence within the a3 Acidic Peptide Region of FVIII
Adherent HEK293 cells were transfected (as described in Example 24) with FVIII-XTEN DNA constructs in which the coding sequence of a B-domain deleted factor VIII contained 2 to 4 XTEN insertions of 144 amino acid residues each, of composition and insertion location as indicated in Table 44, below. At 5 days post-transfection, cell culture supernatants were assayed for FVIII activity by the chromogenic assay (as described in Example 25). Results are shown in Table 44.





TABLE 44



Expression levels of FVIII Activity by CFXTEN variants containing an

XTEN at position 1720 and one, two, or three additional XTEN insertions.







Activity

Construct
Domain, Position, and Type of XTEN Insertion
(mIU/









Name
A1
A2
a3
A3-1
A3-2
mL)











LSD0040.002
26


1720

175


AG144


AG144



LSD0041.008

403

1720

279



AE144

AG144



LSD0045.002


1656
1720

2598




AG144
AG144



PSD080.002
26

1656
1720

1081


AG144

AG144
AG144



PSD083.001

403
1656
1720

789



AE144
AG144
AG144



PSD082.001
26


1720
1900
<LLOQ


AG144


AG144
AE144


PSD090.003
26

1656
1720
1900
316


AG144

AG144
AG144
AE144












































For the purpose of comparison, all FVIII-XTEN constructs had an AG144 XTEN insertion at amino acid position 1720 (numbered relative to full-length factor VIII) within the A3 domain. Expression levels of FVIII-XTEN varians were determined by chromogenic assay and expressed in units of mIU/mL. Constructs with a single additional XTEN insertion at either position 26 in the A1 domain (LSD0040.002) or position 403 in the A2 domain (LSD0041.008) yielded expression levels of 175 and 279 mIU/mL, respectively. In contrast, a construct with a single additional XTEN insertion at position 1656 within the a3 acidic peptide yielded an expression level of 2598 mIU/mL, demonstrating enhancement of expression level for the a3 XTEN insertion construct relative to the A1 and A2 insertion constructs. In addition, in comparison to the FVIII-XTEN construct with XTEN insertions at positions 26 in the A1 domain and 1720 in the A3 domain (LSD0040.002), the construct with an additional XTEN insertion at position 1656 within the a3 acidic peptide region (PSD080.002) yielded significantly higher expression (175 and 1081 mIU/mL, respectively). Consistent with these findings, the construct with XTEN insertions at positions 403 in the A2 domain and 1720 in the A3 domain (LSD0041.008) yielded an expression level of 279 mIU/mL, whereas an additional XTEN insertion at position 1656 within the a3 acidic peptide region (PSD083.001) resulted in an increase in the expression level to 789 mIU/mL. Lastly, the FVIII-XTEN construct with an XTEN insertion at position 26 within the A1 domain and two XTEN insertions at positions 1720 and 1900 within the A3 domain (PSD082.001) did not yield activity above the lower limit of quantitation. However, the FVIII-XTEN construct with an additional XTEN insertion within the a3 acidic peptide region (PSD090.003) resulted in detectable activity, demonstrating that inclusion of an XTEN sequence within the a3 domain can result in recovery of expression (as measured by activity) in FVIII-XTEN constructs that are otherwise expressed at levels below the lower limit of quantitation. Under the conditions of the experiment, the results support the conclusion that insertion of XTEN at the 1656 position and, by extension, within the a3 region, results in enhanced expression of procoagulant FVIII-XTEN compositions.
Example 49
Effect of XTEN Insertion on FVIII Activity Measured by aPTT
A one stage activated partial prothrombin (aPTT) coagulation assay was employed in addition to the chromogenic assay (as described in Example 25) to determine FVIII activity of various FVIII-XTEN fusion proteins.
Method:
The FVIII-XTEN aPTT activity was measured using the Sysmex CA-1500 instrument (Siemens Healthcare Diagnostics Inc., Tarrytown, N.Y.). To create a standard curve for the assay, WHO factor VIII standard was diluted with 2% mock transfection media to 100 mU/mL and a two-fold serial dilution series was then performed, with the last standard being 0.78 mU/mL. FVIII-XTEN cell culture samples were first diluted at 1:50 with aPTT assay buffer, further dilutions were made with 2% mock transfection media when needed.
After dilution, the aPTT assay was performed using Sysmex instrument as follow: 501 of diluted standards and samples were mixed with 50 μl human FVIII deficient plasma and then 501 of aPTT reagent. The mixture was incubated at 37° C. for 4 min, and following incubation, 50 μl of CaCl 2 was added to the mixture, and the clotting time was measured immediately.
To determine test samples FVIII activity, the clotting time of the standards were plotted using semi-log scale (Clotting time: Linear; Standard concentration: Log) to extrapolates the equation between clotting time and FVIII activity, and FVIII-XTEN activity was then calculated against the standard curve. The assay sensitivity was 40 mU/mL factor VIII.
Results:
The results are summarized in FIGS. 44-46 . When single XTEN of 144 or 288 amino acids were inserted into the FVIII, all of the FVIII-XTEN fusion proteins exhibiting activity in the chromogenic assay were also active in aPTT assay. The aPTT activity followed the trend of chromogenic assay, for example, those molecules that showed low FVIII activity in the chromogenic assay also had low aPTT values. Generally, the aPTT results for the fusion proteins were lower than those obtained by the chromogenic assay, with a chromogenic to aPTT ratio of 1.1 up to 2.2, as illustrated in FIG. 44 , for the single XTEN insertions. The FVIII-XTEN fusion proteins with multiple XTEN insertions, in general, showed further reductions in aPTT activity in comparison to chromogenic assay. Assays of FVIII-XTEN with two XTEN insertions showed activity with all constructs, but with chromogenic/aPTT ratios approaching 4, in some instances ( FIG. 45 ). Assays of FVIII-XTEN with some three XTEN insertions also showed activity in both assays, with chromogenic/aPTT ratios approaching 5, in some instances ( FIG. 46 ), while the ratios for the BDD-FVIII control were more comparable (right side of FIG. 46 ). Additionally, the site of XTEN insertion appeared to contribute to the differences seen between aPTT and chromogenic activities. For example, while some molecules with 2 XTEN insertions resulted in up to 4-fold lower activity than chromogenic values, the aPTT activity of other FVII1 molecules with 2 XTEN were fairly comparable to chromogenic activity ( FIG. 45 ). Some molecules with 3 XTEN insertions showed up to 5-fold lower than chromogenic activities, other FVIII molecules with 3 XTEN have aPTT activity less than 2-fold lower than chromogenic activity ( FIG. 45 ). Under the conditions of the experiment, the results support the conclusion that FVIII-XTEN fusion protein constructs do retain procoagulant activity, but that the chromogenic assay generally provides higher activity levels than that in the aPTT assay system employed in the study.
Example 50
Evaluations of the Effect of XTEN Insertion Site Onf FVIII Half-Life Extension
Methods:
Six FVIII-XTEN fusion proteins with single XTEN AG-144 insertions at defined locations were tested in FVIII/VWF DKO mice (as generally described in Example 32) to evaluate the effect of XTEN insertion site on FVIII half-life. Six representative XTEN variants (listed in table 1) with XTEN insertion in either within A1, A2, a3, A3-region1 (A3-R1), A3-region 2 (A3-R2) or at the C-terminus were selected for this study, and BDD-FVIII generated from the base vector was used as the control. FVIII/VWF DKO mice were treated with a single intravenous administration of transient transfaction cell culture media concentrate from the six FVIII-XTEN constructs (or positive control media) at 100-200 IU/kg, and plasma samples were subsequently collected at 5 min, 7 hours and 16 hours post-dosing. Plasma FVIII activity was tested using the FVIII chromogenic assay and FVIII-XTEN half-life was estimated using the WinNonlin program. The study data are summarized in Table 45 and FIG. 47 .
Results:
A significantly longer half-life was observed for all FVIII-XTEN variants tested compared to BDD-FVIII control, but the degree of the half-life increase varied, with the variant with XTEN at the 403 insertion site conferring the least half-life extension at 10-fold (in comparison to control), while the 1900 insertion variant conferred the most half-life extension at 18-fold. The differences of XTEN insertion site on FVIII half-life extension may reflect the roles of different FVIII domains in FVIII clearance in vivo.





TABLE 45



FVIII-XTEN single AG-144 insertion

variants PK in FVIII/VWF DKO mice











BDD-
pSD-
pSD-
pSD-
pSD-
pSD-
pSD-

Treatment
FVIII
050
0003
0039
0010
063
014












Insertion
None
26
403
1656
1720
1900
CT

site








Recovery
21.3
33.8
34.8
36.0
33.6
39.6
32.4

t1/2
0.25
3.15
2.4
3.3
4.28
4.54
3.91

(hr)








t1/2

13
10
13
17
18
16

Increase








(fold)

































Example 51
Evaluations of the Additive Effect of XTEN Ilsertions on FVIII Half-Life Extension
Methods:
To evaluate the effects of multiple XTEN insertions on FVIII-XTEN fusion protein half-life, the half-lives of FVIII-XTEN variants with 1-3 XTEN insertions were determined in FVIII-XTEN DKO mice using the cell culture concentrate from five constructs (as generally described in Example 32). Five FVIII-XTEN variants were tested in the study: pSD-062, with AE144 insertion at position 1900 (numbered relative to full-length factor VIII); pSD-0005 with AE144 in the FVIII B domain (B-domain amino acid position 745); pSD-0019 with AE288 at the FVIII C-terminus (CT); LSD0003.006 with AE144 inserted in the B-domain and AE288 inserted at the C-terminus, and LSD0055.021 with three XTEN of AE144, AE144, and AE288 inserted at position 1900, with the B domain and at the C-terminus. The FVIII-XTEN half-life values were estimated using the WinNonlin program.
Results:
The study results are summarized in Table 46, and the PK curves are shown in FIG. 48 . The study results clearly demonstrated the additive effect of multiple XTEN insertions on FVIII half-life extension. With single XTEN insertions, the half-life of FVIII was extended from 0.25 hr to 3.2-4.0 hr, a 13 to 16-fold increase. When the B and CT XTEN insertions were combined together, the FVIII half-life was further extended to 10.6 hr, a 42-fold prolongation. Finally, in the case of a third XTEN insertion added at position 1900 to the B/CT construct, the half-life reached 16 hr in the FVIII-VWF DKO mice, a 64-fold increase.





TABLE 46



Additive effect of XTEN insertions

on FVIII t 1/2 in FVIII/VWF DKO mice










BDD-
pSD-
pSD-
pSD-
LSD-
LSD-

Treatment
FVIII
062
0005
0019
0003.006
0055.021











XTEN
None
1900
B
CT
B/CT
1900/

Insertion





B/CT

site







Recovery
21.3
35.3
44.9
33.3
39.0
37.2

t1/2
0.25
3.8
3.2
4.0
10.6
16.0

(hr)







t1/2

15
13
16
42
64

Increase







(fold)


































Example 52
Evaluation of FVIII-XTEN Interference with the Binding of Anti-FVIII Antibodies Using the Bethesday Assay
The ability of XTEN insertions in the FVIII molecule to interfer with binding by pre-existing anti-FVIII antibodies to the FVIII-XTEN fusion protein was evaluated in order to determine their utility in treating patients with anti-FVIII inhibitory antibodies.
Methods:
To assess the binding of anti-FVIII antibodies, two FVIII-XTEN variants (PSD088, with 144 XTEN inserted at the locations of 26/403/1656/1900; and PSD-090, with 144 XTEN inserted at the locations of 26/1656/1720/1900) were tested in comparison with Refacto (a marketed rFVIII) against plasma samples from three hemophilia A patients with factor VIII inhibitors (designated 04-483, 05-505, and GK1838-2079), as well as a sheep anti-FVIII poly-clonal antibody from Affinity Biologicals Inc (F8C-EIA-C). The Bethesda titer of the four anti-FVIII ab against the two FVIII-XTEN variants (pSD-088 and pSD-090) and the Refacto control were determined using modified Bethesda assay methods, detailed as follows. Heat inactivated anti-FVIII antibody samples at various dilutions were incubated with 1 IU/mL of each FVIII variant (diluted in 1× in FVIII chromogenic assay buffer) at a 1:1 ratio. The FVIII/antibody mixtures were then incubated for 2 hours in a 37° C. incubator. After the incubation, the samples were diluted for 10-fold with 1×FVIII chromogenic assay buffer, and 25 μL of diluted mixture were then used for a FVIII chromogenic assay, The percentage of remaining FVIII activity was calculated against the post-incubation activity of a known non-neutralizing sample. Bethesda units were calculated using the following formula: BU=dilution factor X (LN(percent of remaining activity)+6.6438).
Results:
The results are listed in Table 47. Decreased Bethesda unit (BU) titers were observed for all four antibodies when tested against the two FVIII-XTEN variants, in comparison with Refacto. A 5 to 8-fold fold decrease against PSD-088 and a 3 to 5-fold decrease against pSD-090, respectively, were obtained. The inhibition curves against FVIII variants for each antibody were plotted ( FIG. 49 ) and compared to Refacto, and demonstrates a clear left-shift of the inhibition curve for the two FVIII-XTEN molecules, with the pSD-088 FVIII-XTEN variant resulting in a further left-shift compared to pSD-090. These results clearly demonstrate that: 1) both FVIII-XTEN variant fusion proteins are more resistant to pre-existing anti-FVIII inhibitory antibodies than Refacto; and 2) PSD-088 is more resistant to anti-FVIII antibodies than PSD-090, which may provide information useful in determining the differences on the XTEN insertion sites in interferring with the binding of anti-FVIII antibodies. Under the conditions of the experiment, the results provide some support for the potential use of FVIII-XTEN compositions for treating hemophilia A patients with factor VIII inhibitors.





TABLE 47



Anti-FVIII antibody Bethesda titer against FVIII-XTEN variants





Anti-FVIII ab.







FVIII
04-483
05-505
GK1838-2079
F8C-EIA-C









pSD-088
2.5
8
47
57

(16/403/1656/1900)





pSD-090
3.4
12
55
96

(26/1656/1720/1900)





Refacto
12
66
268
337
































Example 53
Half-Life Evaluations of FVIII XTEN Fusion Molecules Containing Four XTEN Insertions in Hemophilia A Mice
Methods:
Eight FVIII-XTEN fusion proteins with four XTEN insertions each at defined locations were tested in FVIII/VWF DKO mice to evaluate the effect of the XTEN insertions on FVIII half-life extension: LSD0071.001, contains 403-AG144, 1900-AE144, 745 (B)-AE144, 2332 (CT)-AE288 XTEN insertions (designated as the FVIII amino acid number and the XTEN inserted); LSD0071.002, containing 403-AE144, 1900-AE144, 745(B)-AE144, 2332(CT)-AE288 XTEN insertions; LSD0072.001, containing 403-AG144, 1900-AG144, 745(B)-AE144, 2332(CT)-AE288 XTEN insertions; LSD0072.002, containing 403-AE144, 1900-AG144, 745(B)-AE144, 2332(CT)-AE288 XTEN insertions; pBC0247.004, containing 18-AG144, 403-AE144, 1656-AG144, 2332(CT)-AE288 XTEN insertions; pBC0251.002, containing 18-AG144, 1656-AG144, 1900-AE144, 2332(CT)-AE288 XTEN insertions; pSD088, containing 26-AG144, 403-AE144, 1656)-AG144, 1900-AE144 XTEN insertions and pSD090, containing 26-AG144, 1656-Ag144, 1720-AG144, 1900-AE144 XTEN insertions. FVIII/VWF DKO mice were treated, as generally described in Example 32, with a single intravenous administration of FVIII-XTEN transfection cell media concentrate of the eight constructs at 100-200 IU/kg, and plasma samples were subsequently collected at 5 min, 8 hrs, 24 hrs, 48 hrs, 72 hrs and 96 hrs post-dosing. Plasma FVIII activity was tested using the FVIII chromogenic assay and FVIII-XTEN half-life was estimated using the WinNonlin program.
Results:
All of the eight FVIII XTEN fusion molecules containing four XTEN insertions exhibited longer half-life than unmodified FVIII (results in Table 48). Three molecules with XTEN insertions at positions 403, 1900, B domain, and C-terminal achieved half-life up to 16.3 hrs, which is a 65-fold improvement in comparison to unmodified BDD FVIII. However, the molecules tested with XTEN insertions at 26/403/1656/1900 (pSD088), or at 26/1656/1720/1900 (pSD090) showed half-life of 9.1 hrs and 9.5 hrs, respectively, which, in comparison to BDD FVIII, represents an increase of 36-fold and 38-fold, respectively. pBC247.004 (XTEN insertions at 18/403/1656/CT) and pBC251.002 (XTEN insertions at 18/1900/1656/CT) achieved half-life values of 14.1 hrs and 13 hrs, respectively. The results demonstrate that multiple XTEN insertions (in this case, four XTEN insertions for each FVIII molecule) can significantly improve FVIII half-life. It further shows that the effect of XTEN on FVIII half-life is insertion site dependent, even in the event of multiple XTEN insertions.





TABLE 48



PK of FVIII-XTEN variants with four XTEN

insertions in FVIII/VWF DKO mice








t1/2
t1/2

Treatment
XTEN Insertions
(hr)
Increase (fold)








BDD-FVIII
None
0.25
NA

LSD0071.001
403AG/1900AE/B/CT
16.2
64.8

LSD0071.002
403AE/1900AE/B/CT
16.3
65.2

LSD0072.001
403AG/1900AG/B/CT
11.8
47.2

LSD0072.002
403AE/1900AG/B/CT
16.1
64.4

pBC247.004
18/403/1656/CT
14.1
56.4

pBC251.002
18/1900/1656/CT
13.0
52

pSD088
26/403/1656/1900
9.1
36.4

pSD090
26/1656/1720/1900
9.5
38







































TABLE 49



Exemplary Biological Activity, Exemplary Assays and Preferred Indications






Biologically

Exemplary Activity
Preferred

Active Protein
Biological Activity
Assays
Indication:



Factor VIII
Coagulation factor VIII is a
Chromogenix assay
Hemophilia A;

(Factor VIII;
factor essential for hemostasis.
(Rosen S, Scand J
bleeding;

Octocog alfa;
This gene encodes coagulation
Haematol (1984) 33
Factor VIII

Moroctocog
factor VIII, which participates
(Suppl 40): 139-45);
deficiency;

alfa;
in the intrinsic pathway of
Chromogenix
bleeding

Recombinant
blood coagulation; factor VIII
Coamatic ® Factor
episodes in

Antihemophilic
is a cofactor for factor IXa
VIII assay; one-stage
patients with

factor;
which, in the presence of Ca +
clotting assay
factor VIII

Nordiate;
2 and phospholipids, converts
(Lethagen, S. , et al. ,
inhibitor;

ReFacto;
factor X to the activated
Scandinavian J
Surgery-related

Kogenate;
form Xa. This gene produces
Haematology (1986)
hemorrhagic

Kogenate
two alternatively spliced
37: 448-453.
episodes

SF; Helixate;
transcripts. Transcript variant
One-stage clotting


Recombinate)
I encodes a large glycoprotein,
assay and two-stage



isoform a, which circulates in
clotting assay



plasma and associates with
(Barrowcliffe TW,



von Willebrand factor in a
Semin Thromb



noncovalent complex. This
Hemost. (2002)



protein undergoes multiple
28(3): 247-256);



cleavage events. Transcript
Development of a



variant 2 encodes a puntative
simple chromogenic



small protein, isoform b, which
factor VIII assay



consists primarily of the
for clinical use.



phospholipid binding domain
(Wagenvoord RJ,



of factor VIIIc. This binding
Hendrix HH, Hemker



domain is essential for
HC. Haemostasis



coagulant activity. Defects in
1989; 19(4): 196-204)



this gene results in hemophilia
Bethesda assay



A, a common recessive
(Verbruggen B, et al.



X-linked coagulation disorder.
Improvements in factor




VIII inhibitor detection:




From Bethesda to




Nijmegen. Semin




Thromb Hemost. 2009




Nov; 35(8): 752-759)





























































TABLE 50



Exemplary CFXTEN comprising FVIII and internal/external XTEN sequences (SEQ ID

NOS 1537-1554, respectively, in order of appearance)




CFXTEN


Name
Amino AcidSequence



FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-K127-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AE144-
REKEDDKGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE

V128-N745-
GSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPG

AE288-
SEPATSGSETPGTSTEPSEGSAPGVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL

P1640-
VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA

Y2332)
RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQAS


LEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYD


DDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY


KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQA


SRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRY


YSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRF


LPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGY


TFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTG


DYYEDSYEDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPES


GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE


PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP


ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG


TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLK


RHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEV


EDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTK


DEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS


WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMG


SNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAG


MSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW


IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS


SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF


TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS


MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH


QIALRMEVLGCEAQDLY



FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-A375-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AE576-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

K376-N745-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

AE144-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

P1640-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD

Y2332)
DNSPSFIQIRSVAGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS


TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT


STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG


TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP


SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG


PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST


EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE


SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT


SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS


EGSAPGKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRF


MAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL


PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI


CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIM


HSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE


TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNA


IEPRSFSQNGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS


EGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGP


GSEPATSGSETPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDF


DIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEF


TDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGA


EPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCH


TNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHA


INGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG


VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ


YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMY


SLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELM


GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPK


EWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGN


QDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY



FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-Y1792-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AF144-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

E1793-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

Y2332-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

AE864)
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR


SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG


ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYGGTSTPESGSASPGTSPSGESS


TAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGT


STPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGEEDQRQ


GAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLV


CHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF


HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNL


YPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITA


SGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFI


IMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME


LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN


PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ


GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEE


GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES


ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS


APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS


TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP


ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG


TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP


SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG


PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA


GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS


ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT


SESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS


EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP


GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTE


PSEGSAP



FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-Y2043-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AG144-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

G2044-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

Q2222-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

AG864-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD

V2223-
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY

Y2332)
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR


SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG


ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNET


KTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV


TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLV


MAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKA


GIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGPGSSPSASTGT


GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGT


PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSG


TASSSGGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS


QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR


MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQG


GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPS


ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSST


GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG


SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG


TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG


SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA


SPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG


ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS


PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP


GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG


ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS


PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSST


PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGA


TGSPGSSTPSGATGSPGASPGTSSTGSPGVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS


MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH


QIALRMEVLGCEAQDLY



FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFN

(A1-G1799-
IAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTS

AE144-
QREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLV

A1800-
CREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY

F2093-
VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL

AE42-
LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV

S2094-
RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQR

V2223-
IGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT

AE42-
DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERD

N2224-
LASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLE

AE42-
DPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYE

N2225-
DTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYED

G2278-
ISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE

AE42-
DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGS

K2279-
FTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGGGSEP

Y2332)
ATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG


SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP


GTSTEPSEGSAPGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEK


DVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQ


MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR


KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPL


GMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT


QGARQKFGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPGSSLYISQFIIMYS


LDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMG


CDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVGPAGS


PTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGGNNPKEWLQVDFQKTMKVTGVTT


QGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGGTEPSEGSAPGSPAGSPTSTEEGTSESAT


PESGPGSEPATSGSKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG


CEAQDLY



FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDARGPGSSPSASTGTGPGSSPSASTGTGPGTPGSG

(A1-R28-
TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS

AG144-F29-
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSGFPPRVPKSFPFNTSV

G244-
VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSY

AG288-
WKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL

L245-
VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA

R2090-
RAWPKMHTVNGYVNRSLPGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP

AG576-
SGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT

Q2091-
GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG

Y2332-
SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA

AG864)
STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGLIGCHRKSVY


WHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH


DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK


HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF


KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK


DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQR


GNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDS


LQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP


GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNP


PVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAA


VERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEF TDGSFTQPLYRGELNEHLGLLGPYI


RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM


APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD


ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAGINGYIMDTLPGLVMAQDQRIRWY


LLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGE


HLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWST


KEPFSWIKVDLLAPMIIHGIKTQGARGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS


PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS


GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE


GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA


TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTST


EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS


TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE


GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG


TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA


TPESGPGTSTEPSEGSAPGQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS


GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFT


NMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM


YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQ


IALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST


EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP


AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE


GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG


TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA


TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA


PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE


SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG


SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT


STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT


PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES


ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS


APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI

(A1-T1651-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AG576-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

R1652-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

K1808-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

AG144-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD

P1809-
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY

F2093-
KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL

AG288-
YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI

S2094-
GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA

Y2332)
SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLR


QSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL


RLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDT


TLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGP


ALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP


LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPE


SARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLK


EMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKN


LFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQI


VEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTP


STLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSS


HLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKK


VENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWN


EANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDT


ILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITGPGTPGSGT


ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP


GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASP


GTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST


GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG


SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG


TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG


SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS


TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT


ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSSGRTTLQSDQEEI


DYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRA


QSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS


FYSSLISYEEDQRQGAEPRKNFVKGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG


ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS


PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGPNETKTYFWKVQHHMAPTKD


EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSW


YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS


NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGM


STLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWI


KVDLLAPMIIHGIKTQGARQKFGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST


PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA


TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP


GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS


ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGSSLYISQFII


MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME


LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN


PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ


GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY



FVIII BDD2
ATRRYYLGAVELSWDYMQSDLGELPVDAGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSG

(A1-A28-
ATGSPGPSGPGRFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEV

AG42-F29-
YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEGGPGTPGSGTASSSPG

E124-
SSTPSGATGSPGSSPSASTGTGPGASPGDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYS

AG42-
YLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGGSPSASTGTGPGAS

D125-E124-
PGTSSTGSPGTPGSGTASSSPGSSTPSGAGKSWHSETKNSLMQDRDAASARAWPKMHTVN

AG42-
GYVNSSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQT

D125-P333-
LLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPGSASTGTGPGASPGTSSTGSPGTPGSG

AG42-
TASSSPGSSTPSGATGGQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK

Q334-
HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF

Y2332)
KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK


DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQR


GNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDS


LQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP


GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNP


PVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAA


VERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI


RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM


APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD


ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL


LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH


LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTK


EPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFF


GNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQIT


ASSYFTNMFATWTPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK


SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ


SWVHQIALRMEVLGCEAQDLY



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI

(A1-D345-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AE144-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

Y346-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

D403-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

AE144-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDGGSEPATSGSETPGTSES

R405-
ATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSE

R1797-
TPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGYD

AE288-
DDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDGGT

Q1798-
STPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAE

Y2322)
SPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPG


TSPSGESSTAPGRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLY


GEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTV


EDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVF


DENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS


IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGM


TALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPEN


DIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDS


NNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTI


PSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESG


LMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRK


THIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKN


MEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGP


EKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKI


QEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLN


DSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALK


QFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI


PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNL


SLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKD


LFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLA


WDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWA


KQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR


SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG


ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRGGTSESATPESGPGSE


PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT


STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG


TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT


SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG


PGTSTEPSEGSAPGQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL


EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI


QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR


KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG


MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQG


ARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL


HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHL


QGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQW


TLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY



FVIII (A1-
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

N745)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AE864-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

(P1640-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

Y2332)
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD


LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT


STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP


TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP


GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE


PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES


GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS


TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP


ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG


TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP


SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG


PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA


GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE


SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS


EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTIS


VEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQ


FKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISY


EEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG


LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTF


KENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK


MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHI


RDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFS


SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI


RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNA


WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQN


GKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY



FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-N745)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AE288-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

(P1640-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

Y2332)
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD


LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT


SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT


PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP


GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE


PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQ


EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN


RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP


YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD


LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC


NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT


VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP


LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT


QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI


RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL


HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH


QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ


DLY



FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-S743)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AE288-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

(Q1638-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

Y2332)
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD


LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES


ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES


GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS


ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE


GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQREITRTTLQSDQE


EIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR


AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP


YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD


LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC


NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT


VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP


LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT


QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI


RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL


HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH


QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ


DLY



FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-N745)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AG288_2-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

(P1640-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

Y2332)-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

AG288_2
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG


TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA


STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT


GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA


SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGPPVLKRHQREITRTTLQS


DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL


RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQA


SRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS


DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR


APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGH


VFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC


QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMBH


GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII


ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSK


ARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ


DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGC


EAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG


SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS


PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST


GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP


GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSP


ATG



FVIII BDD9
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI

(A1-S743)-
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

AG288_2-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

(Q1638-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

Y2332)-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

AG288_2
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP


GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST


GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP


GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP


GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGQNPPVLKRHQREITRTTLQS


DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL


RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQA


SRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS


DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR


APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGH


VFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC


QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMBH


GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII


ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSK


ARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ


DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGC


EAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG


SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS


PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST


GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP


GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSP


ATG



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI

BDD10
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

(A1-N745)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

AE288-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

(P1640-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD

AE288
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT


SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT


PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP


GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE


PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQAEITRTTLQSDQ


EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN


RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP


YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD


LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC


NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT


VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP


LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT


QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI


RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL


HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH


QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ


DLYGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG


SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES


GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE


PATSGSETPGTSESATPESGPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI

BDD10
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

(A1-S743)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

AE288-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

(Q1638-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD

AE288
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES


ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES


GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS


ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE


GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQAEITRTTLQSDQE


EIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR


AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP


YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD


LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC


NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT


VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP


LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT


QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI


RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL


HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH


QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ


DLYGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG


SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES


GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE


PATSGSETPGTSESATPESGPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI

BDD10
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

(A1-N745)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

AG288_2-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

(P1640-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD

AG288_2
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG


TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA


STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT


GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA


SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSD


QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR


NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQAS


RPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD


VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA


PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF


TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQT


PLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMBHGIK


TQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARY


IRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL


HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH


QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ


DLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG


TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA


STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT


GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA


SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPAT


G



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI

BDD10
AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ

(A1-S743)-
REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR

AG288_2-
EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN

(Q1638-
RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD

Y2332)-
LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD

AG288_2
DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI


GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA


SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP


FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS


KNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP


GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST


GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP


GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP


GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQNPPVLKRHQAEITRTTLQSD


QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR


NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQAS


RPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD


VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA


PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF


TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQT


PLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIK


TQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARY


IRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL


HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH


QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ


DLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG


TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA


STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT


GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA


SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPAT


G



Sequence name reflects N- to C-terminus configuration of the FVIII segments (amino acid spanning numbers relative to mature sequence) and XTEN components
























































































































































































































































































































































































































































































































































































































































































































TABLE 51



Exemplary CFXTEN comprising FVIII, cleavage sequences and XTEN sequences (SEQ

ID NOS 1555-1590, respectively, in order of appearance)




CFXTEN


Name
Amino AcidSequence



SP-AE288-
MQIELSTCFFLCLLRFCFSGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP

CS-L-
GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA

(FVIII_1-
TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP

745)-
GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS

AE288-
EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQGPEGPSATRRYYLGAVE

(FVIII 1686-
LSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLG

2332)-L-
PTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSH

CS-AE288
TYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI


LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWH


VIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGM


EAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT


WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAI


QHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEI


FKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK


RNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV


AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDF


RNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGTSESATPESGPGSEP


ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST


EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST


EPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE


TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST


EPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD


GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK


NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP


AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD


TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEML


PSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLA


RLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY


RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG


MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKV


TGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLL


TRYLRIHPQSWVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGS


ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS


ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS


APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA


GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS


AP



SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE

CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS

(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP

745)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS

AE576-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG

(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

2332)-L-
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS

CS-AE288
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP


ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS


PRSFQGPSGPATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFV


EFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEY


DDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGA


LLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNG


YVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL


MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD


DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY


KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLY


SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP


LLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI


MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE


TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE


PRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA


PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES


ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA


PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP


SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP


GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS


EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG


TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP


TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHY


FIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLG


PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH


MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD


ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLS


MGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHA


GMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW


IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG


IKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNM


FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE


FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME


VLGCEAQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT


SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP


GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA


TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP


GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



SP-
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV

(FVIII_1-
VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW

745)-
KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK

AE576-
DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW

(FVIII_1686-
PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI

2332)-L-
TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD

CS-AE576
SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN


GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH


GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER


DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED


PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL


TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL


LSKNNAIEPRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS


TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS


TEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS


TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS


APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP


ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS


APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP


ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES


GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPR


SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGE


LNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKT


YFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQ


EFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD


QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE


CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINA


WSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM


VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQ


ITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK


SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS


WVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGSPAGSPTSTEEGTSESATPESGPGTSTEPS


EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG


SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP


TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG


SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP


TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG


TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE


GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT


STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE


SGPGTSTEPSEGSAP



SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE

CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS

(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP

745)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS

AE576-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG

(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

2332)
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS


EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP


ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS


PRSFQGPEGPSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF


VEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE


YDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIG


ALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN


GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL


LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF


DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRK


YKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG


PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI


MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE


TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE


PRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA


PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES


ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA


PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP


SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP


GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS


EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG


TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP


TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHY


FIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLG


PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH


MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD


ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLS


MGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHA


GMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW


IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG


IKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNM


FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE


FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME


VLGCEAQDLY



SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE

CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS

(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP

743)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS

AE288-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG

(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

2332)-L-
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS

CS-AE576
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP


ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPIEP


RSPSGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT


VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDD


QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL


VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY


VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLM


DLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYK


KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSR


RLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI


CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS


INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVF


MSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRS


FSGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE


PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE


EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA


TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP


GTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQS


GSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYS


SLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV


HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDP


TFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK


MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRD


FQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI


SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR


MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVN


NPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQG


NQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGIEPRSPSGSPAGS


PTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP


GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS


EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG


TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP


ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT


STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG


SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE


PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS


TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP



SP-AG288-
MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG

CS-L-
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP

(FVIII_1-
SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT

743)-
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP

AG576-
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSIEPRSPSGSPGATRRYYLGAVEL

(FVIII_1686-
SWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGP

2332)-L-
TIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT

CS-AG288
YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFIL


LFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHV


IGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGME


AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTW


VHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQ


HESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIF


KYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKR


NVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVA


YWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFR


NRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGTPGSGTASSSPGSSTPS


GATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP


GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT


SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP


GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS


GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP


GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS


GATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP


GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA


STGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR


NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP


YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL


EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQ


MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKK


EEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMAS


GHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQK


FSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI


RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAW


RPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKV


KVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQ


PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTP


SGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG


PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG


TSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG


PGTPGSGTASSSPGSSTPSGATGS



SP-AG576-
MQIELSTCFFLCLLRFCFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT

CS-L-
GPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG

(FVIII_1-
SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS

745)-
SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP

AG288-
GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT

(FVIII_1686-
GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASP

2332)-L-
GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG

CS-AE576
SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP


GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG


SQSPRSFQGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF


VEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE


YDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIG


ALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN


GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL


LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF


DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRK


YKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL


YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG


PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI


MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE


TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE


PRSFSQNPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS


SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP


SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT


GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSP


SASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL


RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASR


PYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD


LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI


QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRK


KEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA


SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQ


KFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHY


SIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNA


WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNG


KVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPR


SFQGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS


TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES


GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE


SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS


APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE


SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS


APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST


EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP



SP-
MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV

(FVIII_1-
VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW

743)-
KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK

AG576-
DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW

(FVIII_1686-
PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI

2332)-L-
TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD

CS-AG576
SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN


GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH


GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER


DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED


PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL


TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL


LSKNNAIEPRSFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSST


PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS


SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP


GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG


SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP


GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG


SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG


SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG


SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRS


FQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGEL


NEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTY


FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQE


FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD


QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE


CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINA


WSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM


VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQ


ITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK


SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS


WVHQIALRMEVLGCEAQDLYGSPGQSPRSFQPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST


GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG


ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS


TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG


SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG


ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG


ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT


ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG


SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS


TGTGPGASPGTSSTGS



SP-AG288-
MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG

CS-L-
SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP

(FVIII_1-
SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT

743)-
GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP

AG288-
GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQGPSGPATRRYYLGAV

(FVIII_1686-
ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLL

2332)-L-
GPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGS

CS-AE288
HTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHK


FILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYW


HVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDG


MEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPK


TWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREA


IQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGE


IFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK


RNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV


AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDF


RNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGASPGTSSTGSPGASPG


TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS


PGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS


GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS


PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP


SGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDG


SFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKN


FVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPA


HGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTL


PGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS


KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL


HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG


NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME


SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG


VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR


YLRIHPQSWVHQIALRMEVLGCEAQDLYGPSGPQSPRSFQGTSESATPESGPGSEPATSGSETP


GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA


TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP


GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS


PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



SP-AE576-
MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE

CS-L-
GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS

(FVIII_1-
PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP

743)-
GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS

AG576-
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG

(FVIII_1686-
SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

2332)
GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS


EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP


ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS


PRSFQGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVE


FTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYD


DQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGAL


LVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY


VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLM


DLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD


DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYK


KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSR


RLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI


CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS


INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVF


MSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRS


FSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSS


TPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST


GSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGAS


PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS


SSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS


PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST


GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS


TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS


SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIA


AVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI


RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAP


TKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS


WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS


NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS


TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKV


DLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKH


NIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFAT


WSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLI


SSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL


GCEAQDLY



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA

BDD2
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

S367-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

FXIa-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNSSLPGL

AE42-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF

F368-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSKLT

Y2332-
RAETGEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSGFIQIRSVAKKHPKTWVHY

FXIa-
IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESG

AE864
ILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYK


WTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVIL


FSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY


ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG


MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSD


QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR


AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS


FYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEK


DVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQME


DPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEY


KMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIR


DFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL


YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTL


RMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWTPSKARLHLQGRSNAWRPQV


NNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ


GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGSPAGSP


TSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG


TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE


GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT


STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE


SGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS


TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS


APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP


ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST


EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE


SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES


GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE


SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST


EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA

BDD2
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

N745-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

FIXa-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL

AG288-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF

FIXa-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI

P1640-
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY

Y2332-
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK

FIXa-
HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD

AG864
QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPLGR


IVGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG


SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS


TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG


ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS


TGTGPGTPGSGTASSSPGSSTPSGATGSGPLGRIVGGPPVLKRHQREITRTTLQSDQEEIDYDDT


ISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQ


FKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYE


EDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG


PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN


YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYN


LYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITAS


GQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIM


YSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMG


CDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEW


LQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSF


TPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYPLGRIVGGGASPGTSSTGSPG


SSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS


STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG


TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS


TGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG


ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS


TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG


SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG


ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG


ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT


ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG


SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS


TGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG


SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA

BDD2
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

V128-
EDDKVLQVRIVGGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGPGLQVRIVGG

FVIIa-
FPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQ

AG42-
TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRK

FVIIa-
SVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSH

G2044-
QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK

FVIIa-
KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF

AG144-
KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDF

Y2332-
PILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQ

FVIIa-
IMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSV

AG576
CLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGC


HNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQRE


ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS


SSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF


RNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWA


YFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC


RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHV


FTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP


LGMASGHIRDFQITASGQYGLQVRIVGGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTP


SGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS


PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGLQVRIVGGQWAPKLARLHYSGS


INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT


LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD


AQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG


VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP


QSWVHQIALRMEVLGCEAQDLYLQVRIVGGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST


GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG


ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS


TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG


SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG


ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG


ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT


ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG


SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS


TGTGPGASPGTSSTGS



AE864-
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS

FVIII-
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG

Thrombin-
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP

AE144
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT


SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE


SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS


TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES


GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA


GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA


PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG


SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI


AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE


KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS


LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG


LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL


FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ


IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP


STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY


ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF


KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE


NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT


SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN


KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ


LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ


EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS


LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL


KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI


PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS


LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP


TETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDN


HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT


ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR


HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL


LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH


HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF


DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL


LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH


AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS


WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS


SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN


MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV


KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR


MEVLGCEAQDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGS


PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAP


GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA

BDD3-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

FXIIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AE144
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYGTMTRIVGGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTS


TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES


GPGSEPATSGSETPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA

BDD3-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

Elastase-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AE144
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP


GSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPAT


SGSETPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA

BDD3-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AE144
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST


EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES


GPGSEPATSGSETPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA

BDD3-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

Thrombin-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AE144
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTS


TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES


GPGSEPATSGSETPGTSTEPSEGSAP



AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT

FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP

BDD2-
GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT

MMP-17-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG

AE864
AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL


IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT


VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ


TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV


RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG


RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR


PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL


IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS


NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS


GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN


AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK


TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL


GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV


QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF


TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW


YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH


LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP


FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV


DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF


TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM


YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA


LRMEVLGCEAQDLYGAPLGLRLRGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA


GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE


TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST


EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA


PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA


PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP


SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS


PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP


GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT


SGSETPGTSESATPESGPGTSTEPSEGSAP



AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT

FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP

BDD2-
GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT

FXIIa-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG

AE864
AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL


IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT


VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ


TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV


RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG


RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR


PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL


IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS


NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS


GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN


AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK


TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL


GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV


QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF


TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW


YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH


LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP


FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV


DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF


TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM


YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA


LRMEVLGCEAQDLYGTMTRIVGGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA


GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE


TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST


EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA


PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA


PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP


SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS


PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP


GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT


SGSETPGTSESATPESGPGTSTEPSEGSAP



AG144-
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT

FVIII
GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP

BDD2-
SASTGTGPGASPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT

FXIa-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG

AG576
AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL


IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT


VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ


TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV


RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG


RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR


PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL


IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS


NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS


GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN


AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK


TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL


GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV


QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF


TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW


YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH


LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP


FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV


DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF


TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM


YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA


LRMEVLGCEAQDLYGKLTRAETGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP


SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG


SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPG


SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATG


SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSP


SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG


SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST


PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT


GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP


GTSSTGS



AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT

FXIa-FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP

BDD2-
GTSTEPSEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT

AE864
SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVS


YWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL


VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR


AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEI


SPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL


TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYL


NNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIY


PHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM


ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL


EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYED


TLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISA


YLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ


SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLY


RGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNE


TKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV


TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVM


AQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIW


RVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS


INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT


LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD


AQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG


VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP


QSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA


GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE


TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST


EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES


/ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA


PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA


PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP


SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS


PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP


GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT


SGSETPGTSESATPESGPGTSTEPSEGSAP



AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT

FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP

BDD2-
GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT

Y2332-
LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG

Thrombin-
AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL

AE864
IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT


VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ


TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV


RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG


RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR


PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL


IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS


NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS


GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN


AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK


TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL


GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV


QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF


TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW


YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH


LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP


FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV


DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF


TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM


YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA


LRMEVLGCEAQDLYGLTPRSLLVGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA


GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE


TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST


EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA


PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA


PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP


SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS


PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP


GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT


SGSETPGTSESATPESGPGTSTEPSEGSAP



AE864-
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS

FVIII-
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG

MMP-17-
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP

AE144
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT


SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE


SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS


TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES


GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA


GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA


PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG


SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI


AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE


KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS


LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG


LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL


FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ


IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP


STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY


ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF


KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE


NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT


SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN


KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ


LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ


EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS


LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL


KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI


PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS


LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP


TETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDN


HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT


ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR


HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL


LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH


HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF


DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL


LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH


AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS


WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS


SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN


MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV


KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR


MEVLGCEAQDLYGAPLGLRLRGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGS


PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAP


GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP





AF144-
GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTA

FXIIa-
ESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGT

FVIII-
SPSGESSTAPGTMTRIVGGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV

FXIIa-
VYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW

AF864
KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK


DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW


PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI


TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD


SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN


GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH


GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER


DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED


PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL


TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL


LSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLL


RQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLR


LNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLF


GKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLT


KDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR


MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQ


RTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSR


NLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQN


VEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRI


SPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKE


KGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSG


VQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTS


GKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATE


SSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINE


GQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFD


IYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD


GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK


NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP


AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD


TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEML


PSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLA


RLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY


RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG


MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKV


TGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLL


TRYLRIHPQSWVHQIALRMEVLGCEAQDLYGTMTRIVGGGSTSESPSGTAPGTSPSGESSTAP


GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESP


SGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT


SPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSG


TAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSP


SGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPG


PGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPE


SGPXXXGASASGAPSTX)00(SESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAP


GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTA


ESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGS


TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAES


PGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTST


PESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPG


PGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP



AE864-
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS

FVIII-
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG

FXIa-
TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP

AE144
ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT


SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE


SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS


TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES


GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA


GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA


PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG


SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI


AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE


KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS


LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG


LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL


FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ


IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP


STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY


ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF


KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSP LTESGGPLSLSEE


NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT


SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN


KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ


LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ


EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS


LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL


KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI


PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS


LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP


TETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDN


HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT


ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR


HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL


LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH


HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF


DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL


LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH


AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS


WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS


SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN


MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV


KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR


MEVLGCEAQDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSP


TSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPG


TSESATPESGPGSEPATSGSETPGTSTEPSEGSAP



AE144-
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT

FXIa-FVIII
SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP

BDD9-
GTSTEPSEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT

AE864
SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVS


YWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL


VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR


AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEI


SPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL


TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYL


NNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIY


PHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM


ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL


EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYED


TLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISA


YLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ


SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLY


RGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNE


TKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV


TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVM


AQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIW


RVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS


INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT


LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD


AQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG


VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP


QSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA


GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE


TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST


EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE


TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA


PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES


ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA


PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP


SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP


GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS


PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP


GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT


SGSETPGTSESATPESGPGTSTEPSEGSAP



AE48-
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGKLTRAETGATRRYY

FXIa-FVIII
LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPW

BDD9-
MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVF

AE864
PGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQ


TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRK


SVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSH


QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK


KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF


KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDF


PILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQ


IMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSV


CLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGC


HNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQRE


ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS


SSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF


RNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWA


YFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC


RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHV


FTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP


LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQ


GARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLH


PTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGR


SNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ


NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAG


SPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP


GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS


EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG


TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP


ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT


STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG


SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE


PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS


TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS


ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES


GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE


SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST


EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

BDD9-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AG288_2
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYKLTRAETGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG


SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT


GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP


SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG


SPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

BDD9-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AG864
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTP


SGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS


PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS


GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS


PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG


TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS


PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP


SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS


PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS


GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS


PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP


SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS


PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP


SGATGSPGASPGTSSTGSP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

BDD9 (1-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

745)
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AG288_2-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL

(1640-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF

Y2332)-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI

FXIa-
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY

AG864
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPG


ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG


ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG


ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS


STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG


TPGSGTASSSPGSSTPSGATGSPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE


DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT


QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV


KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH


GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP


GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS


KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL


HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG


NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME


SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG


VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR


YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS


PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS


SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS


TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG


TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP


GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS


SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS


PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG


TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS


PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT


GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS


PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST


GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP


GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

BDD9 (1-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

743)
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AG288_2-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL

(1638-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF

Y2332)-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI

FXIa-
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY

AG864
TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGPGASP


GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG


SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP


GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG


SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPG


SGTASSSPGSSTPSGATGSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE


DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT


QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV


KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH


GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP


GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS


KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL


HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG


NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME


SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG


VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR


YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS


PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS


SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS


TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG


TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP


GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS


SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS


PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG


TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS


PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT


GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS


PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST


GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP


GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP



BDD10 (1-
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

745)
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

AG288_2-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

(1640-
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL

Y2332)-
IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF

FXIa-
CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI

AG864
RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPG


ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG


ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG


ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS


STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG


TPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE


DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT


QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV


KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH


GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP


GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS


KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL


HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG


NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME


SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG


VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR


YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS


PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS


SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS


TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG


TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP


GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS


SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS


PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG


TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS


PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT


GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS


PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST


GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP


GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

BDD10-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AG288_2
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYKLTRAETGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG


ATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG


SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS


TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG


ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATG



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

BDD10-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AG864
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTP


SGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS


PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS


GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS


PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG


TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS


PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP


SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS


PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS


GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS


PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP


SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS


PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP


SGATGSPGASPGTSSTGSP



FVIII
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA

BDD10-
KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK

FXIa-
EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL

AE864
AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL


IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF


CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI


RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY


TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK


HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD


QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD


SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG


LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL


KRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL


WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE


DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF


DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT


ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH


SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV


YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP


MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP


PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS


KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD


GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA


QDLYKLTRAETGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE


PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE


EGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE


PSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSA


PGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA


TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP


GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT


SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP


GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA


TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP


GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS


PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP


GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA


TPESGPGTSTEPSEGSAP



Sequence name reflects N- to C-terminus configuration of the FVIII variant and XTEN components: signal peptide (SP); linker (L); cleavage sequence (CS) may be denoted by protease name active on the sequence, and XTEN components by family name and length, with insertion points for components denoted by FVIII amino acid and numbered positions adjacent to the inserted sequence or A1 being the N-terminus and Y2332 being the C-terminus of the FVIII.