English
Tiếng Việt
(en)The invention provides a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection, comprising an amount of a soluble active agent which interacts with at least one of the binding sites between hIL6 and pS1 and between hIL6 and hepatocytes and other HBV-permissive cells, the active agent being present in sufficient amount to competitively bind to at least one of the sites and thereby to prevent hIL6-mediated HBV infection of hepatocytes and other HBV-permissive cells.
1.ApplicationNumber: US-79547397-A
1.PublishNumber: US-6217858-B1
2.Date Publish: 20010417
3.Inventor: GALUN EITHAN
NAHOR ORIT
BLUM HUBERT E.
4.Inventor Harmonized: GALUN EITHAN(IL)
NAHOR ORIT(IL)
BLUM HUBERT E(DE)
5.Country: US
6.Claims:
(en)The invention provides a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection, comprising an amount of a soluble active agent which interacts with at least one of the binding sites between hIL6 and pS1 and between hIL6 and hepatocytes and other HBV-permissive cells, the active agent being present in sufficient amount to competitively bind to at least one of the sites and thereby to prevent hIL6-mediated HBV infection of hepatocytes and other HBV-permissive cells.
7.Description:
(en)BACKGROUND AND SUMMARY OF THE INVENTION
The present invention relates to pharmaceutical compositions for the treatment of hepatitis B virus (HBV) infection.
HBV infection in humans can cause chronic liver disease which will, in some cases, proceed to hepatocellular carcinoma. The initial steps of HBV attachment to cells and the targeting of the viral genome to the host cell nucleus have yet to be deciphered. The specific receptor for HBV has not so far been identified, even though various serum proteins and cellular membrane glycoproteins have been suggested as mediators of cell penetration or viral receptors. HBV envelope proteins were reported to contain residues which interact with polymerized albumin [P. Pontisso, et al., Journal of Virology , Vol. 63, No. 1981-1, p. 988 (1981)] or with soluble transferrin [M. Gagliardi, et al., Eur. J. Immunol ., Vol. 24, pp. 1372-1376 (1994)], enabling viral penetration of cells via their respective receptors, probably in a non-specific manner.
In a study reported by Neurath, et al. [A. Neurath, et al., J. Exp. Med ., Vol. 175, pp. 461-469 (1992)] hIL-6 was shown to bind the pS1 (aa 21-47) segment of the HBV envelope. Putative candidates for the HBV receptor were recently reported, including Annexin V (endohexin II) [K. Hertogs, et al., Virology , Vol. 197, pp. 549-557 (1993)]; apolipoprotein H [H. Mehdi, et al., Journal of Virology , Vol. 68, pp. 2415-2424 (1994)]; and asialoglycoprotein receptor [U. Treichel, et al., Journal of General Virology , Vol. 75, pp. 3021-3029 (1994)].
Binding experiments have demonstrated that the pre-S1 (pS1) region of the viral envelope protein contains a recognition site for the host cell [A. R. Neurath, et al., Cell , Vol. 46, pp. 429-436 (1986); M. Petit, et al., Virology , Vol. 180, pp. 483-491 (1990); M. Petit, et al., Virology , Vol. 197, pp. 211-222 (1992)]. Although previous studies had suggested that HepG2 cells [R. Bchini, et al., Journal of Virology , Vol. 64, pp. 3025-3032 (1991)] and human hepatocytes [P. Gripon, et al., Journal of Virology , Vol. 62, pp. 4136-4143 (1988); T. Ochiya, et al., Proc. Natl. Acad. Sci. U.S.A ., Vol. 86, pp. 1875-1879 (1989); P. Gripon, et al., Virology , Vol. 192, pp. 534-540 (1993); P. Galle, et al., Gastroenterology , Vol. 106, pp. 664-673 (1994)] could support HBV infection in vitro, no cellular receptor has as yet been defined in either system, and these models were of low experimental reproducibility.
In current reports, it has been shown that a chimeric mouse, generated by using Beige/Nude/X linked immunodeficient (BNX) mice, preconditioned by total body irradiation (12Gy) and reconstituted with severe combined immunodeficient (SCID) mice bone marrow (BM) cells, is permissive for normal human T and B cells [I. Lubin, et al., Science , Vol. 252, pp. 427-431 (1991)], as well as for normal human liver tissue [E. Galun, et al., Journal of Infectious Diseases , Vol. 175, pp. 25-30 (1995)]. Hepatitis C virus (HCV) viremia was detectable for up to two months, after implantation under the kidney capsule of the BNX>SCID chimeric animals of either a human liver fragment with preexisting HCV infection, or normal human liver tissue following incubation ex-vivo of the transplanted liver fragment with HCV-positive sera [E. Galun, et al., ibid.]. Heretofore, one of the major obstacles in elucidating the initial steps of HBV infection and the assessment of antiviral agents, has been the lack of a small animal model. Using the techniques referred to above, it was possible to develop SCID>BNX animals which sustain HBV viremia following the implantation of an ex-vivo HBV DNA-positive sera incubation with liver tissue. The method in which the animals were prepared for the experiments described herein, and the surgical technique for transplantation, are similar to those previously reported [E. Galun, et al., ibid.].
As will be described below, it has now been found, using a chimeric animal model, that human interleukin 6 (hIL6) is essential for HBV infection. Having identified that hIL6 serves as an essential bridge for HBV infection, the invention now provides a pharmaceutical composition for the treatment of hepatitis B virus infection, comprising an amount of a soluble active agent which interacts with at least one of the binding sites between hIL6 and pS1 and between hIL6 and hepatocytes and other HBV-permissive cells, said active agent being present in sufficient amount to competitively bind to at least one of said sites and thereby to prevent hIL6-mediated HBV infection of hepatocytes and other HBV-permissive cells.
In a first preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection, comprising an amount of soluble gp80 and/or gp130 receptor sites sufficient to inhibit the binding of hIL6 to hepatocytes and other HBV-permissive cells.
In a second preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of soluble amino acid sequences corresponding to amino acids 21 to 46 of pS1 to block the interaction of HBV with hIL6.
In a third preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of a soluble ligand selected from the group consisting of peptides LYS41-ALA56, GLY77-GLU95 and GLN153-HIS165 to block the interaction of hIL6 with hepatocytes and other HBV-permissive cells.
In a fourth preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising hIL6 conjugated with an anti-viral agent.
With specific reference now to the examples and figures in detail, it is stressed that the particulars described and shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this context, it is to be noted that only subject matter embraced in the scope of the claims appended hereto, whether in the manner defined in the claims or in a manner similar thereto and involving the main features, as defined in the claims, is intended to be included in the scope of the present invention.
In the drawings:
The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings are provided to the Patent and Trademark Office with payment of the necessary fee.
FIG. 1 illustrates a pre-infected liver fragment from a HBV DNA-positive patient, one month after sub-capsular implantation in a SCID>BNX chimeric mouse, stained for HBsAg;
FIGS. 2A-D shows that hIL6 mediates HBV viremia in SCID>BNX chimeric mice transplanted with human tissue; and
FIG. 3 illustrates the liver histology of a HepG2-hIL-6R tumor, which developed one month following intrasplenic injection into a SCID>BNX chimeric mouse (H and E staining);
FIG. 4 provides the nucleotide sequence for hIL-6 mRNA;
FIGS. 5 a and 5 b provide the nucleotide sequence for hIL-6 receptor mRNA;
FIG. 6 provides the nucleotide sequence for the IL-6 receptor;
FIGS. 7 a and 7 b provide the nucleotide sequence for gp130;
FIG. 8 provides the amino acid sequence for hIL-6 receptor alpha; and
FIG. 9 provides the amino acid sequence for IL-6.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES
Human liver tissue was taken from patients undergoing liver surgery for liver diseases, who had HBV viremia of 10 7 -10 9 parciles/ml with positive HBV DNA in the liver tissue. The liver tissue was implanted under the kidney capsule of the chimeric animals. Although HBsAg was easily detected in pre-infected HBV DNA positive/HBeAg positive transplanted tissue (FIG. 1 ), 1-3 months after liver fragment implantation HBV sequences were undetectable by PCR (applyin primers spanning the viral core gene as well as the envelope region, at the a determinant of the HBsAg) in any of these experiments. Furthermore, intravenous or intraperitoneal (i.p.) injection of 200 μl of high-titer HBV particles (>10 8 /ml) following the transplantation of a normal human liver fragment, failed to generate HBV DNA sequences during the next 30 days (data not shown).
Lymphocytes, positive for HBV DNA by dot blot hybridization, were separated by lymphopheresis (Baxter Fenwell CS-3000 Pulse Blood Cell Separators, Deerfield, Ill., U.S.A.) from a patient with HBV-related chronic liver disease whose sera were positive for HBV DNA and HBeAg. Forty million HBV DNA-positive lymphocytes were injected i.p. to each mouse, subsequent to transplantation of normal human liver at the subcapsular site of the kidney. HBV sequences were not detected in the sera of these animals during the following 21 days.
Although the primary infection site for HBV is hepatocytes, lymphocytes and endothelial cells have both been shown to harbor HBV transcripts and viral-related proteins [J. Romet-Lemonne, et al., Science , Vol. 221, pp. 667-669 (1983); H. Blum, et al., Proc. Natl. Acad. Sci. U.S.A ., Vol. 80, pp. 6685-6688 (1983); E. Galun, et al., American Journal of Pathology , Vol. 145, pp. 1001-1007 (1994)], suggesting a common specific cell membrane receptor mechanism supporting viral penetration. This mechanism would prevent infection of receptor negative cells, despite their being permissive for HBV replication by transfection [E. Galun, et al., Journal of General Virology , Vol. 73, pp. 173-178 (1992)]. All three primary cell types hosting HBV naturally, i.e., hepatocytes, lymphocytes and endothelial cells, respond to hIL6 through the human IL6 receptor (hIL6R) which is expressed on their cell membranes [A. Mackiewicz, et al., The Journal of Immunology , Vol. 149, pp. 2021-2027 (1992); J. Bauer, et al., FEBS Letter , Vol. 249, pp. 27-30 (1989); T. Kishimoto, et al., Science , Vol. 258, p. 593 (1992)]. Furthermore, as previously shown, hIL6 binds to HBV through pS1.
A fragment of normal human liver from a patient with no indication of any HBV-related markers or disease, was incubated ex-vivo with a high titer HBV DNA-positive serum prior to transplantation under the kidney capsule of the chimeric animals. HBV DNA sequences were undetectable by PCR from two different genomic regions in any of these animals during the month following transplantation. Results are shown in FIG. 2 A. These results were reproduced in additional experiments in over 50 mice, using four different HBV DNA-positive sera.
However, when liver tissue originating from the same patients was incubated ex-vivo with the above-mentioned HBV DNA-positive sera togethcr with hIL6, HBV DNA sequences were detected from day 16 to day 31, in sera of about 50% of the transplanted animals. These results are shown in FIG. 2 B.
Similar results were obtained in experiments conducted under the above-stated conditions, using additional HBV DNA sera and liver tissue from different sources. In these experiments, HBV DNA sequences could be detected up to day 60 following transplantation (results not shown).
Pre-exposure of liver tissue to hIL6 prior to incubation with HBV ex-vivo, increased infection to about 90% of the animals. Animals positive for HBV sequences in serum at day 31 were also positive for HBsAg in the implanted hepatocytes, as shown in FIG. 2 D. Liver fragments incubated ex-vivo with HBV under the above conditions and fixed for immunohistochemical analysis prior to transplantation were negative for HBsAg (results not shown).
To further assess the role of hIL6 in supporting HBV infection, a human hepatoblastoma cell line HepG2 (ATCC HB 8065), an HepG2-derived, stably transfected hIL6R cell line, a null hIL6R (a cell line which does not express hIL6R) named HepG2-PDI and an hIL6 producing line named HepG2-hIL6 [S. Rose-John, et al., The Journal of Biological Chemistry , Vol. 268, pp. 22084-22091 (1993)] were incubated with HBV DNA-positive sera, with or without hIL6. Following incubation, the various mixtures were injected intrasplenically to the chimeric mice to generate HCC foci in the liver, as shown in FIG. 3 . The results of these experiments are summarized below in Table 1.
TABLE 1
HBV-DNA as detected by PCR in sera of chimeric mice following
intrasplenic injection of HBV, with or without hIL6, after incubation with
HepG2-derived cell lines
HepG 2
HepG 2
HepG 2
Cell Line
hIL6R
PDI
HepG 2
hIL6
hIL6
+
−
+
−
+
−
+
−
HBV-DNA PCR product
+
−
+
−
+
+/−
+
+
Method:
All cell lines grew in T25 flasks supplemented with DMEM medium, enriched with 10% fetal bovine serum. For infection experiments, cells were trypsinized and washed twice with PBS, followed by incubation with HBV-positive human sera (10 8 virions/ml) in the presence or absence of hlL6 (500 ng/ml) in 1-2 ml of DMEM. After 2-4 h incubation at 37° C., 4×10 6 cells/ml, 0.5 ml/mouse were injected intrasplenically to 8-10 SCID>BNX mice in each group. Animals were splenectomized following the injection.
Mice were bled at two weekly intervals for 3 months, and DNA was extracted from 100 μl sera. The DNA was subjected to PCR amplification. The DNA extraction and the PCR method applied are described in the legend of FIG. 2 . Table 1 summarizes three experiments.
In mice implanted with HepG2-hIL6R cells (which have about one log higher expression of the receptor than HepG2 cells) subsequent to incubation with HBV in the presence of hIL6, HBV-DNA sequences could be detected in serum 13 days after transplantation, whereas HBV sequences were not detected in the sera of mice who underwent the same procedure without the presence of hIL6. Similar results were obtained in experiments using HepG2-PDI cells. These cells do not express the gp 80 binding protein subunit of the hIL6R on the cell membrane [S. Rose-John, et al., ibid.; M. Ehlers, et al., The Journal of Immunology , Vol. 153, p. 1744 (1994)], however, they do express the signal transduction gp 130 subunit of the receptor, which is essential for efficient intemaliztion of hIL6 [E. Dittrich, et al., The Journal of Biological Chemistry , Vol. 269, pp. 10914-19020 (1994)].
In experiments of the same design, using HepG2 cells in the presence and also in the absence of hIL6, HBV sequences could be detected in a number of murine sera. These results are similar to those previously reported by Petit, et al. [R. Bchini, et al., ibid.], showing a low reproducibility in which only three sera supported HBV infection of HepG2 cells in-vitro, out of a total of 55 different serum samples taken from HBV DNA-positive patients. The HepG2-hIL6 cell line, which produces hIL6, generated HBV sequences in mice sera following incubation with the virus, with or without external supplementation of hIL6.
When the liver fragment was incubated ex-vivo with HBV-DNA positive sera in the presence of commercially available human polyclonal anti-HBs viral neutralizing antibodies (HBIG, Hepatect®, Biotest Pharma GmbH, Dreicich. Germany), HBV-DNA was observed at day 11 following transplantation only in 48% (10/21) of mice, as compared to 78% (14/18) of the untreated mice group (Table 2).
TABLE 2
Inhibition of infection - effect of anti-HBs antibodies effect on HBV-DNA
levels in sera of chimeric mice transplanted with human liver fragments
infected ex vivo with HBV
Treatment Group
Mice Positive for HBV-DNA (%)
Untreated
14/18 (78)
HBIG Treatment
10/21 (48)
Method:
For antibody treatment, HBV-DNA positive serum (0.5 ml) was incubated with 100 IU of HBIG for 2 hours at 25° C. Human liver fragments were then added to the untreated or HBIG treated HBV-DNA positive serum according to the same protocol as described above, followed by implantation under the kidney capsule of the chimeric animal. Mice sera were analyzed for the presence of HBV-DNA sequences 11 days after transplantation.
Referring again to the figures, FIG. 1 shows pre-infected liver fragment from a HBV DNA-positive patient, one month after sub-capsular implantation in a SCID>BNX chimeric mouse, stained for HBsAg.
From FIG. 2 it can be seen that hIL6 mediates HBV viremia in SCID>BNX chimeric mice transplanted with human tissue. PCR amplification products of HBV pre-core/core region following DNA extraction from sera of mice, 16 and 31 days after sub-capsular kidney transplantation of normal human liver fragments. The human liver fragments were incubated ex-vivo prior to transplantation with human HBV positive serum (FIG. 2 A); HBV serum and hIL6 simultaneously (FIG. 2 B), or preincubated with hIL6 and later with HBV sera FIG. 2 C). In each of FIGS. 2A to 2 C, the upper panel is an EtBr staining and the lower panel is an 32 P HBV linear insert hybridization result of the same gel. The molecular marker size (m) is indicated by an arrow; numbers at the head of each panel indicate mice identification numbers; + for positive serum control and − for negative serum control.
FIG. 2D shows HBsAg staining of an ex-vivo HBV incubation of a normal liver fragment with hIL6. one month following implantation under the kidney capsule of SCID>BNX mice.
Sera from HBV-positive patients, containing approximately 10 8 virions/ml, were used for infection. Small fragments of normal human liver were incubated with 400 μl sera in 1 ml DMEM supplemented with 2 μg/ml polybrene in the absence (group A) or presence (group B) of hIL6 (500 ng/ml) incubated for 2-4 h at 37° C. In group C, the liver fragments were treated with hIL6 for 2 h at 37° C. before the addition of HBV-positive sera and polybrene. After incubation, 4-5 ml polybrene DMEM were added and the liver fragments were transplanted under the kidney capsule to groups A, B and C of SCID>BNX chimeric mice (10, 19 and 11 mice, respectively). At 2 weekly intervals for 4 months, blood was collected retrobulbarily from each mouse. 100 μl of serum samples were treated with 0.5 mg/ml proteinase K in 10 mM EDTA and 0.25% SDS for 2 h at 55° C. or overnight at 37° C., extracted twice with phenol, once with phenol-CHCl 3 , and once with CHCl 3 . DNA was precipitated with ethanol, using 0.5M NaCl and a DNA microcarrier. DNA was dissolved in 30 μl Tris-EDTA, pH 8.0, and was subjected to PCR amplification.
The 50 μl PCR reaction volume contained 10 ρmole of each oligonucleotide primer in reaction buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.0 mM MgCl 2 , 0.01% (w/v) gelatin, 250 μM of dATP, dGTP, dCTP, dTTP and 0.5 u of Taq polymerase. The reaction mixtures were overlaid with 30 μl of mineral oil. PCR cycles included 94° C. for 1 min., 55° C. for 1 min. and 72° C. for 3 min., 35 repeated cycles. 10 μl of reaction mixture was analyzed on a 2% agarose gel. Oligonucleotides used for the pre-core/core amplification were:
oligo 1, Sense (nt 1778 to 1806):
5′ GGA-GGC-TGT-AGG-CAT-AAA-TTG-GTC-TGC-GC-3′. Sequence ID No. 7
oligo 2, Antisense (nt 2446 to 2408):
5′ CCC-GAG-ATT-GAG-ATC-TTC-TGC-GAC-GCG-GCG-ATT-GAG-ACC-3′. Sequence ID No. 8
Sequence originated from adw subtype; nt numbering starts from EcoRI site. The expected size of the PCR DNA product is 668-bp.
The PCR samples were electrophoresed on 2% agarose gel and transferred to a nylon membrane (Biodynea), hybridized with a nick-translated probe. The autoradiogram was exposed with intensifying screens at −70° C. for 7 h. In order to confirm the PCR results, the mice serum samples were also subjected to PCR amplification with primers spanning the envelope gene region, showing the same results (data not shown).
Reproducible results were obtained from four similar experiments while there were 10-20 mice in each group.
Based on the present discovery that hIL6 acts to mediate HBV infection, it is possible to prepare an antiviral/anti-HBV agent. A pharmaceutical composition for the prevention of HBV infection, comprising an active ingredient having an amino acid sequence similar to hIL6, is thus developed. The hIL6 domain which interacts with hIL6Rα (R for receptor) and/or hiL6Rβ (amino acid residues: 40-60, 70-100 and 135-175) antagonizes hIL6 interaction to prevent HBV infection.
Set forth at FIG. 4 is the nucleotide sequence for human interleukin 6 mRNA (SEQ ID NO 1), as published by L. T. May, et al., “Anti-beta Interferon Antibodies Inhibit the Increased Expression of HLA-B7 mRNA in Tunor Necrosis Factor-Treated Human Fibroblasts: Structural Studies of the beta-2 Interferon Involved,” Proc. Nat'l Acad. Sci. U.S.A. 83 (23), 8957-8960 (1986).
FIGS. 5 a and 5 b depict the nucleotide sequence for the human interleukin 6 receptor mRNA (SEQ ID NO 2), as published by K. Yamasuki, et al., “Cloning and Expression of the Human Interleukin 6 (BSF-2/ISN beta 2) Receptor,” Science 241 (4867), 825-828 (1988).
FIG. 6 depicts the nucleotide sequence for the interleukin-6 receptor (SEQ ID NO 3), as published by H. Schooltink, et al., “Structural and Functional Studies on the Human Nepatic Interleukin-6 Receptor,” Biochem. J. 277:659-664 (1991).
FIGS. 7 a and 7 b depict the nucleotide sequence for the gp 130 interleukin 6 receptor (SEQ ID NO 4), as published by M. Hibi, et al, “Molecular Cloning and Expression of an IL-6 Signal Transducer, gp 130,” Cell 63 (6), 1149-1157 (1990).
FIG. 8 depicts the amino acid sequence for the human interleukin 6 receptor alpha (IL-6R alpha) (SEQ ID NO 5), as published by K. Yamasaki, et al., “Cloning and Expression of the Human Interleukin 6 (BSF-2/ISN beta 2) Receptor,” Science 241 (4867), 825-828 (1988).
FIG. 9 depicts the amino acid sequence for human interleukin 6 (SEQ ID No. 6), as published by C. Nishimura, et al., Biochemistry 35:273-281 (1996).
The molecular analysis of hIL6 binding sites with gp130 and gp80 revealed a number of structural targets on hIL6 which can serve as hIL6 antagonists. The preferable target for an hIL6 antagonist is to disrupt the hIL6/hIL6Rα complex with hIL6Rβ.
Based on previous publication, a number of domains essential for hIL6 activity were reported:
1. Lys41-ala56 (site 2a, also named β2) is involved in the activation of signal-transduction.
2. Gly77-glu95 (site 2c) is important for interaction with hIL6Rα, subunit gp80.
3 Gln153-his165 (site β1), substitution of trp158 to arg or gln160 to glu combined with thr163 to pro-antagonize the biological activity of hIL6.
4. A combined β1 and β2 hIL6 mutant (mhIL6β1+β2) is inactive on XG-1 hIL6 responsive cells, with a weakly antagonizing activity.
5. The addition of two substitutions to the mhIL6 (m for mutant) β1+β2, phe171 to leu and ser177 to arg, resulted in an increase in the affinity to hIL6Rα, while inhibiting its activity on XG-1 cells.
Based on techniques known per se to persons skilled in the art, the proteins and peptides for use in the pharmaceutical compositions of the present invention are readily prepared, e.g., by the following techniques and steps:
Amplification of chosen segment of DNA from plasmid containing HBV DNA (adw2)-adw HTD by PCR, using primers constructed so as to introduce BamH1 and EcoR1 sites compatible to the pGEX-2T (Pharmacia, Uppsala, Sweden, Catalogue No. 27-4801-01) insertion site. This GST fusion vector provides a system in which fusion proteins are easily purified from the bacterial lysates and can be detected directly as a fusion protein or after cleavage with site specific proteases. After introduction of DNA into pGEX-2T, competent E. coli (JM109) are transformed and cloned (LB+10 μg/ml ampicillin). Protein expression is induced by the addition of IPTG (0.1 mM, isopropyl-1-thio-b-D-galactoside) for 1-2 hours. Fusion protein is removed from lysed (sonicated) cells by collection on glutathione-agarose beads (Pharmacia) and eluted from beads using reduced glutathione (5 mM in 50 mM Tris-Cl, pH 8.0). Identification and determination of protein can be done either by use of antibodies to GST or by specific recognition of inserted protein. The complete HBV pS1 protein (aa 1 to aa 119, applying PCR with the sense and anti-sense primers 5′-CGGGATCCATGGGAGGTTGGTCATC-3′[NT 8+2856-2873, EcoR1 as starting site for nt numbering] (Sequence ID No. 9) and 5′-GGAATTCCACTGCATGGC-3′ [nt 6-3210] respectively) (Sequence ID No. 10) and the pS1 attachment site aa21 to aa46 constructs were designed and produced in the pGEX-2T system (compound B in FIG. 1 ).
Truncated soluble forms of gp80 and gp130 are synthesized using the pGEX-2T system as described for the preparation of pS1 (compound A2 and A1, respectively). hIL6 derived peptides (Lys41-ala56, Gly77-glu95 and Gln153-his165, designated C1, C3 and C2, respectively, in FIG. 1) are synthesized by applying a variety of methods including Merrifield solid-phase synthesis and derived methods or other acceptable genetic engineered methods. The compounds produced are linear or cyclic peptides, or parts of large proteins.
Compositions according to the present invention can be administered orally or parenterally, including intravenous, intraperitoneal, intranasal and subcutaneous administration. Implants of the compounds are also useful.
The proteins of the present invention are administered in combination with other drugs, or singly, consistent with good medical practice. The composition is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners. The ‘effective amount’ for purposes herein is thus determined by such considerations as are known in the art.
When administering the compositions parenterally, the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
Proper fluidity can be maintained, for example, by the use of a coating such as lecithin by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters such as isopropyl myristate, may also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, anti-oxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent or additive used would have to be compatible with the compounds.
Sterile injectable solutions can be prepared by incorporating the proteins utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
A pharmacological formulation described and claimed herein can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems, such as polymer matrices, liposomes, and microspheres. An implant suitable for use in the present invention can take the form of a pellet which slowly dissolves after being implanted, or a biocompatible delivery module well-known to those skilled in the art. Such well-known dosage forms and modules are designed such that the active ingredients are slowly released over a period of several days to several weeks.
Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194. which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow, implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are well-known to those skilled in the art.
A pharmacological formulation of the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like, are usable. Known techniques which deliver the new compositions orally or intravenously and retain the biological activity, are preferred.
In one embodiment, the new compositions can be administered initially by intravenous injection. The quantity of the compositions to be administered will vary for the patient being treated, and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day, and preferably will be from 10 μg/kg to 10 mg/kg per day.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
# SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 10
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATTCTGCCCT CGAGCCCACC GGGAACGAAA GAGAAGCTCT ATCTCCCCTC
# 50
CAGGAGCCCA GCTATGAACT CCTTCTCCAC AAGCGCCTTC GGTCCAGTTG
# 100
CCTTCTCCCT GGGGCTGCTC CTGGTGTTGC CTGCTGCCTT CCCTGCCCCA
# 150
GTACCCCCAG GAGAAGATTC CAAAGATGTA GCCGCCCCAC ACAGACAGCC
# 200
ACTCACCTCT TCAGAACGAA TTGACAAACA AATTCGGTAC ATCCTCGACG
# 250
GCATCTCAGC CCTGAGAAAG GAGACATGTA ACAAGAGTAA CATGTGTGAA
# 300
AGCAGCAAAG AGGCACTGGC AGAAAACAAC CTGAACCTTC CAAAGATGGC
# 350
TGAAAAAGAT GGATGCTTCC AATCTGGATT CAATGAGGAG ACTTGCCTGG
# 400
TGAAAATCAT CACTGGTCTT TTGGAGTTTG AGGTATACCT AGAGTACCTC
# 450
CAGAACAGAT TTGAGAGTAG TGAGGAACAA GCCAGAGCTG TCCAGATGAG
# 500
TACAAAAGTC CTGATCCAGT TCCTGCAGAA AAAGGCAAAG AATCTAGATG
# 550
CAATAACCAC CCCTGACCCA ACCACAAATG CCAGCCTGCT GACGAAGCTG
# 600
CAGGCACAGA ACCAGTGGCT GCAGGACATG ACAACTCATC TCATTCTGCG
# 650
CAGCTTTAAG GAGTTCCTGC AGTCCAGCCT GAGGGCTCTT CGGCAAATGT
# 700
AGCATGGGCA CCTCAGATTG TTGTTGTTAA TGGGCATTCC TTCTTCTGGT
# 750
CAGAAACCTG TCCACTGGGC ACAGAACTTA TGTTGTTCTC TATGGAGAAC
# 800
TAAAAGTATG AGCGTTAGGA CACTATTTTA ATTATTTTTA ATTTATTAAT
# 850
ATTTAAATAT GTGAAGCTGA GTTAATTTAT GTAAGTCATA TTTTATATTT
# 900
TTAAGAAGTA CCACTTGAAA CATTTTATGT ATTAGTTTTG AAATAATAAT
# 950
GGAAAGTGGC TATGCAGTTT GAATATCCTT TGTTTCAGAG CCAGATCATT
# 1000
TCTTGGAAAG TGTAGGCTTA CCTCAAATAA ATGGCTAACT TTATACATAT
# 1050
TTTTAAAGAA ATATTTATAT TGTATTTATA TAATGTATAA ATGGTTTTTA
# 1100
TACCAATAAA TGGCATTTTA AAAAATTC
#
# 1128
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3319 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGCGGTCCCC TGTTCTCCCC GCTCAGGTGC GGCGCTGTGG CAGGAAGCCA
# 50
CCCCCTCGGT CGGCCGGTGC GCGGGGCTGT TGCGCCATCC GCTCCGGCTT
# 100
TCGTAACCGC ACCCTGGGAC GGCCCAGAGA CGCTCCAGCG CGAGTTCCTC
# 150
AAATGTTTTC CTGCGTTGCC AGGACCGTCC GCCGCTCTGA GTCATGTGCG
# 200
AGTGGGAAGT CGCACTGACA CTGAGCCGGG CCAGAGGGAG AGGAGCCGAG
# 250
CGCGGCGCGG GGCCGAGGGA CTCGCAGTGT GTGTAGAGAG CCGGGCTCCT
# 300
GCGGATGGGG GCTGCCCCCG GGGCCTGAGC CCGCCTGCCC GCCCACCGCC
# 350
CCGCCCCGCC CCTGCCACCC CTGCCGCCCG GTTCCCATTA GCCTGTCCGC
# 400
CTCTGCGGGA CCATGGAGTG GTAGCCGAGG AGGAAGCATG CTGGCCGTCG
# 450
GCTGCGCGCT GCTGGCTGCC CTGCTGGCCG CGCCGGGAGC GGCGCTGGCC
# 500
CCAAGGCGCT GCCCTGCGCA GGAGGTGGCA AGAGGCGTGC TGACCAGTCT
# 550
GCCAGGAGAC AGCGTGACTC TGACCTGCCC GGGGGTAGAG CCGGAAGACA
# 600
ATGCCACTGT TCACTGGGTG CTCAGGAAGC CGGCTGCAGG CTCCCACCCC
# 650
AGCAGATGGG CTGGCATGGG AAGGAGGCTG CTGCTGAGGT CGGTGCAGCT
# 700
CCACGACTCT GGAAACTATT CATGCTACCG GGCCGGCCGC CCAGCTGGGA
# 750
CTGTGCACTT GCTGGTGGAT GTTCCCCCCG AGGAGCCCCA GCTCTCCTGC
# 800
TTCCGGAAGA GCCCCCTCAG CAATGTTGTT TGTGAGTGGG GTCCTCGGAG
# 850
CACCCCATCC CTGACGACAA AGGCTGTGCT CTTGGTGAGG AAGTTTCAGA
# 900
ACAGTCCGGC CGAAGACTTC CAGGAGCCGT GCCAGTATTC CCAGGAGTCC
# 950
CAGAAGTTCT CCTGCCAGTT AGCAGTCCCG GAGGGAGACA GCTCTTTCTA
# 1000
CATAGTGTCC ATGTGCGTCG CCAGTAGTGT CGGGAGCAAG TTCAGCAAAA
# 1050
CTCAAACCTT TCAGGGTTGT GGAATCTTGC AGCCTGATCC GCCTGCCAAC
# 1100
ATCACAGTCA CTGCCGTGGC CAGAAACCCC CGCTGGCTCA GTGTCACCTG
# 1150
GCAAGACCCC CACTCCTGGA ACTCATCTTT CTACAGACTA CGGTTTGAGC
# 1200
TCAGATATCG GGCTGAACGG TCAAAGACAT TCACAACATG GATGGTCAAG
# 1250
GACCTCCAGC ATCACTGTGT CATCCACGAC GCCTGGAGCG GCCTGAGGCA
# 1300
CGTGGTGCAG CTTCGTGCCC AGGAGGAGTT CGGGCAAGGC GAGTGGAGCG
# 1350
AGTGGAGCCC GGAGGCCATG GGCACGCCTT GGACAGAATC CAGGAGTCCT
# 1400
CCAGCTGAGA ACGAGGTGTC CACCCCCATG CAGGCACTTA CTACTAATAA
# 1450
AGACGATGAT AATATTCTCT TCAGAGATTC TGCAAATGCG ACAAGCCTCC
# 1500
CAGTGCAAGA TTCTTCTTCA GTACCACTGC CCACATTCCT GGTTGCTGGA
# 1550
GGGAGCCTGG CCTTCGGAAC GCTCCTCTGC ATTGCCATTG TTCTGAGGTT
# 1600
CAAGAAGACG TGGAAGCTGC GGGCTCTGAA GGAAGGCAAG ACAAGCATGC
# 1650
ATCCGCCGTA CTCTTTGGGG CAGCTGGTCC CGGAGAGGCC TCGACCCACC
# 1700
CCAGTGCTTG TTCCTCTCAT CTCCCCACCG GTGTCCCCCA GCAGCCTGGG
# 1750
GTCTGACAAT ACCTCGAGCC ACAACCGACC AGATGCCAGG GACCCACGGA
# 1800
GCCCTTATGA CATCAGCAAT ACAGACTACT TCTTCCCCAG ATAGCTGGCT
# 1850
GGGTGGCACC AGCAGCCTGG ACCCTGTGGA TGACAAAACA CAAACGGGCT
# 1900
CAGCAAAAGA TGCTTCTCAC TGCCATGCCA GCTTATCTCA GGGGTGTGCG
# 1950
GCCTTTGGCT TCACGGAAGA GCCTTGCGGA AGGTTCTACG CCAGGGGAAA
# 2000
ATCAGCCTGC TCCAGCTGTT CAGCTGGTTG AGGTTTCAAA CCTCCCTTTC
# 2050
CAAATGCCCA GCTTAAAGGG GTTAGAGTGA ACTTGGGCCA CTGTGAAGAG
# 2100
AACCATATCA AGACTCTTTG GACACTCACA CGGACACTCA AAAGCTGGGC
# 2150
AGGTTGGTGG GGGCCTCGGT GTGGAGAAGC GGCTGGCAGC CCACCCCTCA
# 2200
ACACCTCTGC ACAAGCTGCA CCCTCAGGCA GGTGGGATGG ATTTCCAGCC
# 2250
AAAGCCTCCT CCAGCCGCCA TGCTCCTGGC CCACTGCATC GTTTCATCTT
# 2300
CCAACTCAAA CTCTTAAAAC CCAAGTGCCC TTAGCAAATT CTGTTTTTCT
# 2350
AGGCCTGGGG ACGGCTTTTA CTTAAACGCC AAGGCCTGGG GGAAGAAGCT
# 2400
CTCTCCTCCC TTTCTTCCCT ACAGTTCAAA AACAGCTGAG GGTGAGTGGG
# 2450
TGAATAATAC AGTATGTCAG GGCCTGGTCG TTTTCAACAG AATTATAATT
# 2500
AGTTCCTCAT TAGCAGTTTT GCCTAAATGT GAATGATGAT CCTAGGCATT
# 2550
TGCTGAATAC AGAGGCAACT GCATTGGCTT TGGGTTGCAG GACCTCAGGT
# 2600
GAGAAGCAGA GGAAGGAGAG GAGAGGGGCA CAGGGTCTCT ACCATCCCCT
# 2650
GTAGAGTGGG AGCTGAGTGG GGGATCACAG CCTCTGAAAA CCAATGTTCT
# 2700
CTCTTCTCCA CCTCCCACAA AGGAGAGCTA GCAGCAGGGA GGGCTTCTGC
# 2750
CATTTCTGAG ATCAAAACGG TTTTACTGCA GCTTTGTTTG TTGTCAGCTG
# 2800
AACCTGGGTA ACTAGGGAAG ATAATATTAA GGAAGACAAT GTGAAAAGAA
# 2850
AAATGAGCCT GGCAAGAATG CGTTTAAACT TGGTTTTTAA AAAACTGCTG
# 2900
ACTGTTTTCT CTTGAGAGGG TGGAATATCC AATATTCGCT GTGTCAGCAT
# 2950
AGAAGTAACT TACTTAGGTG TGGGGGAAGC ACCATAACTT TGTTTAGCCC
# 3000
AAAACCAAGT CAAGTGAAAA AGGAGGAAGA GAAAAAATAT TTTCCTGCCA
# 3050
GGCATGGAGG CCCACGCACT TCGGGAGGTC GAGGCAGGAG GATCACTTGA
# 3100
GTCCAGAAGT TTGAGATCAG CCTGGGCAAT GTGATAAAAC CCCATCTCTA
# 3150
CAAAAAGCAT AAAAATTAGC CAAGTGTGGT AGAGTGTGCC TGAAGTCCCA
# 3200
GATACTTGGG GGGCTGAGGT GGGAGGATCT CTTGAGCCTG GGAGGTCAAG
# 3250
GCTGCAGTGA GCCGAGATTG CACCACTGCA CTCCAGCCTG GGGTGACAGA
# 3300
GCAAGTGAGA CCCTGTCTC
#
# 331
#9
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1486 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATTAGCCTGT CCGCCTCTGC GGGACCATGG AGTGGTAGCC GAGGAGGAAG
# 50
CATGCTGGCC GTCGGCTGCG CGCTGCTGGC TGCCCTGCTG GCCGCGCCGG
# 100
GAGCGGCGCT GGCCCCAAGG CGCTGCCCTG CGCAGGAGGT GGCGAGAGGC
# 150
GTGCTGACCA GTCTGCCAGG AGACAGCGTG ACTCTGACCT GCCCGGGGGT
# 200
AGAGCCGGAA GACAATGCCA CTGTTCACTG GGTGCTCAGG AAGCCGGCTG
# 250
CAGGCTCCCA CCCCAGCAGA TGGGCTGGCA TGGGAAGGAG GCTGCTGCTG
# 300
AGGTCGGTGC AGCTCCACGA CTCTGGAAAC TATTCATGCT ACCGGGCCGG
# 350
CCGCCCAGCT GGGACTGTGC ACTTGCTGGT GGATGTTCCC CCCGAGGAGC
# 400
CCCAGCTCTC CTGCTTCCGG AAGAGCCCCC TCAGCAATGT TGTTTGTGAG
# 450
TGGGGTCCTC GGAGCACCCC ATCCCTGACG ACAAAGGCTG TGCTCTTGGT
# 500
GAGGAAGTTT CAGAACAGTC CGGCCGAAGA CTTCCAGGAG CCGTGCCAGT
# 550
ATTCCCAGGA GTCCCAGAAG TTCTCCTGCC AGTTAGCAGT CCCGGAGGGA
# 600
GACAGCTCTT TCTACATAGT GTCCATGTGC GTCGCCAGTA GTGTCGGGAG
# 650
CAAGTTCAGC AAAACTCAAA CCTTTCAGGG TTGTGGAATC TTGCAGCCTG
# 700
ATCCGCCTGC CAACATCACA GTCACTGCCG TGGCCAGAAA CCCCCGCTGG
# 750
CTCAGTGTCA CCTGGCAAGA CCCCCACTCC TGGAACTCAT CTTTCTACAG
# 800
ACTACGGTTT GAGCTCAGAT ATCGGGCTGA ACGGTCAAAG ACATTCACAA
# 850
CATGGATGGT CAAGGACCTC CAGCATCACT GTGTCATCCA CGACGCCTGG
# 900
AGCGGCCTGA GGCACGTGGT GCAGCTTCGT GCCCAGGAGG AGTTCGGGCA
# 950
AGGCGAGTGG AGCGAGTGGA GCCCGGAGGC CATGGGCACG CCTTGGACAG
# 1000
AATCCAGGAG TCCTCCAGCT GAGAACGAGG TGTCCACCCC CATGCAGGCA
# 1050
CTTACTACTA ATAAAGACGA TGATAATATT CTCTTCAGAG ATTCTGCAAA
# 1100
TGCGACAAGC CTCCCAGTGC AAGATTCTTC TTCAGTACCA CTGCCCACAT
# 1150
TCCTGGTTGC TGGAGGGAGC CTGGCCTTCG GAACGCTCCT CTGCATTGCC
# 1200
ATTGTTCTGA GGTTCAAGAA GACGTGGAAG CTGCGGGCTC TGAAGGAAGG
# 1250
CAAGACAAGC ATGCATCCGC CGTACTCTTT GGGGCAGCTG GTCCCGGAGA
# 1300
GGCCTCGACC CACCCCAGTG CTTGTTCCTC TCATCTCCCC ACCGGTGTCC
# 1350
CCCAGCAGCC TGGGGTCTGA CAATACCTCG AGCCACAACC GACCAGATGC
# 1400
CAGGGACCCA CGGAGCCCTT ATGACATCAG CAATACAGAC TACTTCTTCC
# 1450
CCAGATAGCT GGCTGGGTGG CACCAGCAGC CTGGAC
#
# 1486
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3085 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#4:
GAGCAGCCAA AAGGCCCGCG GAGTCGCGCT GGGCCGCCCC GGCGCAGCTG
# 50
AACCGGGGGC CGCGCCTGCC AGGCCGACGG GTCTGGCCCA GCCTGGCGCC
# 100
AAGGGGTTCG TGCGCTGTGG AGACGCGGAG GGTCGAGGCG GCGCGGCCTG
# 150
AGTGAAACCC AATGGAAAAA GCATGACATT TAGAAGTAGA AGACTTAGCT
# 200
TCAAATCCCT ACTCCTTCAC TTACTAATTT TGTGATTTGG AAATATCCGC
# 250
GCAAGATGTT GACGTTGCAG ACTTGGGTAG TGCAAGCCTT GTTTATTTTC
# 300
CTCACCACTG AATCTACAGG TGAACTTCTA GATCCATGTG GTTATATCAG
# 350
TCCTGAATCT CCAGTTGTAC AACTTCATTC TAATTTCACT GCAGTTTGTG
# 400
TGCTAAAGGA AAAATGTATG GATTATTTTC ATGTAAATGC TAATTACATT
# 450
GTCTGGAAAA CAAACCATTT TACTATTCCT AAGGAGCAAT ATACTATCAT
# 500
AAACAGAACA GCATCCAGTG TCACCTTTAC AGATATAGCT TCATTAAATA
# 550
TTCAGCTCAC TTGCAACATT CTTACATTCG GACAGCTTGA ACAGAATGTT
# 600
TATGGAATCA CAATAATTTC AGGCTTGCCT CCAGAAAAAC CTAAAAATTT
# 650
GAGTTGCATT GTGAACGAGG GGAAGAAAAT GAGGTGTGAG TGGGATGGTG
# 700
GAAGGGAAAC ACACTTGGAG ACAAACTTCA CTTTAAAATC TGAATGGGCA
# 750
ACACACAAGT TTGCTGATTG CAAAGCAAAA CGTGACACCC CCACCTCATG
# 800
CACTGTTGAT TATTCTACTG TGTATTTTGT CAACATTGAA GTCTGGGTAG
# 850
AAGCAGAGAA TGCCCTTGGG AAGGTTACAT CAGATCATAT CAATTTTGAT
# 900
CCTGTATATA AAGTGAAGCC CAATCCGCCA CATAATTTAT CAGTGATCAA
# 950
CTCAGAGGAA CTGTCTAGTA TCTTAAAATT GACATGGACC AACCCAAGTA
# 1000
TTAAGAGTGT TATAATACTA AAATATAACA TTCAATATAG GACCAAAGAT
# 1050
GCCTCAACTT GGAGCCAGAT TCCTCCTGAA GACACAGCAT CCACCCGATC
# 1100
TTCATTCACT GTCCAAGACC TTAAACCTTT TACAGAATAT GTGTTTAGGA
# 1150
TTCGCTGTAT GAAGGAAGAT GGTAAGGGAT ACTGGAGTGA CTGGAGTGAA
# 1200
GAAGCAAGTG GGATCACCTA TGAAGATAGA CCATCTAAAG CACCAAGTTT
# 1250
CTGGTATAAA ATAGATCCAT CCCATACTCA AGGCTACAGA ACTGTACAAC
# 1300
TCGTGTGGAA GACATTGCCT CCTTTTGAAG CCAATGGAAA AATCTTGGAT
# 1350
TATGAAGTGA CTCTCACAAG ATGGAAATCA CATTTACAAA ATTACACAGT
# 1400
TAATGCCACA AAACTGACAG TAAATCTCAC AAATGATCGC TATCTAGCAA
# 1450
CCCTAACAGT AAGAAATCTT GTTGGCAAAT CAGATGCAGC TGTTTTAACT
# 1500
ATCCCTGCCT GTGACTTTCA AGCTACTCAC CCTGTAATGG ATCTTAAAGC
# 1550
ATTCCCCAAA GATAACATGC TTTGGGTGGA ATGGACTACT CCAAGGGAAT
# 1600
CTGTAAAGAA ATATATACTT GAGTGGTGTG TGTTATCAGA TAAAGCACCC
# 1650
TGTATCACAG ACTGGCAACA AGAAGATGGT ACCGTGCATC GCACCTATTT
# 1700
AAGAGGGAAC TTAGCAGAGA GCAAATGCTA TTTGATAACA GTTACTCCAG
# 1750
TATATGCTGA TGGACCAGGA AGCCCTGAAT CCATAAAGGC ATACCTTAAA
# 1800
CAAGCTCCAC CTTCCAAAGG ACCTACTGTT CGGACAAAAA AAGTAGGGAA
# 1850
AAACGAAGCT GTCTTAGAGT GGGACCAACT TCCTGTTGAT GTTCAGAATG
# 1900
GATTTATCAG AAATTATACT ATATTTTATA GAACCATCAT TGGAAATGAA
# 1950
ACTGCTGTGA ATGTGGATTC TTCCCACACA GAATATACAT TGTCCTCTTT
# 2000
GACTAGTGAC ACATTGTACA TGGTACGAAT GGCAGCATAC ACAGATGAAG
# 2050
GTGGGAAGGA TGGTCCAGAA TTCACTTTTA CTACCCCAAA GTTTGCTCAA
# 2100
GGAGAAATTG AAGCCATAGT CGTGCCTGTT TGCTTAGCAT TCCTATTGAC
# 2150
AACTCTTCTG GGAGTGCTGT TCTGCTTTAA TAAGCGAGAC CTAATTAAAA
# 2200
AACACATCTG GCCTAATGTT CCAGATCCTT CAAAGAGTCA TATTGCCCAG
# 2250
TGGTCACCTC ACACTCCTCC AAGGCACAAT TTTAATTCAA AAGATCAAAT
# 2300
GTATCCAGAT GGCAATTTCA CTGATGTAAG TGTTGTGGAA ATAGAAGCAA
# 2350
ATGACAAAAA GCCTTTTCCA GAAGATCTGA AATCATTGGA CCTGTTCAAA
# 2400
AAGGAAAAAA TTAATACTGA AGGACACAGC AGTGGTATTG GGGGGTCTTC
# 2450
ATGCATGTCA TCTTCTAGGC CAAGCATTTC TAGCAGTGAT GAAAATGAAT
# 2500
CTTCACAAAA CACTTCGAGC ACTGTCCAGT ATTCTACCGT GGTACACAGT
# 2550
GGCTACAGAC ACCAAGTTCC GTCAGTCCAA GTCTTCTCAA GATCCGAGTC
# 2600
TACCCAGCCC TTGTTAGATT CAGAGGAGCG GCCAGAAGAT CTACAATTAG
# 2650
TAGATCATGT AGATGGCGGT GATGGTATTT TGCCCAGGCA ACAGTACTTC
# 2700
AAACAGAACT GCAGTCAGCA TGAATCCAGT CCAGATATTT CACATTTTGA
# 2750
AAGGTCAAAG CAAGTTTCAT CAGTCAATGA GGAAGATTTT GTTAGACTTA
# 2800
AACAGCAGAT TTCAGATCAT ATTTCACAAT CCTGTGGATC TGGGCAAATG
# 2850
AAAATGTTTC AGGAAGTTTC TGCAGCAGAT GCTTTTGGTC CAGGTACTGA
# 2900
GGGACAAGTA GAAAGATTTG AAACAGTTGG CATGGAGGCT GCGACTGATG
# 2950
AAGGCATGCC TAAAAGTTAC TTACCACAGA CTGTACGGCA AGGCGGCTAC
# 3000
ATGCCTCAGT GAAGGACTAG TAGTTCCTGC TACAACTTCA GCAGTACCTA
# 3050
TAAAGTAAAG CTAAAATGAT TTTATCTGTG AATTC
#
# 3085
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 amino
#acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#5:
Met Leu Ala Val Gly Cys Ala Leu Leu Ala Al
#a Leu Leu Ala Ala Pro
5
#
#10
#15
Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Al
#a Gln Glu Val Ala Arg
20
# 25
# 30
Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Va
#l Thr Leu Thr Cys Pro
35
# 40
# 45
Gly Val Glu Pro Glu Asp Asn Ala Thr Val Hi
#s Trp Val Leu Arg Lys
50
# 55
# 60
Pro Ala Ala Gly Ser His Pro Ser Arg Trp Al
#a Gly Met Gly Arg Arg
65
# 70
# 75
#80
Leu Leu Leu Arg Ser Val Gln Leu His Asp Se
#r Gly Asn Tyr Ser Cys
85
# 90
# 95
Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val Hi
#s Leu Leu Val Asp Val
100
# 105
# 110
Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Ar
#g Lys Ser Pro Leu Ser
115
# 120
# 125
Asn Val Val Cys Glu Trp Gly Pro Arg Ser Th
#r Pro Ser Leu Thr Thr
130
# 135
# 140
Lys Ala Val Leu Leu Val Arg Lys Phe Gln As
#n Ser Pro Ala Glu Asp
145 1
#50 1
#55 1
#60
Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Se
#r Gln Lys Phe Ser Cys
165
# 170
# 175
Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Ph
#e Tyr Ile Val Ser Met
180
# 185
# 190
Cys Val Ala Ser Ser Val Gly Ser Lys Phe Se
#r Lys Thr Gln Thr Phe
195
# 200
# 205
Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pr
#o Ala Asn Ile Thr Val
210
# 215
# 220
Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Se
#r Val Thr Trp Gln Asp
225 2
#30 2
#35 2
#40
Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Le
#u Arg Phe Glu Leu Arg
245
# 250
# 255
Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Th
#r Trp Met Val Lys Asp
260
# 265
# 270
Leu Gln His His Cys Val Ile His Asp Ala Tr
#p Ser Gly Leu Arg His
275
# 280
# 285
Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gl
#y Gln Gly Glu Trp Ser
290
# 295
# 300
Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Tr
#p Thr Glu Ser Arg Ser
305 3
#10 3
#15 3
#20
Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Me
#t Gln Ala Leu Thr Thr
325
# 330
# 335
Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg As
#p Ser Ala Asn Ala Thr
340
# 345
# 350
Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pr
#o Leu Pro Thr Phe Leu
355
# 360
# 365
Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Le
#u Leu Cys Ile Ala Ile
370
# 375
# 380
Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Ar
#g Ala Leu Lys Glu Gly
385 3
#90 3
#95 4
#00
Lys Thr Ser Met His Pro Pro Tyr Ser Leu Gl
#y Gln Leu Val Pro Glu
405
# 410
# 415
Arg Pro Arg Pro Thr Pro Val Leu Val Pro Le
#u Ile Ser Pro Pro Val
420
# 425
# 430
Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Se
#r Ser His Asn Arg Pro
435
# 440
# 445
Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp Il
#e Ser Asn Thr Asp Tyr
450
# 455
# 460
Phe Phe Pro Arg
465
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 ami
#no acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#6:
Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pr
#o Val Ala Phe Ser Leu
1 5
# 10
# 15
Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pr
#o Ala Pro Val Pro Pro
20
# 25
# 30
Gly Glu Asp Ser Lys Asp Val Ala Ala Pro Hi
#s Arg Gln Pro Leu Thr
35
# 40
# 45
Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Ty
#r Ile Leu Asp Gly Ile
50
# 55
# 60
Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Se
#r Asn Met Cys Glu Ser
65
# 70
# 75
# 80
Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu As
#n Leu Pro Lys Met Ala
85
# 90
# 95
Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe As
#n Glu Glu Thr Cys Leu
100
# 105
# 110
Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Gl
#u Val Tyr Leu Glu Tyr
115
# 120
# 125
Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gl
#n Ala Arg Ala Val Gln
130
# 135
# 140
Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gl
#n Lys Lys Ala Lys Asn
145 1
#50 1
#55 1
#60
Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Th
#r Asn Ala Ser Leu Leu
165
# 170
# 175
Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gl
#n Asp Met Thr Thr His
180
# 185
# 190
Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gl
#n Ser Ser Leu Arg Ala
195
# 200
# 205
Leu Arg Gln Met
210
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GGAGGCTGTA GGCATAAATT GGTCTGCGC
#
# 29
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CCCGAGATTG AGATCTTCTG CGACGCGGCG ATTGAGACC
#
# 39
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) SEQUENCE DESCRIPTION:
#SEQ ID NO:9:
CGGGATCCAT GGGAGGTTGG TCATC
#
# 25
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGAATTCCAC TGCATGGC
#
#
# 18
1.PublishNumber: US-6217858-B1
2.Date Publish: 20010417
3.Inventor: GALUN EITHAN
NAHOR ORIT
BLUM HUBERT E.
4.Inventor Harmonized: GALUN EITHAN(IL)
NAHOR ORIT(IL)
BLUM HUBERT E(DE)
5.Country: US
6.Claims:
(en)The invention provides a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection, comprising an amount of a soluble active agent which interacts with at least one of the binding sites between hIL6 and pS1 and between hIL6 and hepatocytes and other HBV-permissive cells, the active agent being present in sufficient amount to competitively bind to at least one of the sites and thereby to prevent hIL6-mediated HBV infection of hepatocytes and other HBV-permissive cells.
7.Description:
(en)BACKGROUND AND SUMMARY OF THE INVENTION
The present invention relates to pharmaceutical compositions for the treatment of hepatitis B virus (HBV) infection.
HBV infection in humans can cause chronic liver disease which will, in some cases, proceed to hepatocellular carcinoma. The initial steps of HBV attachment to cells and the targeting of the viral genome to the host cell nucleus have yet to be deciphered. The specific receptor for HBV has not so far been identified, even though various serum proteins and cellular membrane glycoproteins have been suggested as mediators of cell penetration or viral receptors. HBV envelope proteins were reported to contain residues which interact with polymerized albumin [P. Pontisso, et al., Journal of Virology , Vol. 63, No. 1981-1, p. 988 (1981)] or with soluble transferrin [M. Gagliardi, et al., Eur. J. Immunol ., Vol. 24, pp. 1372-1376 (1994)], enabling viral penetration of cells via their respective receptors, probably in a non-specific manner.
In a study reported by Neurath, et al. [A. Neurath, et al., J. Exp. Med ., Vol. 175, pp. 461-469 (1992)] hIL-6 was shown to bind the pS1 (aa 21-47) segment of the HBV envelope. Putative candidates for the HBV receptor were recently reported, including Annexin V (endohexin II) [K. Hertogs, et al., Virology , Vol. 197, pp. 549-557 (1993)]; apolipoprotein H [H. Mehdi, et al., Journal of Virology , Vol. 68, pp. 2415-2424 (1994)]; and asialoglycoprotein receptor [U. Treichel, et al., Journal of General Virology , Vol. 75, pp. 3021-3029 (1994)].
Binding experiments have demonstrated that the pre-S1 (pS1) region of the viral envelope protein contains a recognition site for the host cell [A. R. Neurath, et al., Cell , Vol. 46, pp. 429-436 (1986); M. Petit, et al., Virology , Vol. 180, pp. 483-491 (1990); M. Petit, et al., Virology , Vol. 197, pp. 211-222 (1992)]. Although previous studies had suggested that HepG2 cells [R. Bchini, et al., Journal of Virology , Vol. 64, pp. 3025-3032 (1991)] and human hepatocytes [P. Gripon, et al., Journal of Virology , Vol. 62, pp. 4136-4143 (1988); T. Ochiya, et al., Proc. Natl. Acad. Sci. U.S.A ., Vol. 86, pp. 1875-1879 (1989); P. Gripon, et al., Virology , Vol. 192, pp. 534-540 (1993); P. Galle, et al., Gastroenterology , Vol. 106, pp. 664-673 (1994)] could support HBV infection in vitro, no cellular receptor has as yet been defined in either system, and these models were of low experimental reproducibility.
In current reports, it has been shown that a chimeric mouse, generated by using Beige/Nude/X linked immunodeficient (BNX) mice, preconditioned by total body irradiation (12Gy) and reconstituted with severe combined immunodeficient (SCID) mice bone marrow (BM) cells, is permissive for normal human T and B cells [I. Lubin, et al., Science , Vol. 252, pp. 427-431 (1991)], as well as for normal human liver tissue [E. Galun, et al., Journal of Infectious Diseases , Vol. 175, pp. 25-30 (1995)]. Hepatitis C virus (HCV) viremia was detectable for up to two months, after implantation under the kidney capsule of the BNX>SCID chimeric animals of either a human liver fragment with preexisting HCV infection, or normal human liver tissue following incubation ex-vivo of the transplanted liver fragment with HCV-positive sera [E. Galun, et al., ibid.]. Heretofore, one of the major obstacles in elucidating the initial steps of HBV infection and the assessment of antiviral agents, has been the lack of a small animal model. Using the techniques referred to above, it was possible to develop SCID>BNX animals which sustain HBV viremia following the implantation of an ex-vivo HBV DNA-positive sera incubation with liver tissue. The method in which the animals were prepared for the experiments described herein, and the surgical technique for transplantation, are similar to those previously reported [E. Galun, et al., ibid.].
As will be described below, it has now been found, using a chimeric animal model, that human interleukin 6 (hIL6) is essential for HBV infection. Having identified that hIL6 serves as an essential bridge for HBV infection, the invention now provides a pharmaceutical composition for the treatment of hepatitis B virus infection, comprising an amount of a soluble active agent which interacts with at least one of the binding sites between hIL6 and pS1 and between hIL6 and hepatocytes and other HBV-permissive cells, said active agent being present in sufficient amount to competitively bind to at least one of said sites and thereby to prevent hIL6-mediated HBV infection of hepatocytes and other HBV-permissive cells.
In a first preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection, comprising an amount of soluble gp80 and/or gp130 receptor sites sufficient to inhibit the binding of hIL6 to hepatocytes and other HBV-permissive cells.
In a second preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of soluble amino acid sequences corresponding to amino acids 21 to 46 of pS1 to block the interaction of HBV with hIL6.
In a third preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of a soluble ligand selected from the group consisting of peptides LYS41-ALA56, GLY77-GLU95 and GLN153-HIS165 to block the interaction of hIL6 with hepatocytes and other HBV-permissive cells.
In a fourth preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising hIL6 conjugated with an anti-viral agent.
With specific reference now to the examples and figures in detail, it is stressed that the particulars described and shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this context, it is to be noted that only subject matter embraced in the scope of the claims appended hereto, whether in the manner defined in the claims or in a manner similar thereto and involving the main features, as defined in the claims, is intended to be included in the scope of the present invention.
In the drawings:
The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings are provided to the Patent and Trademark Office with payment of the necessary fee.
FIG. 1 illustrates a pre-infected liver fragment from a HBV DNA-positive patient, one month after sub-capsular implantation in a SCID>BNX chimeric mouse, stained for HBsAg;
FIGS. 2A-D shows that hIL6 mediates HBV viremia in SCID>BNX chimeric mice transplanted with human tissue; and
FIG. 3 illustrates the liver histology of a HepG2-hIL-6R tumor, which developed one month following intrasplenic injection into a SCID>BNX chimeric mouse (H and E staining);
FIG. 4 provides the nucleotide sequence for hIL-6 mRNA;
FIGS. 5 a and 5 b provide the nucleotide sequence for hIL-6 receptor mRNA;
FIG. 6 provides the nucleotide sequence for the IL-6 receptor;
FIGS. 7 a and 7 b provide the nucleotide sequence for gp130;
FIG. 8 provides the amino acid sequence for hIL-6 receptor alpha; and
FIG. 9 provides the amino acid sequence for IL-6.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES
Human liver tissue was taken from patients undergoing liver surgery for liver diseases, who had HBV viremia of 10 7 -10 9 parciles/ml with positive HBV DNA in the liver tissue. The liver tissue was implanted under the kidney capsule of the chimeric animals. Although HBsAg was easily detected in pre-infected HBV DNA positive/HBeAg positive transplanted tissue (FIG. 1 ), 1-3 months after liver fragment implantation HBV sequences were undetectable by PCR (applyin primers spanning the viral core gene as well as the envelope region, at the a determinant of the HBsAg) in any of these experiments. Furthermore, intravenous or intraperitoneal (i.p.) injection of 200 μl of high-titer HBV particles (>10 8 /ml) following the transplantation of a normal human liver fragment, failed to generate HBV DNA sequences during the next 30 days (data not shown).
Lymphocytes, positive for HBV DNA by dot blot hybridization, were separated by lymphopheresis (Baxter Fenwell CS-3000 Pulse Blood Cell Separators, Deerfield, Ill., U.S.A.) from a patient with HBV-related chronic liver disease whose sera were positive for HBV DNA and HBeAg. Forty million HBV DNA-positive lymphocytes were injected i.p. to each mouse, subsequent to transplantation of normal human liver at the subcapsular site of the kidney. HBV sequences were not detected in the sera of these animals during the following 21 days.
Although the primary infection site for HBV is hepatocytes, lymphocytes and endothelial cells have both been shown to harbor HBV transcripts and viral-related proteins [J. Romet-Lemonne, et al., Science , Vol. 221, pp. 667-669 (1983); H. Blum, et al., Proc. Natl. Acad. Sci. U.S.A ., Vol. 80, pp. 6685-6688 (1983); E. Galun, et al., American Journal of Pathology , Vol. 145, pp. 1001-1007 (1994)], suggesting a common specific cell membrane receptor mechanism supporting viral penetration. This mechanism would prevent infection of receptor negative cells, despite their being permissive for HBV replication by transfection [E. Galun, et al., Journal of General Virology , Vol. 73, pp. 173-178 (1992)]. All three primary cell types hosting HBV naturally, i.e., hepatocytes, lymphocytes and endothelial cells, respond to hIL6 through the human IL6 receptor (hIL6R) which is expressed on their cell membranes [A. Mackiewicz, et al., The Journal of Immunology , Vol. 149, pp. 2021-2027 (1992); J. Bauer, et al., FEBS Letter , Vol. 249, pp. 27-30 (1989); T. Kishimoto, et al., Science , Vol. 258, p. 593 (1992)]. Furthermore, as previously shown, hIL6 binds to HBV through pS1.
A fragment of normal human liver from a patient with no indication of any HBV-related markers or disease, was incubated ex-vivo with a high titer HBV DNA-positive serum prior to transplantation under the kidney capsule of the chimeric animals. HBV DNA sequences were undetectable by PCR from two different genomic regions in any of these animals during the month following transplantation. Results are shown in FIG. 2 A. These results were reproduced in additional experiments in over 50 mice, using four different HBV DNA-positive sera.
However, when liver tissue originating from the same patients was incubated ex-vivo with the above-mentioned HBV DNA-positive sera togethcr with hIL6, HBV DNA sequences were detected from day 16 to day 31, in sera of about 50% of the transplanted animals. These results are shown in FIG. 2 B.
Similar results were obtained in experiments conducted under the above-stated conditions, using additional HBV DNA sera and liver tissue from different sources. In these experiments, HBV DNA sequences could be detected up to day 60 following transplantation (results not shown).
Pre-exposure of liver tissue to hIL6 prior to incubation with HBV ex-vivo, increased infection to about 90% of the animals. Animals positive for HBV sequences in serum at day 31 were also positive for HBsAg in the implanted hepatocytes, as shown in FIG. 2 D. Liver fragments incubated ex-vivo with HBV under the above conditions and fixed for immunohistochemical analysis prior to transplantation were negative for HBsAg (results not shown).
To further assess the role of hIL6 in supporting HBV infection, a human hepatoblastoma cell line HepG2 (ATCC HB 8065), an HepG2-derived, stably transfected hIL6R cell line, a null hIL6R (a cell line which does not express hIL6R) named HepG2-PDI and an hIL6 producing line named HepG2-hIL6 [S. Rose-John, et al., The Journal of Biological Chemistry , Vol. 268, pp. 22084-22091 (1993)] were incubated with HBV DNA-positive sera, with or without hIL6. Following incubation, the various mixtures were injected intrasplenically to the chimeric mice to generate HCC foci in the liver, as shown in FIG. 3 . The results of these experiments are summarized below in Table 1.
TABLE 1
HBV-DNA as detected by PCR in sera of chimeric mice following
intrasplenic injection of HBV, with or without hIL6, after incubation with
HepG2-derived cell lines
HepG 2
HepG 2
HepG 2
Cell Line
hIL6R
PDI
HepG 2
hIL6
hIL6
+
−
+
−
+
−
+
−
HBV-DNA PCR product
+
−
+
−
+
+/−
+
+
Method:
All cell lines grew in T25 flasks supplemented with DMEM medium, enriched with 10% fetal bovine serum. For infection experiments, cells were trypsinized and washed twice with PBS, followed by incubation with HBV-positive human sera (10 8 virions/ml) in the presence or absence of hlL6 (500 ng/ml) in 1-2 ml of DMEM. After 2-4 h incubation at 37° C., 4×10 6 cells/ml, 0.5 ml/mouse were injected intrasplenically to 8-10 SCID>BNX mice in each group. Animals were splenectomized following the injection.
Mice were bled at two weekly intervals for 3 months, and DNA was extracted from 100 μl sera. The DNA was subjected to PCR amplification. The DNA extraction and the PCR method applied are described in the legend of FIG. 2 . Table 1 summarizes three experiments.
In mice implanted with HepG2-hIL6R cells (which have about one log higher expression of the receptor than HepG2 cells) subsequent to incubation with HBV in the presence of hIL6, HBV-DNA sequences could be detected in serum 13 days after transplantation, whereas HBV sequences were not detected in the sera of mice who underwent the same procedure without the presence of hIL6. Similar results were obtained in experiments using HepG2-PDI cells. These cells do not express the gp 80 binding protein subunit of the hIL6R on the cell membrane [S. Rose-John, et al., ibid.; M. Ehlers, et al., The Journal of Immunology , Vol. 153, p. 1744 (1994)], however, they do express the signal transduction gp 130 subunit of the receptor, which is essential for efficient intemaliztion of hIL6 [E. Dittrich, et al., The Journal of Biological Chemistry , Vol. 269, pp. 10914-19020 (1994)].
In experiments of the same design, using HepG2 cells in the presence and also in the absence of hIL6, HBV sequences could be detected in a number of murine sera. These results are similar to those previously reported by Petit, et al. [R. Bchini, et al., ibid.], showing a low reproducibility in which only three sera supported HBV infection of HepG2 cells in-vitro, out of a total of 55 different serum samples taken from HBV DNA-positive patients. The HepG2-hIL6 cell line, which produces hIL6, generated HBV sequences in mice sera following incubation with the virus, with or without external supplementation of hIL6.
When the liver fragment was incubated ex-vivo with HBV-DNA positive sera in the presence of commercially available human polyclonal anti-HBs viral neutralizing antibodies (HBIG, Hepatect®, Biotest Pharma GmbH, Dreicich. Germany), HBV-DNA was observed at day 11 following transplantation only in 48% (10/21) of mice, as compared to 78% (14/18) of the untreated mice group (Table 2).
TABLE 2
Inhibition of infection - effect of anti-HBs antibodies effect on HBV-DNA
levels in sera of chimeric mice transplanted with human liver fragments
infected ex vivo with HBV
Treatment Group
Mice Positive for HBV-DNA (%)
Untreated
14/18 (78)
HBIG Treatment
10/21 (48)
Method:
For antibody treatment, HBV-DNA positive serum (0.5 ml) was incubated with 100 IU of HBIG for 2 hours at 25° C. Human liver fragments were then added to the untreated or HBIG treated HBV-DNA positive serum according to the same protocol as described above, followed by implantation under the kidney capsule of the chimeric animal. Mice sera were analyzed for the presence of HBV-DNA sequences 11 days after transplantation.
Referring again to the figures, FIG. 1 shows pre-infected liver fragment from a HBV DNA-positive patient, one month after sub-capsular implantation in a SCID>BNX chimeric mouse, stained for HBsAg.
From FIG. 2 it can be seen that hIL6 mediates HBV viremia in SCID>BNX chimeric mice transplanted with human tissue. PCR amplification products of HBV pre-core/core region following DNA extraction from sera of mice, 16 and 31 days after sub-capsular kidney transplantation of normal human liver fragments. The human liver fragments were incubated ex-vivo prior to transplantation with human HBV positive serum (FIG. 2 A); HBV serum and hIL6 simultaneously (FIG. 2 B), or preincubated with hIL6 and later with HBV sera FIG. 2 C). In each of FIGS. 2A to 2 C, the upper panel is an EtBr staining and the lower panel is an 32 P HBV linear insert hybridization result of the same gel. The molecular marker size (m) is indicated by an arrow; numbers at the head of each panel indicate mice identification numbers; + for positive serum control and − for negative serum control.
FIG. 2D shows HBsAg staining of an ex-vivo HBV incubation of a normal liver fragment with hIL6. one month following implantation under the kidney capsule of SCID>BNX mice.
Sera from HBV-positive patients, containing approximately 10 8 virions/ml, were used for infection. Small fragments of normal human liver were incubated with 400 μl sera in 1 ml DMEM supplemented with 2 μg/ml polybrene in the absence (group A) or presence (group B) of hIL6 (500 ng/ml) incubated for 2-4 h at 37° C. In group C, the liver fragments were treated with hIL6 for 2 h at 37° C. before the addition of HBV-positive sera and polybrene. After incubation, 4-5 ml polybrene DMEM were added and the liver fragments were transplanted under the kidney capsule to groups A, B and C of SCID>BNX chimeric mice (10, 19 and 11 mice, respectively). At 2 weekly intervals for 4 months, blood was collected retrobulbarily from each mouse. 100 μl of serum samples were treated with 0.5 mg/ml proteinase K in 10 mM EDTA and 0.25% SDS for 2 h at 55° C. or overnight at 37° C., extracted twice with phenol, once with phenol-CHCl 3 , and once with CHCl 3 . DNA was precipitated with ethanol, using 0.5M NaCl and a DNA microcarrier. DNA was dissolved in 30 μl Tris-EDTA, pH 8.0, and was subjected to PCR amplification.
The 50 μl PCR reaction volume contained 10 ρmole of each oligonucleotide primer in reaction buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.0 mM MgCl 2 , 0.01% (w/v) gelatin, 250 μM of dATP, dGTP, dCTP, dTTP and 0.5 u of Taq polymerase. The reaction mixtures were overlaid with 30 μl of mineral oil. PCR cycles included 94° C. for 1 min., 55° C. for 1 min. and 72° C. for 3 min., 35 repeated cycles. 10 μl of reaction mixture was analyzed on a 2% agarose gel. Oligonucleotides used for the pre-core/core amplification were:
oligo 1, Sense (nt 1778 to 1806):
5′ GGA-GGC-TGT-AGG-CAT-AAA-TTG-GTC-TGC-GC-3′. Sequence ID No. 7
oligo 2, Antisense (nt 2446 to 2408):
5′ CCC-GAG-ATT-GAG-ATC-TTC-TGC-GAC-GCG-GCG-ATT-GAG-ACC-3′. Sequence ID No. 8
Sequence originated from adw subtype; nt numbering starts from EcoRI site. The expected size of the PCR DNA product is 668-bp.
The PCR samples were electrophoresed on 2% agarose gel and transferred to a nylon membrane (Biodynea), hybridized with a nick-translated probe. The autoradiogram was exposed with intensifying screens at −70° C. for 7 h. In order to confirm the PCR results, the mice serum samples were also subjected to PCR amplification with primers spanning the envelope gene region, showing the same results (data not shown).
Reproducible results were obtained from four similar experiments while there were 10-20 mice in each group.
Based on the present discovery that hIL6 acts to mediate HBV infection, it is possible to prepare an antiviral/anti-HBV agent. A pharmaceutical composition for the prevention of HBV infection, comprising an active ingredient having an amino acid sequence similar to hIL6, is thus developed. The hIL6 domain which interacts with hIL6Rα (R for receptor) and/or hiL6Rβ (amino acid residues: 40-60, 70-100 and 135-175) antagonizes hIL6 interaction to prevent HBV infection.
Set forth at FIG. 4 is the nucleotide sequence for human interleukin 6 mRNA (SEQ ID NO 1), as published by L. T. May, et al., “Anti-beta Interferon Antibodies Inhibit the Increased Expression of HLA-B7 mRNA in Tunor Necrosis Factor-Treated Human Fibroblasts: Structural Studies of the beta-2 Interferon Involved,” Proc. Nat'l Acad. Sci. U.S.A. 83 (23), 8957-8960 (1986).
FIGS. 5 a and 5 b depict the nucleotide sequence for the human interleukin 6 receptor mRNA (SEQ ID NO 2), as published by K. Yamasuki, et al., “Cloning and Expression of the Human Interleukin 6 (BSF-2/ISN beta 2) Receptor,” Science 241 (4867), 825-828 (1988).
FIG. 6 depicts the nucleotide sequence for the interleukin-6 receptor (SEQ ID NO 3), as published by H. Schooltink, et al., “Structural and Functional Studies on the Human Nepatic Interleukin-6 Receptor,” Biochem. J. 277:659-664 (1991).
FIGS. 7 a and 7 b depict the nucleotide sequence for the gp 130 interleukin 6 receptor (SEQ ID NO 4), as published by M. Hibi, et al, “Molecular Cloning and Expression of an IL-6 Signal Transducer, gp 130,” Cell 63 (6), 1149-1157 (1990).
FIG. 8 depicts the amino acid sequence for the human interleukin 6 receptor alpha (IL-6R alpha) (SEQ ID NO 5), as published by K. Yamasaki, et al., “Cloning and Expression of the Human Interleukin 6 (BSF-2/ISN beta 2) Receptor,” Science 241 (4867), 825-828 (1988).
FIG. 9 depicts the amino acid sequence for human interleukin 6 (SEQ ID No. 6), as published by C. Nishimura, et al., Biochemistry 35:273-281 (1996).
The molecular analysis of hIL6 binding sites with gp130 and gp80 revealed a number of structural targets on hIL6 which can serve as hIL6 antagonists. The preferable target for an hIL6 antagonist is to disrupt the hIL6/hIL6Rα complex with hIL6Rβ.
Based on previous publication, a number of domains essential for hIL6 activity were reported:
1. Lys41-ala56 (site 2a, also named β2) is involved in the activation of signal-transduction.
2. Gly77-glu95 (site 2c) is important for interaction with hIL6Rα, subunit gp80.
3 Gln153-his165 (site β1), substitution of trp158 to arg or gln160 to glu combined with thr163 to pro-antagonize the biological activity of hIL6.
4. A combined β1 and β2 hIL6 mutant (mhIL6β1+β2) is inactive on XG-1 hIL6 responsive cells, with a weakly antagonizing activity.
5. The addition of two substitutions to the mhIL6 (m for mutant) β1+β2, phe171 to leu and ser177 to arg, resulted in an increase in the affinity to hIL6Rα, while inhibiting its activity on XG-1 cells.
Based on techniques known per se to persons skilled in the art, the proteins and peptides for use in the pharmaceutical compositions of the present invention are readily prepared, e.g., by the following techniques and steps:
Amplification of chosen segment of DNA from plasmid containing HBV DNA (adw2)-adw HTD by PCR, using primers constructed so as to introduce BamH1 and EcoR1 sites compatible to the pGEX-2T (Pharmacia, Uppsala, Sweden, Catalogue No. 27-4801-01) insertion site. This GST fusion vector provides a system in which fusion proteins are easily purified from the bacterial lysates and can be detected directly as a fusion protein or after cleavage with site specific proteases. After introduction of DNA into pGEX-2T, competent E. coli (JM109) are transformed and cloned (LB+10 μg/ml ampicillin). Protein expression is induced by the addition of IPTG (0.1 mM, isopropyl-1-thio-b-D-galactoside) for 1-2 hours. Fusion protein is removed from lysed (sonicated) cells by collection on glutathione-agarose beads (Pharmacia) and eluted from beads using reduced glutathione (5 mM in 50 mM Tris-Cl, pH 8.0). Identification and determination of protein can be done either by use of antibodies to GST or by specific recognition of inserted protein. The complete HBV pS1 protein (aa 1 to aa 119, applying PCR with the sense and anti-sense primers 5′-CGGGATCCATGGGAGGTTGGTCATC-3′[NT 8+2856-2873, EcoR1 as starting site for nt numbering] (Sequence ID No. 9) and 5′-GGAATTCCACTGCATGGC-3′ [nt 6-3210] respectively) (Sequence ID No. 10) and the pS1 attachment site aa21 to aa46 constructs were designed and produced in the pGEX-2T system (compound B in FIG. 1 ).
Truncated soluble forms of gp80 and gp130 are synthesized using the pGEX-2T system as described for the preparation of pS1 (compound A2 and A1, respectively). hIL6 derived peptides (Lys41-ala56, Gly77-glu95 and Gln153-his165, designated C1, C3 and C2, respectively, in FIG. 1) are synthesized by applying a variety of methods including Merrifield solid-phase synthesis and derived methods or other acceptable genetic engineered methods. The compounds produced are linear or cyclic peptides, or parts of large proteins.
Compositions according to the present invention can be administered orally or parenterally, including intravenous, intraperitoneal, intranasal and subcutaneous administration. Implants of the compounds are also useful.
The proteins of the present invention are administered in combination with other drugs, or singly, consistent with good medical practice. The composition is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners. The ‘effective amount’ for purposes herein is thus determined by such considerations as are known in the art.
When administering the compositions parenterally, the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
Proper fluidity can be maintained, for example, by the use of a coating such as lecithin by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters such as isopropyl myristate, may also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, anti-oxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent or additive used would have to be compatible with the compounds.
Sterile injectable solutions can be prepared by incorporating the proteins utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
A pharmacological formulation described and claimed herein can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems, such as polymer matrices, liposomes, and microspheres. An implant suitable for use in the present invention can take the form of a pellet which slowly dissolves after being implanted, or a biocompatible delivery module well-known to those skilled in the art. Such well-known dosage forms and modules are designed such that the active ingredients are slowly released over a period of several days to several weeks.
Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194. which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow, implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are well-known to those skilled in the art.
A pharmacological formulation of the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like, are usable. Known techniques which deliver the new compositions orally or intravenously and retain the biological activity, are preferred.
In one embodiment, the new compositions can be administered initially by intravenous injection. The quantity of the compositions to be administered will vary for the patient being treated, and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day, and preferably will be from 10 μg/kg to 10 mg/kg per day.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
# SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 10
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATTCTGCCCT CGAGCCCACC GGGAACGAAA GAGAAGCTCT ATCTCCCCTC
# 50
CAGGAGCCCA GCTATGAACT CCTTCTCCAC AAGCGCCTTC GGTCCAGTTG
# 100
CCTTCTCCCT GGGGCTGCTC CTGGTGTTGC CTGCTGCCTT CCCTGCCCCA
# 150
GTACCCCCAG GAGAAGATTC CAAAGATGTA GCCGCCCCAC ACAGACAGCC
# 200
ACTCACCTCT TCAGAACGAA TTGACAAACA AATTCGGTAC ATCCTCGACG
# 250
GCATCTCAGC CCTGAGAAAG GAGACATGTA ACAAGAGTAA CATGTGTGAA
# 300
AGCAGCAAAG AGGCACTGGC AGAAAACAAC CTGAACCTTC CAAAGATGGC
# 350
TGAAAAAGAT GGATGCTTCC AATCTGGATT CAATGAGGAG ACTTGCCTGG
# 400
TGAAAATCAT CACTGGTCTT TTGGAGTTTG AGGTATACCT AGAGTACCTC
# 450
CAGAACAGAT TTGAGAGTAG TGAGGAACAA GCCAGAGCTG TCCAGATGAG
# 500
TACAAAAGTC CTGATCCAGT TCCTGCAGAA AAAGGCAAAG AATCTAGATG
# 550
CAATAACCAC CCCTGACCCA ACCACAAATG CCAGCCTGCT GACGAAGCTG
# 600
CAGGCACAGA ACCAGTGGCT GCAGGACATG ACAACTCATC TCATTCTGCG
# 650
CAGCTTTAAG GAGTTCCTGC AGTCCAGCCT GAGGGCTCTT CGGCAAATGT
# 700
AGCATGGGCA CCTCAGATTG TTGTTGTTAA TGGGCATTCC TTCTTCTGGT
# 750
CAGAAACCTG TCCACTGGGC ACAGAACTTA TGTTGTTCTC TATGGAGAAC
# 800
TAAAAGTATG AGCGTTAGGA CACTATTTTA ATTATTTTTA ATTTATTAAT
# 850
ATTTAAATAT GTGAAGCTGA GTTAATTTAT GTAAGTCATA TTTTATATTT
# 900
TTAAGAAGTA CCACTTGAAA CATTTTATGT ATTAGTTTTG AAATAATAAT
# 950
GGAAAGTGGC TATGCAGTTT GAATATCCTT TGTTTCAGAG CCAGATCATT
# 1000
TCTTGGAAAG TGTAGGCTTA CCTCAAATAA ATGGCTAACT TTATACATAT
# 1050
TTTTAAAGAA ATATTTATAT TGTATTTATA TAATGTATAA ATGGTTTTTA
# 1100
TACCAATAAA TGGCATTTTA AAAAATTC
#
# 1128
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3319 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGCGGTCCCC TGTTCTCCCC GCTCAGGTGC GGCGCTGTGG CAGGAAGCCA
# 50
CCCCCTCGGT CGGCCGGTGC GCGGGGCTGT TGCGCCATCC GCTCCGGCTT
# 100
TCGTAACCGC ACCCTGGGAC GGCCCAGAGA CGCTCCAGCG CGAGTTCCTC
# 150
AAATGTTTTC CTGCGTTGCC AGGACCGTCC GCCGCTCTGA GTCATGTGCG
# 200
AGTGGGAAGT CGCACTGACA CTGAGCCGGG CCAGAGGGAG AGGAGCCGAG
# 250
CGCGGCGCGG GGCCGAGGGA CTCGCAGTGT GTGTAGAGAG CCGGGCTCCT
# 300
GCGGATGGGG GCTGCCCCCG GGGCCTGAGC CCGCCTGCCC GCCCACCGCC
# 350
CCGCCCCGCC CCTGCCACCC CTGCCGCCCG GTTCCCATTA GCCTGTCCGC
# 400
CTCTGCGGGA CCATGGAGTG GTAGCCGAGG AGGAAGCATG CTGGCCGTCG
# 450
GCTGCGCGCT GCTGGCTGCC CTGCTGGCCG CGCCGGGAGC GGCGCTGGCC
# 500
CCAAGGCGCT GCCCTGCGCA GGAGGTGGCA AGAGGCGTGC TGACCAGTCT
# 550
GCCAGGAGAC AGCGTGACTC TGACCTGCCC GGGGGTAGAG CCGGAAGACA
# 600
ATGCCACTGT TCACTGGGTG CTCAGGAAGC CGGCTGCAGG CTCCCACCCC
# 650
AGCAGATGGG CTGGCATGGG AAGGAGGCTG CTGCTGAGGT CGGTGCAGCT
# 700
CCACGACTCT GGAAACTATT CATGCTACCG GGCCGGCCGC CCAGCTGGGA
# 750
CTGTGCACTT GCTGGTGGAT GTTCCCCCCG AGGAGCCCCA GCTCTCCTGC
# 800
TTCCGGAAGA GCCCCCTCAG CAATGTTGTT TGTGAGTGGG GTCCTCGGAG
# 850
CACCCCATCC CTGACGACAA AGGCTGTGCT CTTGGTGAGG AAGTTTCAGA
# 900
ACAGTCCGGC CGAAGACTTC CAGGAGCCGT GCCAGTATTC CCAGGAGTCC
# 950
CAGAAGTTCT CCTGCCAGTT AGCAGTCCCG GAGGGAGACA GCTCTTTCTA
# 1000
CATAGTGTCC ATGTGCGTCG CCAGTAGTGT CGGGAGCAAG TTCAGCAAAA
# 1050
CTCAAACCTT TCAGGGTTGT GGAATCTTGC AGCCTGATCC GCCTGCCAAC
# 1100
ATCACAGTCA CTGCCGTGGC CAGAAACCCC CGCTGGCTCA GTGTCACCTG
# 1150
GCAAGACCCC CACTCCTGGA ACTCATCTTT CTACAGACTA CGGTTTGAGC
# 1200
TCAGATATCG GGCTGAACGG TCAAAGACAT TCACAACATG GATGGTCAAG
# 1250
GACCTCCAGC ATCACTGTGT CATCCACGAC GCCTGGAGCG GCCTGAGGCA
# 1300
CGTGGTGCAG CTTCGTGCCC AGGAGGAGTT CGGGCAAGGC GAGTGGAGCG
# 1350
AGTGGAGCCC GGAGGCCATG GGCACGCCTT GGACAGAATC CAGGAGTCCT
# 1400
CCAGCTGAGA ACGAGGTGTC CACCCCCATG CAGGCACTTA CTACTAATAA
# 1450
AGACGATGAT AATATTCTCT TCAGAGATTC TGCAAATGCG ACAAGCCTCC
# 1500
CAGTGCAAGA TTCTTCTTCA GTACCACTGC CCACATTCCT GGTTGCTGGA
# 1550
GGGAGCCTGG CCTTCGGAAC GCTCCTCTGC ATTGCCATTG TTCTGAGGTT
# 1600
CAAGAAGACG TGGAAGCTGC GGGCTCTGAA GGAAGGCAAG ACAAGCATGC
# 1650
ATCCGCCGTA CTCTTTGGGG CAGCTGGTCC CGGAGAGGCC TCGACCCACC
# 1700
CCAGTGCTTG TTCCTCTCAT CTCCCCACCG GTGTCCCCCA GCAGCCTGGG
# 1750
GTCTGACAAT ACCTCGAGCC ACAACCGACC AGATGCCAGG GACCCACGGA
# 1800
GCCCTTATGA CATCAGCAAT ACAGACTACT TCTTCCCCAG ATAGCTGGCT
# 1850
GGGTGGCACC AGCAGCCTGG ACCCTGTGGA TGACAAAACA CAAACGGGCT
# 1900
CAGCAAAAGA TGCTTCTCAC TGCCATGCCA GCTTATCTCA GGGGTGTGCG
# 1950
GCCTTTGGCT TCACGGAAGA GCCTTGCGGA AGGTTCTACG CCAGGGGAAA
# 2000
ATCAGCCTGC TCCAGCTGTT CAGCTGGTTG AGGTTTCAAA CCTCCCTTTC
# 2050
CAAATGCCCA GCTTAAAGGG GTTAGAGTGA ACTTGGGCCA CTGTGAAGAG
# 2100
AACCATATCA AGACTCTTTG GACACTCACA CGGACACTCA AAAGCTGGGC
# 2150
AGGTTGGTGG GGGCCTCGGT GTGGAGAAGC GGCTGGCAGC CCACCCCTCA
# 2200
ACACCTCTGC ACAAGCTGCA CCCTCAGGCA GGTGGGATGG ATTTCCAGCC
# 2250
AAAGCCTCCT CCAGCCGCCA TGCTCCTGGC CCACTGCATC GTTTCATCTT
# 2300
CCAACTCAAA CTCTTAAAAC CCAAGTGCCC TTAGCAAATT CTGTTTTTCT
# 2350
AGGCCTGGGG ACGGCTTTTA CTTAAACGCC AAGGCCTGGG GGAAGAAGCT
# 2400
CTCTCCTCCC TTTCTTCCCT ACAGTTCAAA AACAGCTGAG GGTGAGTGGG
# 2450
TGAATAATAC AGTATGTCAG GGCCTGGTCG TTTTCAACAG AATTATAATT
# 2500
AGTTCCTCAT TAGCAGTTTT GCCTAAATGT GAATGATGAT CCTAGGCATT
# 2550
TGCTGAATAC AGAGGCAACT GCATTGGCTT TGGGTTGCAG GACCTCAGGT
# 2600
GAGAAGCAGA GGAAGGAGAG GAGAGGGGCA CAGGGTCTCT ACCATCCCCT
# 2650
GTAGAGTGGG AGCTGAGTGG GGGATCACAG CCTCTGAAAA CCAATGTTCT
# 2700
CTCTTCTCCA CCTCCCACAA AGGAGAGCTA GCAGCAGGGA GGGCTTCTGC
# 2750
CATTTCTGAG ATCAAAACGG TTTTACTGCA GCTTTGTTTG TTGTCAGCTG
# 2800
AACCTGGGTA ACTAGGGAAG ATAATATTAA GGAAGACAAT GTGAAAAGAA
# 2850
AAATGAGCCT GGCAAGAATG CGTTTAAACT TGGTTTTTAA AAAACTGCTG
# 2900
ACTGTTTTCT CTTGAGAGGG TGGAATATCC AATATTCGCT GTGTCAGCAT
# 2950
AGAAGTAACT TACTTAGGTG TGGGGGAAGC ACCATAACTT TGTTTAGCCC
# 3000
AAAACCAAGT CAAGTGAAAA AGGAGGAAGA GAAAAAATAT TTTCCTGCCA
# 3050
GGCATGGAGG CCCACGCACT TCGGGAGGTC GAGGCAGGAG GATCACTTGA
# 3100
GTCCAGAAGT TTGAGATCAG CCTGGGCAAT GTGATAAAAC CCCATCTCTA
# 3150
CAAAAAGCAT AAAAATTAGC CAAGTGTGGT AGAGTGTGCC TGAAGTCCCA
# 3200
GATACTTGGG GGGCTGAGGT GGGAGGATCT CTTGAGCCTG GGAGGTCAAG
# 3250
GCTGCAGTGA GCCGAGATTG CACCACTGCA CTCCAGCCTG GGGTGACAGA
# 3300
GCAAGTGAGA CCCTGTCTC
#
# 331
#9
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1486 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATTAGCCTGT CCGCCTCTGC GGGACCATGG AGTGGTAGCC GAGGAGGAAG
# 50
CATGCTGGCC GTCGGCTGCG CGCTGCTGGC TGCCCTGCTG GCCGCGCCGG
# 100
GAGCGGCGCT GGCCCCAAGG CGCTGCCCTG CGCAGGAGGT GGCGAGAGGC
# 150
GTGCTGACCA GTCTGCCAGG AGACAGCGTG ACTCTGACCT GCCCGGGGGT
# 200
AGAGCCGGAA GACAATGCCA CTGTTCACTG GGTGCTCAGG AAGCCGGCTG
# 250
CAGGCTCCCA CCCCAGCAGA TGGGCTGGCA TGGGAAGGAG GCTGCTGCTG
# 300
AGGTCGGTGC AGCTCCACGA CTCTGGAAAC TATTCATGCT ACCGGGCCGG
# 350
CCGCCCAGCT GGGACTGTGC ACTTGCTGGT GGATGTTCCC CCCGAGGAGC
# 400
CCCAGCTCTC CTGCTTCCGG AAGAGCCCCC TCAGCAATGT TGTTTGTGAG
# 450
TGGGGTCCTC GGAGCACCCC ATCCCTGACG ACAAAGGCTG TGCTCTTGGT
# 500
GAGGAAGTTT CAGAACAGTC CGGCCGAAGA CTTCCAGGAG CCGTGCCAGT
# 550
ATTCCCAGGA GTCCCAGAAG TTCTCCTGCC AGTTAGCAGT CCCGGAGGGA
# 600
GACAGCTCTT TCTACATAGT GTCCATGTGC GTCGCCAGTA GTGTCGGGAG
# 650
CAAGTTCAGC AAAACTCAAA CCTTTCAGGG TTGTGGAATC TTGCAGCCTG
# 700
ATCCGCCTGC CAACATCACA GTCACTGCCG TGGCCAGAAA CCCCCGCTGG
# 750
CTCAGTGTCA CCTGGCAAGA CCCCCACTCC TGGAACTCAT CTTTCTACAG
# 800
ACTACGGTTT GAGCTCAGAT ATCGGGCTGA ACGGTCAAAG ACATTCACAA
# 850
CATGGATGGT CAAGGACCTC CAGCATCACT GTGTCATCCA CGACGCCTGG
# 900
AGCGGCCTGA GGCACGTGGT GCAGCTTCGT GCCCAGGAGG AGTTCGGGCA
# 950
AGGCGAGTGG AGCGAGTGGA GCCCGGAGGC CATGGGCACG CCTTGGACAG
# 1000
AATCCAGGAG TCCTCCAGCT GAGAACGAGG TGTCCACCCC CATGCAGGCA
# 1050
CTTACTACTA ATAAAGACGA TGATAATATT CTCTTCAGAG ATTCTGCAAA
# 1100
TGCGACAAGC CTCCCAGTGC AAGATTCTTC TTCAGTACCA CTGCCCACAT
# 1150
TCCTGGTTGC TGGAGGGAGC CTGGCCTTCG GAACGCTCCT CTGCATTGCC
# 1200
ATTGTTCTGA GGTTCAAGAA GACGTGGAAG CTGCGGGCTC TGAAGGAAGG
# 1250
CAAGACAAGC ATGCATCCGC CGTACTCTTT GGGGCAGCTG GTCCCGGAGA
# 1300
GGCCTCGACC CACCCCAGTG CTTGTTCCTC TCATCTCCCC ACCGGTGTCC
# 1350
CCCAGCAGCC TGGGGTCTGA CAATACCTCG AGCCACAACC GACCAGATGC
# 1400
CAGGGACCCA CGGAGCCCTT ATGACATCAG CAATACAGAC TACTTCTTCC
# 1450
CCAGATAGCT GGCTGGGTGG CACCAGCAGC CTGGAC
#
# 1486
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3085 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#4:
GAGCAGCCAA AAGGCCCGCG GAGTCGCGCT GGGCCGCCCC GGCGCAGCTG
# 50
AACCGGGGGC CGCGCCTGCC AGGCCGACGG GTCTGGCCCA GCCTGGCGCC
# 100
AAGGGGTTCG TGCGCTGTGG AGACGCGGAG GGTCGAGGCG GCGCGGCCTG
# 150
AGTGAAACCC AATGGAAAAA GCATGACATT TAGAAGTAGA AGACTTAGCT
# 200
TCAAATCCCT ACTCCTTCAC TTACTAATTT TGTGATTTGG AAATATCCGC
# 250
GCAAGATGTT GACGTTGCAG ACTTGGGTAG TGCAAGCCTT GTTTATTTTC
# 300
CTCACCACTG AATCTACAGG TGAACTTCTA GATCCATGTG GTTATATCAG
# 350
TCCTGAATCT CCAGTTGTAC AACTTCATTC TAATTTCACT GCAGTTTGTG
# 400
TGCTAAAGGA AAAATGTATG GATTATTTTC ATGTAAATGC TAATTACATT
# 450
GTCTGGAAAA CAAACCATTT TACTATTCCT AAGGAGCAAT ATACTATCAT
# 500
AAACAGAACA GCATCCAGTG TCACCTTTAC AGATATAGCT TCATTAAATA
# 550
TTCAGCTCAC TTGCAACATT CTTACATTCG GACAGCTTGA ACAGAATGTT
# 600
TATGGAATCA CAATAATTTC AGGCTTGCCT CCAGAAAAAC CTAAAAATTT
# 650
GAGTTGCATT GTGAACGAGG GGAAGAAAAT GAGGTGTGAG TGGGATGGTG
# 700
GAAGGGAAAC ACACTTGGAG ACAAACTTCA CTTTAAAATC TGAATGGGCA
# 750
ACACACAAGT TTGCTGATTG CAAAGCAAAA CGTGACACCC CCACCTCATG
# 800
CACTGTTGAT TATTCTACTG TGTATTTTGT CAACATTGAA GTCTGGGTAG
# 850
AAGCAGAGAA TGCCCTTGGG AAGGTTACAT CAGATCATAT CAATTTTGAT
# 900
CCTGTATATA AAGTGAAGCC CAATCCGCCA CATAATTTAT CAGTGATCAA
# 950
CTCAGAGGAA CTGTCTAGTA TCTTAAAATT GACATGGACC AACCCAAGTA
# 1000
TTAAGAGTGT TATAATACTA AAATATAACA TTCAATATAG GACCAAAGAT
# 1050
GCCTCAACTT GGAGCCAGAT TCCTCCTGAA GACACAGCAT CCACCCGATC
# 1100
TTCATTCACT GTCCAAGACC TTAAACCTTT TACAGAATAT GTGTTTAGGA
# 1150
TTCGCTGTAT GAAGGAAGAT GGTAAGGGAT ACTGGAGTGA CTGGAGTGAA
# 1200
GAAGCAAGTG GGATCACCTA TGAAGATAGA CCATCTAAAG CACCAAGTTT
# 1250
CTGGTATAAA ATAGATCCAT CCCATACTCA AGGCTACAGA ACTGTACAAC
# 1300
TCGTGTGGAA GACATTGCCT CCTTTTGAAG CCAATGGAAA AATCTTGGAT
# 1350
TATGAAGTGA CTCTCACAAG ATGGAAATCA CATTTACAAA ATTACACAGT
# 1400
TAATGCCACA AAACTGACAG TAAATCTCAC AAATGATCGC TATCTAGCAA
# 1450
CCCTAACAGT AAGAAATCTT GTTGGCAAAT CAGATGCAGC TGTTTTAACT
# 1500
ATCCCTGCCT GTGACTTTCA AGCTACTCAC CCTGTAATGG ATCTTAAAGC
# 1550
ATTCCCCAAA GATAACATGC TTTGGGTGGA ATGGACTACT CCAAGGGAAT
# 1600
CTGTAAAGAA ATATATACTT GAGTGGTGTG TGTTATCAGA TAAAGCACCC
# 1650
TGTATCACAG ACTGGCAACA AGAAGATGGT ACCGTGCATC GCACCTATTT
# 1700
AAGAGGGAAC TTAGCAGAGA GCAAATGCTA TTTGATAACA GTTACTCCAG
# 1750
TATATGCTGA TGGACCAGGA AGCCCTGAAT CCATAAAGGC ATACCTTAAA
# 1800
CAAGCTCCAC CTTCCAAAGG ACCTACTGTT CGGACAAAAA AAGTAGGGAA
# 1850
AAACGAAGCT GTCTTAGAGT GGGACCAACT TCCTGTTGAT GTTCAGAATG
# 1900
GATTTATCAG AAATTATACT ATATTTTATA GAACCATCAT TGGAAATGAA
# 1950
ACTGCTGTGA ATGTGGATTC TTCCCACACA GAATATACAT TGTCCTCTTT
# 2000
GACTAGTGAC ACATTGTACA TGGTACGAAT GGCAGCATAC ACAGATGAAG
# 2050
GTGGGAAGGA TGGTCCAGAA TTCACTTTTA CTACCCCAAA GTTTGCTCAA
# 2100
GGAGAAATTG AAGCCATAGT CGTGCCTGTT TGCTTAGCAT TCCTATTGAC
# 2150
AACTCTTCTG GGAGTGCTGT TCTGCTTTAA TAAGCGAGAC CTAATTAAAA
# 2200
AACACATCTG GCCTAATGTT CCAGATCCTT CAAAGAGTCA TATTGCCCAG
# 2250
TGGTCACCTC ACACTCCTCC AAGGCACAAT TTTAATTCAA AAGATCAAAT
# 2300
GTATCCAGAT GGCAATTTCA CTGATGTAAG TGTTGTGGAA ATAGAAGCAA
# 2350
ATGACAAAAA GCCTTTTCCA GAAGATCTGA AATCATTGGA CCTGTTCAAA
# 2400
AAGGAAAAAA TTAATACTGA AGGACACAGC AGTGGTATTG GGGGGTCTTC
# 2450
ATGCATGTCA TCTTCTAGGC CAAGCATTTC TAGCAGTGAT GAAAATGAAT
# 2500
CTTCACAAAA CACTTCGAGC ACTGTCCAGT ATTCTACCGT GGTACACAGT
# 2550
GGCTACAGAC ACCAAGTTCC GTCAGTCCAA GTCTTCTCAA GATCCGAGTC
# 2600
TACCCAGCCC TTGTTAGATT CAGAGGAGCG GCCAGAAGAT CTACAATTAG
# 2650
TAGATCATGT AGATGGCGGT GATGGTATTT TGCCCAGGCA ACAGTACTTC
# 2700
AAACAGAACT GCAGTCAGCA TGAATCCAGT CCAGATATTT CACATTTTGA
# 2750
AAGGTCAAAG CAAGTTTCAT CAGTCAATGA GGAAGATTTT GTTAGACTTA
# 2800
AACAGCAGAT TTCAGATCAT ATTTCACAAT CCTGTGGATC TGGGCAAATG
# 2850
AAAATGTTTC AGGAAGTTTC TGCAGCAGAT GCTTTTGGTC CAGGTACTGA
# 2900
GGGACAAGTA GAAAGATTTG AAACAGTTGG CATGGAGGCT GCGACTGATG
# 2950
AAGGCATGCC TAAAAGTTAC TTACCACAGA CTGTACGGCA AGGCGGCTAC
# 3000
ATGCCTCAGT GAAGGACTAG TAGTTCCTGC TACAACTTCA GCAGTACCTA
# 3050
TAAAGTAAAG CTAAAATGAT TTTATCTGTG AATTC
#
# 3085
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 amino
#acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#5:
Met Leu Ala Val Gly Cys Ala Leu Leu Ala Al
#a Leu Leu Ala Ala Pro
5
#
#10
#15
Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Al
#a Gln Glu Val Ala Arg
20
# 25
# 30
Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Va
#l Thr Leu Thr Cys Pro
35
# 40
# 45
Gly Val Glu Pro Glu Asp Asn Ala Thr Val Hi
#s Trp Val Leu Arg Lys
50
# 55
# 60
Pro Ala Ala Gly Ser His Pro Ser Arg Trp Al
#a Gly Met Gly Arg Arg
65
# 70
# 75
#80
Leu Leu Leu Arg Ser Val Gln Leu His Asp Se
#r Gly Asn Tyr Ser Cys
85
# 90
# 95
Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val Hi
#s Leu Leu Val Asp Val
100
# 105
# 110
Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Ar
#g Lys Ser Pro Leu Ser
115
# 120
# 125
Asn Val Val Cys Glu Trp Gly Pro Arg Ser Th
#r Pro Ser Leu Thr Thr
130
# 135
# 140
Lys Ala Val Leu Leu Val Arg Lys Phe Gln As
#n Ser Pro Ala Glu Asp
145 1
#50 1
#55 1
#60
Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Se
#r Gln Lys Phe Ser Cys
165
# 170
# 175
Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Ph
#e Tyr Ile Val Ser Met
180
# 185
# 190
Cys Val Ala Ser Ser Val Gly Ser Lys Phe Se
#r Lys Thr Gln Thr Phe
195
# 200
# 205
Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pr
#o Ala Asn Ile Thr Val
210
# 215
# 220
Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Se
#r Val Thr Trp Gln Asp
225 2
#30 2
#35 2
#40
Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Le
#u Arg Phe Glu Leu Arg
245
# 250
# 255
Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Th
#r Trp Met Val Lys Asp
260
# 265
# 270
Leu Gln His His Cys Val Ile His Asp Ala Tr
#p Ser Gly Leu Arg His
275
# 280
# 285
Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gl
#y Gln Gly Glu Trp Ser
290
# 295
# 300
Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Tr
#p Thr Glu Ser Arg Ser
305 3
#10 3
#15 3
#20
Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Me
#t Gln Ala Leu Thr Thr
325
# 330
# 335
Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg As
#p Ser Ala Asn Ala Thr
340
# 345
# 350
Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pr
#o Leu Pro Thr Phe Leu
355
# 360
# 365
Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Le
#u Leu Cys Ile Ala Ile
370
# 375
# 380
Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Ar
#g Ala Leu Lys Glu Gly
385 3
#90 3
#95 4
#00
Lys Thr Ser Met His Pro Pro Tyr Ser Leu Gl
#y Gln Leu Val Pro Glu
405
# 410
# 415
Arg Pro Arg Pro Thr Pro Val Leu Val Pro Le
#u Ile Ser Pro Pro Val
420
# 425
# 430
Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Se
#r Ser His Asn Arg Pro
435
# 440
# 445
Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp Il
#e Ser Asn Thr Asp Tyr
450
# 455
# 460
Phe Phe Pro Arg
465
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 ami
#no acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:
#6:
Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pr
#o Val Ala Phe Ser Leu
1 5
# 10
# 15
Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pr
#o Ala Pro Val Pro Pro
20
# 25
# 30
Gly Glu Asp Ser Lys Asp Val Ala Ala Pro Hi
#s Arg Gln Pro Leu Thr
35
# 40
# 45
Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Ty
#r Ile Leu Asp Gly Ile
50
# 55
# 60
Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Se
#r Asn Met Cys Glu Ser
65
# 70
# 75
# 80
Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu As
#n Leu Pro Lys Met Ala
85
# 90
# 95
Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe As
#n Glu Glu Thr Cys Leu
100
# 105
# 110
Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Gl
#u Val Tyr Leu Glu Tyr
115
# 120
# 125
Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gl
#n Ala Arg Ala Val Gln
130
# 135
# 140
Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gl
#n Lys Lys Ala Lys Asn
145 1
#50 1
#55 1
#60
Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Th
#r Asn Ala Ser Leu Leu
165
# 170
# 175
Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gl
#n Asp Met Thr Thr His
180
# 185
# 190
Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gl
#n Ser Ser Leu Arg Ala
195
# 200
# 205
Leu Arg Gln Met
210
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GGAGGCTGTA GGCATAAATT GGTCTGCGC
#
# 29
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CCCGAGATTG AGATCTTCTG CGACGCGGCG ATTGAGACC
#
# 39
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) SEQUENCE DESCRIPTION:
#SEQ ID NO:9:
CGGGATCCAT GGGAGGTTGG TCATC
#
# 25
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base
#pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGAATTCCAC TGCATGGC
#
#
# 18
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Expert Pharmaceutical composition for treating hepatitis B virus (HBV) infection
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