Patent application title: ANTI-PRE-S1 HBV ANTIBODIES
Inventors:
Jianhua Sui (Waltham, MA, US)
Dan Li (Beijing, CN)
Dan Li (Beijing, CN)
Wenhui Li (Beijing, CN)
IPC8 Class: AC07K1608FI
USPC Class:
1 1
Class name:
Publication date: 2022-09-01
Patent application number: 20220275060
Abstract:
Provided are human antibodies that specifically bind to HBV Pre-S1 domain
ligand and inhibit HBV or HDV infection, antibodies binding to a set of
amino acid residues that are critical for viral receptor engagement, and
uses of these antibodies to prevent, or treat or diagnose HBV or HDV
infection.Claims:
1.-10. (canceled)
11. An antibody, or antigen-binding fragment thereof, comprising: a) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 4; b) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 8, c) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 12; d) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 15 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 16; e) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 20; f) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 23 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; g) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 27 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 28; h) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 30 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; i) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 32 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; j) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 34 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; k) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 36 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; l) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; m) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; n) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 42 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; o) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 44 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; p) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 46 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; q) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 48 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; r) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 50; s) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 52; t) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 54; u) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 56; or v) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 58.
12. The antibody, or antigen-binding fragment thereof of, of claim 11, wherein the antibody or antigen-binding fragment thereof further comprises a Fc domain.
13. The antibody, or antigen-binding fragment thereof of, of claim 11, wherein the antibody or antigen-binding fragment thereof exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity in an ADCC assay.
14. The antibody, or antigen-binding fragment thereof of, of claim 11, wherein the antibody or antigen-binding fragment thereof binds to Pre-S1 of Hepatitis B virus (HBV).
15. The antibody, or antigen-binding fragment thereof of, of claim 11, wherein the antibody is a monoclonal antibody.
16. The antibody, or antigen-binding fragment thereof of, of claim 15, wherein the antibody is a human monoclonal antibody.
17. A nucleotide sequence encoding the antibody or antigen-binding fragment thereof of claim 11.
18. An expression vector comprising the nucleotide sequence of claim 17.
19. A cultured cell expressing the antibody or antigen-binding fragment thereof of claim 11.
20. A method of treating or preventing Hepatitis B virus (HBV) or Hepatitis D virus (HDV) infection in a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof of claim 11, wherein the subject is determined to have HBV or HDV infection or has been exposed to HBV or HDV.
21. The method of claim 20, wherein the antibody or antigen-binding fragment thereof is administered by injection.
22. An antibody, or antigen-binding fragment thereof, that binds Pre-S1 of HBV, wherein the antibody or antigen-binding fragment thereof binds an epitope on Pre-S1 comprising at least residues 20, 21, and 23 of 59C peptide, wherein the 59C peptide corresponds to residues 20-48 of SEQ ID NO: 147.
23. The antibody, or antigen-binding fragment thereof of, of claim 22, wherein the antibody or antigen-binding fragment thereof binds an epitope on Pre-S1 comprising at least residues 20-27 of the 59C peptide.
24. The antibody, or antigen-binding fragment thereof of, of claim 22, wherein the antibody is a monoclonal antibody.
25. The antibody, or antigen-binding fragment thereof of, of claim 24, wherein the antibody is a human monoclonal antibody.
26. A method of treating or preventing Hepatitis B virus (HBV) or Hepatitis D virus (HDV) infection in a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof of claim 22, wherein the subject is determined to have HBV or HDV infection or has been exposed to HBV or HDV.
27. The method of claim 26, wherein the antibody or antigen-binding fragment thereof is administered by injection.
Description:
INTRODUCTION
[0001] More than one third of the world population has been infected by Hepatitis B virus (HBV), and 240 million people are presently chronically infected. HBV infection and related diseases result in about one million deaths annually.
[0002] The surface antigen of HBV is composed of Large (L), Middle (M) and Small (S) proteins. The L and M proteins have additional domains at their N terminal as compared to the S protein which only has the S domain. L contains Pre-S1, Pre-S2, and S domains; M contains Pre-S2 and S domains; S protein contains only the S domain. The pre-S1 domain in L protein is the target molecule of HBV receptor(s) expressed on human hepatic cell surface, and antibodies to the pre-S1 domain of HBV have been reported, e.g. Watashi et al, Int. J. Mol. Sci. 2014, 15, 2892-2905, refs 22-27. Relevant literature includes descriptions of the HBV receptor in WO2013159243A1, a humanized antibody from mouse hybridoma, KR127 in U.S. Pat. No. 7,115,723, and pre-S1 peptides in U.S. Pat. No. 7,892,754.
SUMMARY OF THE INVENTION
[0003] The invention provides methods and compositions for immune-activation by inhibiting HBV and/or HDV. In one aspect, the invention provides an antibody antigen binding domain which specifically binds HBV Pre-S1, and comprises complementarity determining region (CDR) 1, CDR2 and CDR3, in a combination selected from (a)-(r) as follows, wherein the antibody (Ab), heavy chain (HC) or light chain (LC) and CDR nomenclature system (Kabat, IMGT or composite) from which the CDR combinations derive are shown in the first column, and residues in bold text are Kabat system, and residues underlined are IMGT system:
HCDRs of Unique HBV Pre-S1 Specific Antibodies
TABLE-US-00001
[0004] MAbs CDR1 CDR2 CDR3 m36-HC GFTFDDYAMH GTSWNSGSIGYADSVKG AKTSYGGAFDI K: SEQ ID NO: 59, res. 6-10 K: SEQ ID NO: 60 K: SEQ ID NO: 61, res. 3-11 I: SEQ ID NO: 59, res. 1-8 I: SEQ ID NO: 60, res. 2-9 I: SEQ ID NO: 61 C: SEQ ID NO: 59 C: SEQ ID NO: 60 C: SEQ ID NO: 61 m36-LC SGNTSNIGSYYAY DNNQRPS ATWDDSLNGPV K: SEQ ID NO: 62 K: SEQ ID NO: 63 K: SEQ ID NO: 64 I: SEQ ID NO: 62, res. 4-11 I: SEQ ID NO: 63, res. 1-3 I: SEQ ID NO: 64 C: SEQ ID NO: 62 C: SEQ ID NO: 63 C: SEQ ID NO: 64 71-HC GYTTGYYIH RINPNSGGTN AREGRGGMDV K: SEQ ID NO: 65, res. 5-9 K: SEQ ID NO: 67, res. 3-10 K: SEQ ID NO: 66 I: SEQ ID NO: 65, res. 1-7 I: SEQ ID NO: 66 I: SEQ ID NO: 67 C: SEQ ID NO: 65 C: SEQ ID NO: 66 C: SEQ ID NO: 67 71-LC RSSQSLLHSNGYNY LGSNRAS MQGLQPPIT K: SEQ ID NO: 68, res. 1-12 K: SEQ ID NO: 69 K: SEQ ID NO: 70 I: SEQ ID NO: 68, res. 4-14 I: SEQ ID NO: 69 I: SEQ ID NO: 70 C: SEQ ID NO: 68 C: SEQ ID NO: 69 C: SEQ ID NO: 70 76-HC GFTFSSYAMH VISYDGSNKYYADSVKG ASGAFDI K: SEQ ID NO: 71, res. 6-10 K: SEQ ID NO: 72 K: SEQ ID NO: 73, res. 3-7 I: SEQ ID NO: 71, res. 1-8 I: SEQ ID NO: 72, res. 2-9 I: SEQ ID NO: 73 C: SEQ ID NO: 71 C: SEQ ID NO: 72 C: SEQ ID NO: 73 76-LC RSSHSLVYSDGNTYLS KVSNRDF MQGTHWPGT K: SEQ ID NO: 74 K: SEQ ID NO: 75 K: SEQ ID NO: 76 I: SEQ ID NO: 74, res. 4-14 I: SEQ ID NO: 75, res. 1-3 I: SEQ ID NO: 76 C: SEQ ID NO: 74 C: SEQ ID NO: 75 C: SEQ ID NO: 76 147-HC GDSVSSNSVAWN RTYYRSKWYNDYAVSVKS ARADGSRGGGYDQ K: SEQ ID NO: 77, res. 6-12 K: SEQ ID NO: 78 K: SEQ ID NO: 79, res. 3-13 I: SEQ ID NO: 77, res. 1-10 I: SEQ ID NO: 78, res. 2-10 I: SEQ ID NO: 79 C: SEQ ID NO: 77 C: SEQ ID NO: 78 C: SEQ ID NO: 79 T47-LC KSSQSILYRSNNKNYLA WASTRES QQYYTTPQT K: SEQ ID NO: 80 K: SEQ ID NO: 81 K: SEQ TD NO: 82 I: SEQ ID NO: 80, res. 4-15 I: SEQ ID NO: 81, res. 1-3 I: SEQ ID NO: 82, res. 1-8 C: SEQ ID NO: 80 C: SEQ ID NO: 81 C: SEQ ID NO: 82 m1Q-HC GFTFSSYAMH VISYDGSNKYYVDSVKG ARSTYGMDV K: SEQ ID NO: 83, res. 6-10 K: SEQ ID NO: 84 K: SEQ ID NO: 85, res. 3-9 I: SEQ ID NO: 83, res. 1-8 I: SEQ ID NO: 84, res. 2-9 I: SEQ ID NO: 85 C: SEQ ID NO: 83 C: SEQ ID NO: 84 C: SEQ ID NO: 85 m1Q-LC RSSQSLVHSDGNTYLN KVSNRDS MQGTHWWT K: SEQ ID NO: 86 K: SEQ ID NO: 87 K: SEQ ID NO: 88 I: SEQ ID NO: 86, res. 4-14 I: SEQ ID NO: 87, res. 1-3 I: SEQ ID NO: 88 C: SEQ ID NO: 86 C: SEQ ID NO: 87 C: SEQ ID NO: 88 2H5-HC GDSVSSKSAAWN RTYYRSKWHNDYAVS ARGQMGALDV K: SEQ ID NO: 89, res. 6-12 K: SEQ ID NO: 90 K: SEQ TD NO: 91, res. 3-10 I: SEQ ID NO: 89, res. 1-10 I: SEQ ID NO: 90, res. 3-10 I: SEQ ID NO: 91 C: SEQ ID NO: 89 C: SEQ ID NO: 90 C: SEQ ID NO: 91 2H5-LC SGSSSNIGSYYVYWY GNNQRPS QSYDSSLSGVI K: SEQ ID NO: 92 K: SEQ ID NO: 93 K: SEQ TD NO: 94 I: SEQ ID NO: 92, res. 4-11 I: SEQ ID NO: 93, res. 1-3 I: SEQ ID NO: 94 C: SEQ ID NO: 92 C: SEQ ID NO: 93 C: SEQ ID NO: 94 m150-HC GFTFSSYAMH VI SYDGSNKYYADSVKG ARLVAGRSAFDI K: SEQ ID NO: 95, res. 6-10 K: SEQ ID NO: 96 K: SEQ ID NO: 97, res. 3-12 I: SEQ ID NO: 95, res. 1-8 I: SEQ ID NO: 96, res. 2-9 I: SEQ ID NO: 97 C: SEQ ID NO: 95 C: SEQ ID NO: 96 C: SEQ ID NO: 97 m150-LC RASQSVSSNLA GASTRAT QQYNNWPPIT K: SEQ ID NO: 98 K: SEQ ID NO: 99 K: SEQ ID NO: 100 I: SEQ ID NO: 98, res. 4-9 I: SEQ ID NO: 99, res. 1-3 I: SEQ ID NO: 100 C: SEQ ID NO: 98 C: SEQ ID NO: 99 C: SEQ ID NO: 100
HCDRs of Antibodies Derived from 2H5 VH-Chain Shuffled Libraries
TABLE-US-00002
[0005] MAbs HCDR1 HCDR2 HCDR3 #4 VH GDSVSSKSVTWN RTYYRSKWFNDYAVS ARAKMGGMDV K: SEQ ID NO: 101, res 6-12 K: SEQ ID NO: 102 K: SEQ ID NO: 103, res 3-10 I: SEQ ID NO: 101, res. 1-10 I: SEQ ID NO: 102, res. 2-10 I: SEQ ID NO: 103 C: SEQ ID NO: 101 C: SEQ ID NO: 102 C: SEQ ID NO: 103 #31 VH GDSVSSNSAAWN RTYYRSKWYNDYAVS TRQSWHGMEV K: SEQ ID NO: 104, res 6-12 K: SEQ ID NO: 105 K: SEQ ID NO: 106, res 3-10 I: SEQ ID NO: 104, res. 1-10 I: SEQ ID NO: 105, res. 2-10 I: SEQ ID NO: 106 C: SEQ ID NO: 104 C: SEQ ID NO: 105 C: SEQ ID NO: 106 #32 VH GDSVSSNSAAWN RTYYRSKWYNDYAVS ARSIATGTDY K: SEQ ID NO: 107, res 6-12 K: SEQ ID NO: 108 K: SEQ ID NO: 109, res 3-10 I: SEQ ID NO: 107, res. 1-10 I: SEQ ID NO: 108, res. 2-10 I: SEQ ID NO: 109 C: SEQ ID NO: 107 C: SEQ ID NO: 108 C: SEQ ID NO: 109 #69 VH GDSVSSSRATWN RTYYRSKWFNDYAVS ARAKMGGMDV K: SEQ ID NO: 110, res 6-12 K: SEQ ID NO: 111 K: SEQ ID NO: 112, res 3-10 I: SEQ ID NO: 110, res. 1-10 I: SEQ ID NO: 111, res. 2-10 I: SEQ ID NO: 112 C: SEQ ID NO: 110 C: SEQ ID NO: 111 C: SEQ ID NO: 112 A14 VH GDSVSSNSAAWN RTYYRSKWYNDYAVS ARGTRWGMDV K: SEQ ID NO: 113, res 6-12 K: SEQ ID NO: 114 K: SEQ ID NO: 115, res 3-10 I: SEQ ID NO: 113, res. 1-10 I: SEQ ID NO: 114, res. 2-10 I: SEQ ID NO: 115 C: SEQ ID NO: 113 C: SEQ ID NO: 114 C: SEQ ID NO: 115 A21 VH GDSVSSNSAAWN RTYYRSKWYNDYAVS ARAKVYGVDV K: SEQ ID NO: 116, res 6-12 K: SEQ ID NO: 117 K: SEQ ID NO: 118, res 3-10 I: SEQ ID NO: 116, res. 1-10 I: SEQ ID NO: 117, res. 2-10 I: SEQ ID NO: 118 C: SEQ ID NO: 116 C: SEQ ID NO: 117 C: SEQ ID NO: 118 B103 VH GDSVSSKSATWN RTYYRSRWFNDYAVS ARGNMGAMDV K: SEQ ID NO: 119, res 6-12 K: SEQ ID NO: 120 K: SEQ ID NO: 121, res 3-10 I: SEQ ID NO: 119, res. 1-10 I: SEQ ID NO: 120, res. 2-10 I: SEQ ID NO: 121 C: SEQ ID NO: 119 C: SEQ ID NO: 120 C: SEQ ID NO: 121 B129 VH GDRVSSNRAAWN RTYYRSQWYNDYAVS ARGTAMG-DA K: SEQ ID NO: 122, res 6-12 K: SEQ ID NO: 123 K: SEQ ID NO: 124, res 3-9 I: SEQ ID NO: 122, res. 1-10 I: SEQ ID NO: 123, res. 2-10 I: SEQ ID NO: 124 C: SEQ ID NO: 122 C: SEQ ID NO: 123 C: SEQ ID NO: 124 B139 VH GDSVSSNSAAWN RTYYRSKWYNDYAVS ARQASNGFDI K: SEQ ID NO: 125, res 6-12 K: SEQ ID NO: 126 K: SEQ ID NO: 127, res 3-10 I: SEQ ID NO: 125, res. 1-10 I: SEQ ID NO: 126, res. 2-10 I: SEQ ID NO: 127 C: SEQ ID NO: 125 C: SEQ ID NO: 126 C: SEQ ID NO: 127 B172 VH GDSVSSNSAAWN RTYYRSKWYNDYAVS ARQGTTGFDY K: SEQ ID NO: 128, res 6-12 K: SEQ ID NO: 129 K: SEQ ID NO: 130, res 3-10 I: SEQ ID NO: 128, res. 1-10 I: SEQ ID NO: 129, res. 2-10 I: SEQ ID NO: 130 C: SEQ ID NO: 128 C: SEQ ID NO: 129 C: SEQ ID NO: 130
HCDRs of Antibodies Derived from A14 VL-Chain Shuffled Libraries
TABLE-US-00003
[0006] MAbs LCDR1 HCDR2 HCDR3 #8 VL SGSSSNIGNYYVSWY DNAKRPS QSYDNSLSGLV K: SEQ ID NO: 131 K: SEQ ID NO: 132 K: SEQ ID NO: 133 I: SEQ ID NO: 131, res. 4-11 I: SEQ ID NO: 132, res. 1-3 I: SEQ ID NO: 133 C: SEQ ID NO: 131 C: SEQ ID NO: 132 C: SEQ ID NO: 133 #20 VL SGTSSNIGSKYVYWY TNDQRPS QSYDSSLRAVV K: SEQ ID NO: 134 K: SEQ ID NO: 135 K: SEQ ID NO: 136 I: SEQ ID NO: 134, res. 4-11 I: SEQ ID NO: 135, res. 1-3 I: SEQ ID NO: 136 C: SEQ ID NO: 134 C: SEQ ID NO: 135 C: SEQ ID NO: 136 #20-m1 VL SGTSSNIGSFYVYWY TNDQRPS QSYDSSLRAVV K: SEQ ID NO: 137 K: SEQ ID NO: 138 K: SEQ ID NO: 139 I: SEQ ID NO: 137, res. 4-11 I: SEQ ID NO: 138, res. 1-3 I: SEQ ID NO: 139 C: SEQ ID NO: 137 C: SEQ ID NO: 138 C: SEQ ID NO: 139 #20-m2 VL SGTSSNIGSFYVYWY TNDQRPS QSYDSSLRAVV K: SEQ ID NO: 140 K: SEQ ID NO: 141 K: SEQ ID NO: 142 I: SEQ ID NO: 140, res. 4-11 I: SEQ ID NO: 141, res. 1-3 I: SEQ ID NO: 142 C: SEQ ID NO: 140 C: SEQ ID NO: 141 C: SEQ ID NO: 142 #20-m3 VL SGTSSNIGSYYVYWY TNDQRPS QSYDSSLRAVV K: SEQ ID NO: 143 K: SEQ ID NO: 144 K: SEQ ID NO: 145 I: SEQ ID NO: 143, res. 4-11 I: SEQ ID NO: 144, res. 1-3 I: SEQ ID NO: 145 C: SEQ ID NO: 143 C: SEQ ID NO: 144 C: SEQ ID NO: 145
[0007] In embodiments the invention provides an antibody antigen binding domain comprising a heavy chain variable region (Vh) comprising a CDR1, CDR2 and CDR3 combination and a light chain variable region (Vl) comprising a CDR1, CDR2 and CDR3 combination, or comprising a heavy chain variable region (Vh) and/or a light chain variable region (VI), selected from: m36, 71, 76, T47, m1Q, 2H5, m150; and 4, 31, 32, 69, A14, A21, B103, B129, B139, B172; and 8, 20, 20-m1, 20-m2, 20-m3.
[0008] In embodiments the antibody antigen binding domain specifically binds aa11-28 or aa19-25 of pre-S1.
[0009] The invention also provides antibodies, particularly monoclonal antibodies, and F(ab) or F(ab)2 comprising a subject binding domain.
[0010] The invention also provides novel polynucleotides such as cDNAs and expression vectors, encoding a subject antigen binding domain, and cells comprising such polynucleotides, and non-human animals comprising such cells. The polynucleotides may be operably linked to a heterologous transcription regulating sequence for expression, and may be incorporated into such vectors, cells, etc.
[0011] The invention provides methods of using the subject domains to treat HBV or HDV infection, or to induce antibody-dependent cell-mediated cytotoxicity (ADCC), comprising administering the domain to a person determined to have HBV or HDV infection, to have been exposed to HBV or HDV, to be at high risk for HBV or HDV exposure or infection, to be in need of Pre-S1 domain antagonism, or to be otherwise in need thereof. The invention further provides the use of subject compositions for the manufacture of a medicament for HBV or HDV infection, optionally in conjunction with a virus replication inhibitor.
[0012] The invention includes all combinations of the recited particular embodiments. Further embodiments and the full scope of applicability of the invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.
BRIEF DESCRIPTION OF THE DRAWING
[0013] FIG. 1. HBV neutralization by 10 antibodies from 2H5 VH-chain shuffled library selections.
DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
[0014] Unless the context indicates otherwise, the term "antibody" is used in the broadest sense and specifically covers antibodies (including full length monoclonal antibodies) and antibody fragments so long as they recognize HBV/HDV Pre-S1 or otherwise inhibit HBV/HDV. An antibody molecule is usually monospecific, but may also be described as idiospecific, heterospecific, or polyspecific. Antibody molecules bind by means of specific binding sites to specific antigenic determinants or epitopes on antigens. "Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
[0015] Natural and engineered antibody structures are well known in the art, e.g. Strohl et al., Therapeutic antibody engineering: Current and future advances driving the strongest growth area in the pharmaceutical industry, Woodhead Publishing Series in Biomedicine No. 11, October 2012; Holliger et al. Nature Biotechnol 23, 1126-1136 (2005); Chames et al. Br J Pharmacol. 2009 May; 157(2): 220-233.
[0016] Monoclonal antibodies (MAbs) may be obtained by methods known to those skilled in the art. See, for example Kohler et al (1975); U.S. Pat. No. 4,376,110; Ausubel et al (1987-1999); Harlow et al (1988); and Colligan et al (1993). The mAbs of the invention may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
[0017] An "isolated polynucleotide" refers to a polynucleotide segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA, which is part of a hybrid gene encoding additional polypeptide sequence.
[0018] A "construct" means any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a polynucleotide molecule where one or more polynucleotide molecule has been linked in a functionally operative manner, i.e. operably linked. A recombinant construct will typically comprise the polynucleotides of the invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the invention.
[0019] A "vector" refers any recombinant polynucleotide construct that may be used for the purpose of transformation, i.e. the introduction of heterologous DNA into a host cell. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors".
[0020] An "expression vector" as used herein refers to a nucleic acid molecule capable of replication and expressing a gene of interest when transformed, transfected or transduced into a host cell. The expression vectors comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desired, provide amplification within the host. The expression vector further comprises a promoter to drive the expression of the polypeptide within the cells. Suitable expression vectors may be plasmids derived, for example, from pBR322 or various pUC plasmids, which are commercially available. Other expression vectors may be derived from bacteriophage, phagemid, or cosmid expression vectors.
EXAMPLES
[0021] Human Monoclonal Antibodies Block Viral Infection of Hepatitis B and D Virus
[0022] Here we disclose human monoclonal antibodies that can block HDV and HBV viral infections. These antibodies were identified from a large phage display antibody library, which was established using peripheral blood mononuclear cells from 93 healthy donors. By selection and screening of the antibody library using pre-S1 domain of HBV envelope protein as a target, a panel of human monoclonal antibodies with neutralizing activities against HBV and HDV infections were identified. Among them, 2H5, showed best neutralizing activities against HBV and HDV infections. The co-crystal structure of 2H5 in complex with its target (8 amino acids of the Pre-S1 domain) was solved. By optimizing 2H5 by chain shuffling approach we developed even more potent neutralizing antibodies. These antibodies recognize similar epitope as 2H5 and the epitope is highly conserved among different genotypes of HBV. An exemplary antibody, A14 was tested in mice bearing humanized NTCP and provided complete protection of mice from HDV infection, and animal studies confirmed protection against HBV infection.
[0023] Antigen Target:
[0024] pre-S1 peptides. As antigen for selection we used two peptides derived from the pre-S1 domain of HBV. They were synthesized by Scilight-peptide (Beijing, China) at purity greater than 95%. NC36b: a peptide comprising of residues 4-38 of the pre-S1 domain of HBV L protein with a biotin modification at its C-terminus. m47b: a myristoylated lipopeptide comprising of amino acids 2-48 of pre-S1 domain with a biotin modification at the C-terminus and a myristoylation modification at the N-terminus.
TABLE-US-00004 Pre-S1 GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG (SEQ ID NO: 146) (2-48) m47b Myr-G.......................................... K-Biotin NC36b N...............................K-Biotin
[0025] Human Monoclonal Antibodies Against Pre-S1 Peptides were Generated Based on Phage Display Antibody Technology with Modifications [1, 2].
[0026] Phage Display Antibody Library.
[0027] A human non-immune scFv (Single-chain variable fragment) antibody library was constructed from peripheral blood mononuclear cells (PBMCs) of 93 healthy donors. The library has a size of a total of 1.1.times.10.sup.10 members.
[0028] Selection and Screening of Phage Antibody Library.
[0029] Phage particles expressing scFv on their surface (phage-scFv) were prepared from the library and used for selection of scFvs against the synthesized NC36b and m47b. The peptides were captured on streptavidin-conjugated magnetic M-280 Dynabeads.RTM. (Life Technologies) and then incubated with 5.times.10.sup.12 phage particles prepared from the library, respectively. For each peptide, two rounds of selection were performed. For each round of selection, in order to obtain high affinity antibodies, the amount of peptides captured onto the magnetic beads was optimized and extensive washing steps were applied. In addition, to recover high affinity binders from the magnetic beads and increase the diversity of phage-scFvs recovered, two elution methods including peptide competition elution and conventional basic triethanolamine solution were used. Subsequently, a total of about 2000 single clones were picked and rescued to produce phage-scFvs in the bacterial culture supernatant, and screened for specific binding to m47b and/or NC36b by enzyme-linked immunosorbent assay (ELISA). Clones that bound to m47b and/or NC36b with values of optical density at 450 nm>1.0 were scored as positive, whereas negative clones gave values of <0.1. For m47b and/or NC36b specific binding clones, the genes of variable regions of heavy (VH) and light (VL) chain were sequenced and their corresponding amino acid sequences were aligned to eliminate repeated clones and identify antibodies with different sequence for further characterization. A total of 109 clones with unique sequence were identified.
[0030] Further Characterization of the Antibodies with Unique Antibody Sequences to Identify the Best Antibody Candidate.
[0031] The antibody clones with unique sequence were either produced as purified phage-scFv particles or converted to scFv-Fc minibodies or full-length human IgG1s, and then tested for their binding activities by ELISA, and HBV and HDV neutralization activities in cell cultures. By these assays, antibodies were ranked based on their binding activity and neutralization activity. The top antibody with the highest neutralization activity was chosen for further development.
[0032] Preparation of Purified Phage-scFvs for ELISA or Neutralization Assay.
[0033] The phage-scFvs in the supernatant of 10-30 mL bacterial culture were precipitated by PEG/NaCL and then quantified by a spectrometer. Activities of different phage-scFvs for antigen binding or neutralizing viral infection were evaluated based on the dose-response of serial diluted phage-Abs that was normalized to the same concentration.
[0034] Preparation of scFv-Fc Minibodies.
[0035] ScFv encoding gene from the phage-scFv expressing vector was subcloned into an expression vector containing human IgG1 Fc fragment at C-terminus of the scFv. To produce scFv-Fc, 293F (Life Technologies) or 293T cells (ATCC) were transiently transfected with the scFv-Fc expression plasmid, 72 hours after transfection, the cell culture supernatant were harvested and scFv-Fc was purified by Protein A affinity chromatography (Protein A Sepharose CL-4B, GE Healthcare).
[0036] Preparation of Full-Length IgG1 Antibody.
[0037] The VH and VL coding sequence of a scFv were separately subcloned into antibody heavy chain (HC) expression vector and light chain (LC) expression vector. To make IgG1 antibody, 293F or 293T cells were transiently co-transfected with the two expression plasmids (HC+LC plasmids) at a 1:1 ratio. 72 hours after transfection, the cell culture supernatant were harvested for purification of IgG1 by Protein A affinity chromatography.
[0038] ELISA Assay.
[0039] 5 .mu.g/mL of streptavidin (Sigma) in phosphate buffered saline (PBS) was coated in U-bottom 96-well plate (Nunc, MaxiSorp.TM.), 100 .mu.L per well, at 4.degree. C. overnight or 37.degree. C. for 1 hour. 2 .mu.g/mL (370 nM) of m47b or NC36b peptides at 100 .mu.L per well were then captured onto the plates by incubation at 30'C for 0.5-1 hour. For phage-scFv based ELISA, serial diluted phage-scFvs in PBS containing 2% nonfat milk were added to each well at 100 .mu.L per well. Specific bound phage-scFvs were detected by adding HRP-conjugated mouse anti-M13 antibody (GE Healthcare) and incubated for 30 mins at 30.degree. C. In between each incubation step, the ELISA plate was washed for 6 times with PBST solution (0.05% Tween20 containing PBS) at 200 .mu.L per well. Followed by HRP-conjugated antibody incubation, the ELISA signal was developed by incubating with TMB substrate (Sigma) for 5-10 mins at 30.degree. C. and then stop the reaction with 2M H.sub.2SO.sub.4 at 25 .mu.L per well. The absorbance at 450 nm was read by a microplate reader (Bio-Rad). For scFv-Fc or IgG1 based ELISA, the method was basically the same as described above for phage-scFvs except the hound antibodies were detected by HRP-conjugated mouse anti-human IgG Fc antibody (Sigma).
[0040] Preparation of HBV and HDV Viruses.
[0041] HBV and HDV were produced as previously described [3]. HDV. Briefly, a plasmid containing a head to tail trimer of 1.0.times.HDV cDNA of a genotype I virus (Genebank accession number: AF425644.1) under the control of a CMV promoter was constructed with de novo synthesized HDV cDNA for the production of HDV RNPs. A pUC18 plasmid containing nucleotide 2431.about.4990 of HBV (Genotype D, Genebank accession number: U95551.1), was used for expressing HBV envelope proteins under the control of endogenous HBV promoter. HDV virions were produced by transfection of the plasmids in Huh-7 as previously described by Sureau et al [4]. The transfected cell culture supernatant was harvested and directly used for HDV neutralization assay. HBV. HBV genotype B, C and D viruses were produced by transfection of Huh-7 cells with a plasmid containing 1.05 copies of HBV genome under the control of a CMV promoter. Genotype B or C HBV viruses were also from plasma of HBV patients.
[0042] HBV and HDV Neutralization Assays.
[0043] The neutralization assays were performed as previously described [3, 5] with minor modifications. HepG2-hNTCP cells (a HepG2 cell line stably expressing HBV and HDV receptor hNTCP (human sodium taurocholate cotransporting polypeptide)) were used in these assays. HepG2-hNTCP cells were cultured in PMM medium [3] for 12-24 hours in a 48-well plate before viral infection. About 500 multiplicities of genome equivalents (mge) of HDV or 200 mge of HBV mixed with different forms of antibodies: phage-scFvs, scFv-Fc or IgG1 were inoculated with HepG2-hNTCP cells in the presence of 5% PEG8000 and incubated for 16 hours. Cells were then washed with medium for three times and maintained in PMM. Cell culture medium was changed with fresh PMM medium every 2-3 days. For HDV infection, at 7 days post infection (dpi), HDV infected cells were fixed with 100% methanol at room temperature for 10 min, intracellular delta antigen was stained with 5 .mu.g/mL of FITC conjugated 4G5 (a mouse anti-HDV Delta antigen monoclonal antibody) and nuclear were stained with DAPI. Images were collected by a Fluorescence Microscope (Nikon). The neutralization activity against HDV was determined based on the stained Delta antigen amount and strength. For HBV infection, at dpi 3, 5 and 7, the culture supernatant were collected and tested for HBV secreted viral antigen HBsAg and/or HBeAg with commercial ELISA kits (Wantai, Beijing, China). The levels of HBeAg and/or HBsAg were used to evaluate HBV neutralization activity of the antibodies.
[0044] Through the above described ELISA and HBV neutralization assays we identified some top antibodies, which showed specific binding with NC36b as well as m47b and 47b (a peptide similar to m47b but without the myristoylation and showed neutralization activities in HBV.
[0045] Among these top antibodies, m36, 2H5 and m1Q were the top three antibodies showing best HBV (genotype D) neutralization activity. m36 was excluded from further testing as it showed reduced expression when converted into full-length IgG1. 2H5 and m1Q were further compared for HDV neutralization activity, 2H5 showed better activity in neutralizing HDV infection. Based on the high binding activity with the peptide and potent neutralizing activity against HBV and HDV, 2H5 was chosen for further development. In addition, 2H5 showed greater HBV and HDV neutralization activity than a previously published pre-S1 peptide antibody KR127 [6-8]. In HBV infection assay, 2H5-IgG1 is 11-fold more potent than KR127 as indicated by the IC.sub.50 (the antibody concentration resulting 50% inhibition of HBV infection); 2H5 also showed greater inhibitory effect on HDV infection assay.
[0046] Mapping the Binding Epitope of 2H5 Antibody.
[0047] To map the epitope of 2H5 on pre-S1 region, we synthesized short peptides covering different regions of the pre-S1 domain and tested their ability to compete for the binding of 2H5 to m47b by competition ELISA assay. The shortest peptide that can compete for the binding is the LN16 peptide (corresponding to the NT amino acid (aa) 11-28 of the pre-S1 domain of HBV L protein (Genotype D), indicating the binding epitope of 2H5 is located within this region. LD15 and LA15 peptides also showed some degree of competition activity but at lower level than LN16. The common amino acids shared by the three peptides, LN16, LD15 and LA15, are aa19-25 of pre-S1. We therefore tested LN16 peptides each carrying a single alanine mutation at position 19, 20, 22 and 23, LN16-L19A, -D20A, -P21A, -F23A, for their competition activity, the result showed that all of them had reduced competition activity (LN16-L19A) or completely lost this activity (LN16-D20A, -P21A, -F23A), indicating these amino acids are critically important for pre-S1 binding to 2H5.
[0048] The 2H5 Epitope is Highly Conserved Among the Majority of HBV Genotypes.
[0049] Sequence alignment of pre-S1 peptides of eight HBV genotypes showed that the epitope is highly conserved among them. The major variable amino acid is at position 24: glycine in genotype A and C, a lysine or arginine in genotype D and other genotypes. To test if this amino acid change will affect 2H5 binding to pre-S1 peptide, the NC36b peptide containing an arginine at position 24 was synthesized and test for binding with 2H5 by ELISA. The result showed that this amino acid change had only minimal effect on the binding. This is consistent with the HBV and HDV viral neutralization result that 2H5 neutralized HBV of genotype D and HDV carrying HBV genotype D envelopes.
[0050] Structural Characterization of the 2H5 scFv and Pre-S1 Peptide Complex.
[0051] We also determined the crystal structure of 2H5 (as the scFv fragment fused with a His.sub.6 tag at its N-terminal) in complex with a pre-S1 peptide, 59C. The amino acid sequence of 59C corresponds to aa-10.about.48 of pre-S1 of genotype C: GGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQV (SEQ ID NO:147). 2H5-scFv and 59C were co-expressed in E. coli. The complex was purified as a complex by Immobilized Metal Ion Affinity Chromatography (IMAC) using Ni-NTA agarose beads (QIAGEN) followed by Size Exclusion Chromatography-HPLC (SEC-HPLC) with Superdex 5200 10/300 column (GE Healthcare). The purified 2H5-scFv/59C complex was then concentrated and crystallized at 20.degree. C. using the hanging-drop vapor-diffusion method by mixing 1 .mu.L of protein (29 mg/mL in 10 mM VI Tris-HCl pH 8.0 and 100 mM NaCl) and 1 .mu.L of reservoir solution containing 2.8 M sodium acetate, pH 7.0. Needle-shaped crystals appeared after 10 days. The X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility beamline BL17U and processed by HKL2000 [9]. The structure was determined at 2.7 A.degree. resolution by molecular replacement in Phaser [10, 11] using VH and VL derived from the structure of Herceptin-Fab complex (PDB 3H0T) [12] as starting model. Initial model from molecular replacement was further refined in Phenix [13] and manually rebuilt with Coot [14]. The final model includes 220 residues of 2H5 scFv, residues 20-27 of the 59C peptide. RAMPAGE analysis shows that 96.71% of residues are in the favored region and 3.29% of residues are in the allowed region [15]. The structure revealed that both VH and VL of 2H5 scFv participate in the interaction with the peptide. The eight amino acids of the peptide included in the structure are D.sub.20-P.sub.21A.sub.22F.sub.23G24N.sub.25A.sub.26S.sub.27. Among them, D.sub.20, P.sub.21, A.sub.22, F.sub.23, A.sub.26 and S.sub.27 make interactions with 2H5. Three amino acids, D.sub.20, P.sub.21 and F.sub.23 make critical interactions for 2H5 binding.
[0052] Improvement of 2H5 Affinity and Neutralization Activity by VH-Chain Shuffling.
[0053] Identification of Four Top Antibodies from VH-Chain Shuffled Library of 2H5.
[0054] We next used chain shuffling to improve 2H5's binding affinity and neutralization activity, in which one of the two chains (VH and VL) is fixed and combined with a repertoire of the other chain to yield a secondary library that can be selected for superior activity. First, we did VH chain shuffling, in which VL of 2H5 was fixed and paired with a library of VH chains. Two VH-Lib/2H5VL phage display libraries were constructed. One library size is .about.2.times.10.sup.8, the other one is about 9.times.10.sup.8. By using peptides captured on streptavidin-conjugated magnetic M-280 Dynabeads.RTM. (Life Technologies) as target, the two VH-Lib/2H5VL libraries were separately selected for one round each. At the end of the one round of selection from both libraries, total 576 individual clones were randomly picked and screened for binding with m47b by ELISA. Positive clones in ELISA were selected and sequenced. 10 clones with unique VH sequences (Table 1) and showed equal or stronger binding activity to m47.sub.b in phage antibody form than 2H5 were identified. These 10 clones were then converted into full-length human IgG1 and validated for binding to m47b by ELISA, neutralizing HBV (genotype D) (FIG. 1) and HDV by in vitro neutralization assays. Four top antibodies, #31, #32, A14 and A21 were selected based on their overall activities in binding to m47b, neutralizing HBV and HDV.
TABLE-US-00005 TABLE 1 VH sequence alignment of 10 antibodies from 2H5 VH-chain shuffled library selections. QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWY #4 VH ......................G........K.VT.....E..TG..............F #31 VH ............................................................ #32 VH ............................................................ #69 VH ...........M...................SR.T.....E..TG..............F 2H5 VH ......................G........K...........................H A14 VH ............................................................ A21 VH ............................................................ B103 VH ......................G........K..T...V...A..............R.F B129 VH ...........L...............R....R.....V..................Q.. 3172 VH ............................................................ B139 VH ..................T...V..................................... NDYAVSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARGKMGGMDVWGQGTTVTVSS (SEQ ID NO: 148) #4 VH ............V....................RG......A.................. #31 VH .......................................T.QSWH..E............ #32 VH ..............S...........K..............SIAT.T.Y.....L..... #69 VH ............V....................RG......A.................. 2H5 VH ..........................................Q..AL............. A14 VH ..........................................TRW.........L..... A21 VH .........................................A.VY.V............. B103 VH ............VK...............S.....I......N..A.............. B129 VH ..........V..S..A....V....................TAM.-.A.....L..... B172 VH .........................................QGTT.F.Y........... 8139 VH ......L..................................QASN.F.I.....M.....
[0055] FIG. 1. shows HBV neutralization by 10 antibodies from 2H5 VH-chain shuffled library selections. HepG2-hNTCP cells were infected by incubation with HBV (genotype D) in the presence of antibodies at different concentrations for 16 hours. Antibody and viruses were washed away afterwards and continued to culture for 7 days, cell culture medium was changed every 2 days. The secreted HBeAg was detected by ELISA at 7 days post infection. Based on the reduction of HBeAg level, the HBV neutralization activity was calculated and expressed as the percentage changes for infected cells in the presence of antibodies relative to the control (cells infected in the presence of a control antibody).
[0056] Epitope Mapping of the Four Top Antibodies from 2H5 VH-Chain Shuffled Libraries.
[0057] As described above, we used peptide competition ELISA method to map the binding epitope of the four top antibodies identified from 2H5 VH-chain shuffled libraries. The LN16 peptide (corresponding to the NT amino acid (aa) 11-28 of pre-S1 domain), and LN16 peptide mutants, LN16-L19A, -D20A, -P21A, -F23A were used to compete for binding of these antibodies to m47b peptide. Our data revealed that all of them had similar peptide competition pattern as 2H5, amino acids, L19, D20, P21 and F23 are important for these antibodies' binding. The D20 and F23 are most important for all antibodies, whereas L19 and P21 played slightly variable role for different antibodies.
[0058] Further Characterize the Four Top Antibodies from 2H5 VH-Chain Shuffled Libraries.
[0059] These antibodies have more than 15-20 fold improved HBV (genotype D) neutralization activity as compared to the parental 2H5 antibody. The 1050 for these antibodies are around .about.10-40 pM. A representative antibody out of these 4 antibodies, A14, was further compared to Hepatitis B Immune Globulin in neutralizing HBV (genotype D) infection. HBIG is prepared from the plasma of donors who have high antibody levels of the hepatitis B surface antigen (HBsAg) and used as a post exposure prophylaxis for people at risk to develop hepatitis B in clinic. A14 showed more than 1000-fold greater neutralization activity than HBIG. Furthermore, A14 showed broadly neutralization activity against other two HBV genotypes, B and C. The IC50 for genotype B, C and D are 80 pM, 30 pM and 10 pM, respectively. A14 was also examined for neutralizing six HB V genotype C viruses from plasma of HB V infected patients. Again A14 was at least several hundreds to 1000-fold more potent than HBIG in neutralizing these viruses.
[0060] A14 is the one with the highest Fab melting temperatures (Tm) of 80.2.degree. C., reflecting the best thermostability of its variable domains. A14 is stabilized by approximately 2.degree. C. comparing to the original 2H5, whereas other three nAbs all have slightly reduced thermostability. The thermostability was measured using differential scanning calorimetry (DSC).
[0061] Using primary human hepatocytes (PHH), we also demonstrated the potent neutralization activity of A14 against two HBV clinical strains from HBV patient plasma samples. One virus is genotype B; the other virus is a genotype C virus. HBsAg or HBeAg secreted to cell culture supernatants was examined every two days over the entire infection course using commercial kits (Autobio Diagnostics Co., Ltd.).
[0062] A14 competed with pre-S1 for binding to NTCP expressed on cells. A14 effectively competed with pre-S1 (FITC labeled pre-S1 peptide: m59) for binding to NTCP expressed on HepG2 cells in a dose-dependent manner.
[0063] A14 has no cross reactivity with 12 different cell lines representing 6 different tissues. This was analyzed by Western blotting and immunostaining assays.
[0064] A14 has antibody mediated cytotoxicity (ADCC) activity against cells carrying its epitope on cell surface and HBV producing cells as well as infected cells. In the ADCC assay, the epitope of A14 was stably expressed on CHO cell surface, HBV producing DE19 cells, and infected HepG2-hNTCP cells were used as target cells. A human NK cell line (NK92-MI expressing CD16 (V158 allele) and FcRgamma chain was used as effector cells. The effector cells and target cells (E/T) were co-cultured at a ratio of 6:1 for 6 hours in the presence of A14 or its Fc mutant. The cell killing was determined by using LDH release assay kit form Promega. The ADCC assay showed that A14 exhibited strong specific killing of CHO cells expressing the epitope, HBV producing cells, and HBV-infected HepG2-hNTCP cells but not the control cells lacking of the epitope expression, non-HBV producing cells and non-HBV infected cells. Meanwhile, the A14's Fc mutant (D265A/N297A) that lacks the ADCC activity but retains the same binding activity had no ADCC activity.
[0065] ADCC activity is common to antibodies having the same or similar epitope as A14, including 2H5, and its VH chain shuffled derived ones: 4, 31, 32, 69, A14, A21, B103, B129, B139, B172, and the VL chain shuffled clones #8, 20, 20-m1, 20-m2, 20-m3, and antibodies having distinct epitopes, such as m36, 71, 76, T47, m150, m1Q can also present ADCC activity; for example, m1Q, also showed ADCC activity, its epitope is approximate to the C-terminal of A14's epitope on preS1.
[0066] A14 Protected Mice from HDV Infection.
[0067] We previously revealed that the molecular determinant restricting mouse NTCP (mNTCP) to support viral entry of HBV and HDV is located within the residues 84-87 of mNTCP. When residues 84-87 were replaced by the human NTCP counterparts, it can effectively support viral infections in cell cultures [16]. Based on this, we have established a mouse model (background of FVB strain) that can support HDV infection by replacing mNTCP's residues at 84-87 with the corresponding residues of hNTCP using a genome editing method, TALEN [17, 18]. Using this mouse model, we tested if A14 can protect mice from HDV infection. FVB mice (age of 9 days after birth) with aa84-87 of mNTCP modified homozygotes were administered A14 mAb at 10 mg/kg of body weight. At 1 hour after mAb administration, mice were challenged with HDV viruses. At day 6 after HDV challenge, mice were sacrificed and liver tissues were harvested in liquid nitrogen immediately after collection. Mouse liver samples were then homogenized and lysed by Trizol.RTM. reagent to extract the total RNA. The RNA samples were reverse transcribed into cDNA with Prime Script RT-PCR Kit (Takara). To quantify HDV total RNA (genome equivalent) and edited NTCP RNA copies, the cDNA obtained from 20 ng RNA was used as template for real time PCR assay. Real time PCR was performed on an ABI Fast 7500 real time system instrument (Applied Biosystems, USA). The edited NTCP and HDV viral genome equivalent copies were calculated with a standard curve and the cellular GAPDH RNA was used as an internal control. A14 mAb completely blocked HDV infection, whereas HDV infection reached 1-10.times.10.sup.6 copies/20 ng liver RNA in the control group. Mice in both groups had comparable NTCP mRNA copies in the liver tissue.
[0068] A14 Protected Mice from HBV Infection in a Prophylaxis Mouse Model and inhibited HBV infection in a treatment mouse model.
[0069] A mouse HBV infection model has been established using FRG (Fah-/-Rag2-/-/IL2rg-/-) triple knock-out mice transplanted with human hepatocytes [19, 20]. The FRG mice allows transplanted human hepatocytes replicating in mouse liver to form a chimeric liver with up to 98% human hepatocytes, as such the liver humanized FRG mice (FRGC) are highly susceptible to HBV infection. To test the prophylactic effect of A14, 10 FRGC mice were divided into two groups, five mice each. A14 prophylaxis group mice were injected with A14 at 15 mg/kg dosage by a single IP administration one day prior to HBV virus challenge, while mice in the control group were injected with same volume of PBS. On day 0, all mice were injected with 10e9 GE (genome equivalent) HBV each via tail vein. To test the therapeutic effect of A14, FRGC mice were challenged with 10e9 GE/mice of HBV via tail vein on day 0, on day 5 post-infection, the mice were treated with entecavir (ETV) control or A14 or HBIG. ETV was orally given at 0.1 mg/kg daily; A14 or HBIG were administrated every three days by I.P. injection at 20 mg/kg and 72 mg/kg (40 IU/kg), respectively. For both prophylaxis and treatment model, blood samples were collected every 3 days from all mice for measuring HBsAg and HBV DNA titer in serum. The mice were scarified at the end of the experiment, dpi35 and the liver tissues were preserved for immunohistochemical staining (THC) of HBsAg and HBcAg. A14 showed 100% protection of FRGC mice from HBV infection in the prophylaxis model; it also showed significant inhibition of HBV infection in the treatment model.
[0070] Taken together, the results clearly demonstrated that A14 mAb is a potent HDV and HBV entry inhibitor in animal model. A14 mAb can be used to replace HBIG for prevention of HDV and HBV infection. On the other hand, A14 treatment of an established HBV infection in mice significantly inhibited HBV infection, moreover A14 showed specific ADCC activity against HBV-infected cells but not the non-HBV infected cells. These results indicate that A14 mAb may be combined with ETV to treat patient who are chronically infected by HBV. As A14 blocks new viral entry into host cells and has ADCC activity against infected cells, whereas ETV inhibits viral replication, combination of A14 with a viral replication inhibitor such as ETV, lamivudine, adefovir, tenofovir, telbivudine or other nucleoside and nucleotide analogues (NUCs) provide new therapeutic and prophylactic options for patients and can achieve better viremia control and HBsAg reduction.
[0071] Improvement of A14 Affinity and Neutralization Activity by VL-Chain Shuffling.
[0072] To further improve A14 activity, we made an A14-VL chain shuffled phage display library, in which VH of A14 was fixed and paired with a library of VL chains. The final library (A14VH/VLlib) constructed had a size of .about.3.times.10.sup.8. By using m47b peptide captured on streptavidin-conjugated magnetic M-280 Dynabeads.RTM. (Life Technologies) as target, the A14VH/VLlib library was selected for two rounds. 196 clones were screened for binding with m47b by ELISA. All clones were positive but 24 clones with highest OD450 reading were picked for sequencing. Two clones, #8 and #20, with different VL chain sequences than A14's VL were identified. These two antibodies were converted into full-length human IgG1 and tested for binding to m47b by ELISA. They both showed stronger binding activity to m47b than A14. In the HBV neutralization assay of HBV (genotype D), #8 showed 5-fold improvement in neutralizing HBV infection, whereas #20 showed similar activity as A14. Further mutagenesis of the VL of #20 (#20-m1, -m2, -m3) improved its neutralization activity by .about.3-5-fold than A14, reached to the similar level as #8. The elevated HDV neutralization activities of these #20 mutants compared to A14 were demonstrated. Thus these A14-derived antibodies with further improved activities can be used similarly as A14 as described above, either alone or in combination with a viral replication inhibitor.
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[0092] 20. Bissig, K. D., et al., Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J Clin Invest, 2010. 120(3): p. 924-30. Antibody Sequences of 7 Antibodies Derived from Naive Library
TABLE-US-00006
[0092] m36 m36 VH DNA: (SEQ ID NO: 01) CAAGTTCCTTTATGTGCTGTCTCATCATTTTGGCAAGAATTCGCCACCATGAAACATCTGTGGT TCTTCCTTCTCCTGGTGGCAGCGGCCCAGCCGGCCATGGCCCAGATGCAGCTGGTGCAGTCTGG GGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT GATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA TTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAG AGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTG TATTACTGTGCAAAAACGTCCTACGGGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCA CCGTCTCCTCA m36 VL DNA: (SEQ ID NO: 02) CAGCCTGTGCTGACTCAATCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAAACACTTCCAACATCGGAAGTTATTATGCATACTGGTATCAGCAACTCCCAGGAAC GOCCCCCAAACTCCTCATCTATGATAATAATCAGCGGCCCTCOGGGATCCCTOCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCAG ATTATTACTGTGCAACATGGGATGACAGCCTGAATGGTCCGGTGTTCGGCGGAGGGACCAAGGT CACCGTCCTA m36 VH Amino acid: (SEQ ID NO: 03) QMQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSV KGRFTTSRDNAKNSLYLQMNSLRAEDTALYYCAKTSYGGAFDIWGQGTMVTVSS m36 VL Amino acid: (SEQ ID NO: 04) QPVLTQSPSASGTPGQRVTTSCSGNTSNIGSYYAYWYQQLPGTAPKLLIYDNNQRPSGTPARFS GSKSGTSASLAISGLQSEDEADYYCATWDDSLNGPVFGGGTKVTVL 71: 71 VH DNA: (SEQ ID NO: 05) CAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGATACACCTTCACCGGCTACTATATACATTGGGTGCGACAGGCCCCTGGACA AGGGCTTGAGTGGATGGGACGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTT CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGGACGGCCTACATGGAACTGAGTACAC TGACATCTGACGACACGGCCOTTTATTACTGTGCGAGAGAAGGAAGGGGCGGCATGGACGTCTG GOGCCAAGGGACCACGOTCACCGTCTCCTCA 71 VL DNA: (SEQ ID NO: 06) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCT CCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCA GAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTOGGTTCTAATCGGGCCTCCGGGOTCCCT CACAGGTTCACTCCCACTCCATCACGCACACATTTTACACTGAAAATCACCACAGTGGAGCCTC ACCATGTTCCCATTTATTACTOCATGCAACCTCTACAACCTCCCATCACCTTCCGCCACGCCAC ACGACTGGAGATTAAA 71 VH Amino acid: (SEQ ID NO: 07) QVQLVESGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGRINPNSGGTNYAQKF QGRVTMTRDTSIRTAYMELSTLTSDDTAVYYCAREGRGGMDVWGQGTTVTVSS 71 VL Amino acid: (SEQ ID NO: 08) DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP DRFSGSGSGTDFTLKISRVEAEDVGTYYCMQGLQPPITFGQGTRLEIK 76: 76 VH DNA: (SEQ ID NO: 09) GAGGTGCAGCTGTTGGAGACCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAA GGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTG AACCGCCGATTCACCATCTCCACAGACAATTCCAAGAACACCCTCTATCTCCAAATGAACACCC TCAGACCTGACCACACCCCTCTCTATTACTUTCCGACTCGTCCTTTTCATATCTGCGCCCAACC GACAATGGTCACCGTCTCTTCA 76 VL DNA: (SEQ ID NO: 10) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCT CCTGCAGGTCTAGTCACAGCCTCGTATACAGTGATGGAAACACCTACTTGAGTTGGTTTCACCA GAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAATCGGGACTTTGGGGTCCCA GACAGATTCAGCGGCAGTGGGTCAGGCACTGACTTCACACTGAAGATCAGCAGGGTGGAGGCTG AGGATGTTGGAGTTTATTACTGCATGCAAGGTACACACTGGCCTGGGACGTTCGGCCAGGGGAC CAAACTGGATATCAAA 76 VH Amino acid: (SEQ ID NO: 11) EVQLLETGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSV KGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCASGAFDIWGQGTMVTVSS 76 VL Amino acid: (SEQ ID NO: 12) DVVMTQSPLSLPVTLGQPASISCRSSHSLVYSDGNTYLSWFHQRPGQSPRRLIYKVSNRDFGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPGTFGQGTKLDIK T47: T47 VH DNA: (SEQ ID NO: 13) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCTCCT GTGCCATCTCCGGGGACAGTGTCTCCAGCAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTCTCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAGCTCTGTGACTCCCGAGGACACGGCTGTATATTACTGTGCAAGAGCCGATGGTTCGCGAGG GGGAGGGTATGACCAGTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA T47 VL DNA: (SEQ ID NO: 14) GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCA AATGCAAGTCCAGTCAGTCTATTTTATACAGGTCCAACAATAAGAACTACTTAGCTTGGTACCA ACACAAACCAGGACAGCCTCCTAAGCTGCTCATTTCCTGGGCATCTACCCGGGAATCCGGGGTC CCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGG CTGAAGATGTGGCGGTTTATTACTGTCAGCAATATTATACTACTCCTCAGACTTTTGGCCAGGG GACCAAGGTGGAGATCAAA 147 V14 Amino acid: (SEQ ID NO: 15) QVQLQQSGPGLVKPSQTLSLSCAISGDSVSSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNPDTSKNQFSLQLSSVTPEDTAVYYCARADGSRGGGYDQWGQGTLVTVSS T47 VL Amino acid: (SEQ ID NO: 16) DIVMTQSDDSLAVSLGERATTKCKSSQSILYRSNNKNYLAWYQHKPGQDDKLLISWASTRESGV DDRFSCSGSCTDFTLTTNSLQAEDVAVYYCQQYYTTDQTFCQCTKVEIK m1Q m1Q VH DNA (SEQ ID NO: 17) CAGGTCCAGTTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAA GGGGCTGGAGCAGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGTAGACTCCGTG AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGATCTACATACGGTATGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCA m1Q-VL DNA (SEQ ID NO: 18) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGTCGGCCTCCATCT CCTGCACGTCTACTCAAAGCCTCGTACACAGTCATGGAAACACCTACTTGAATTGOTTTCACCA GAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAATCGGGACTCCGGGGTCCCA GACAGATTCAGCGGCAGTGGGTCAGACACTGATTTCACACTGGAAATCAGCAGGGTGGAGGCCG ACGATCTTGGCATTTATTACTCCATGCAAGGTACACACTGGTCCACCTTCGCCCAAGCCACCAA GCTGGATATCAAA m10 VH Amino acid: (SEQ ID NO: 19) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEQVAVISYDGSNKYYVDSV KGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCARSTYGMDVWGQGTTVIVSS m1Q Vk Amino acid: (SEQ ID NO: 20) DVVMTQSPLSLPVTLGQSASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVP DRFSGSGSDTDFTLEISRVEAEDVGTYYCMQGTHWWTFGQGTKLDIK 2H5: 2H5 VH DNA: (SEQ ID NO: 21) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGGCATCTCCGGGGACAGTGTCTCTAGCAAGAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC TTCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGCATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTTTCCCTGCAGC TGAACTCTGTGACCCCCGAAGACACGGCTGTGTATTATTGTGCGCGCGGCCAGATGGGAGCTTT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 2H5 VL DNA: (SEQ ID NO: 22) CAGTCTGTGTTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAAGCAGCTCCAACATCGGAAGTTATTATGTATACTGGTACCAGCAATTCCCAGGAAC GGCCCCCAAACTCCTCATCTATGGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGTCAGTCCTATGACAGCAGCCTGAGTGGTGTGATATTCGGCGGAGGGACCAAGCT GACCGTCCTA 2H5 VH Amino acid: (SEQ ID NO: 23) QVQLQQSGPGLVKPSQTLSLTCGTSGDSVSSKSAAWNWIRQSPSRGLEWLGRTYYRSKWHNDYA VSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARGQMGALDVWGQGTTVTVSS 2H5 VL Amino acid: (SEQ ID NO: 24) QSVLTQPPSASGTPGQRVTTSCSGSSSNIGSYYVYWYQQFPGTAPKLLIYGNNQRPSGVPDRFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGVIFGGGTKLTVL
m150 m150 VH DNA: (SEQ ID NO: 25) GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAA GGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTG AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGTTGGTGGCTGGTCGAAGTGCTTTTGA TATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA m150 VK DNA: (SEQ ID NO: 26) GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGC TCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGC AGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTT ATTACTGTCAGCAGTATAATAACTGGCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGAT TAAA m150 VH Amino acid: (SEQ ID NO: 27) EVQLVQSGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSV KGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCARLVAGRSAFDIWGQGTTVTVSS m150 NK Amino acid: (SEQ ID NO: 28) EIVLTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGTPARFSG SGSGTEFILTTSSLQSEDFAVYYCQQYNNWPPITEGQGTRLEIK
Antibody Sequences of 10 Antibodies Derived from 2H5 VH-Chain Shuffled Library Selection.
[0093] Note, these antibodies have the same VL sequence as 2H5, therefore only VH sequences of these antibodies were listed below.
TABLE-US-00007 #4 #4 VH DNA: (SEQ ID NO: 29) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGGCATCTCCGGGGACAGTGTCTCTAGCAAGAGTGTTACTTGGAACTGGATCAGGGAGTCTCC AACGGGAGGCCTTGAGTGGCTGGGCAGGACATACTATAGGTCCAAGTGGTTTAATGATTATGCA GTATCTGTGAAAAGTCGAATAACTGTCAACCCAGACACATCCAAGAACCAGTTTTCCCTGCAGC TAAACTCTGTGACTCCCGAGGACAGGGGTGTCTATTACTGCGCACGCGCCAAGATGGGAGGTAT GGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCTTCA #4 VH Amino Acid: (SEQ ID NO: 30) QVQLQQSGPGLVKPSQTLSLTCGTSGDSVSSKSVTWNWIRESPIGGLEWLGRTYYRSKWFNDYA VSVKSRITVNPDTSKNQFSLQLNSVTPEDRGVYYCARAKMGGMDVWGQGTTVTVSS #31 VH DNA: (SEQ ID NO: 31) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TCAACTCTCTGACTCCCCACCACACGCCTCTTTATTACTCTACAACACACACTTGCCACGCTAT CCAAGTCTCCCCCCAACCCACCACCGTCACCCTCTCCTCA #31 VH Amino acid: (SEQ ID NO: 32) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCTRQSWHGMEVWGQGTTVTVSS #32 VH DNA: (SEQ ID NO: 33) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACTCAGACACATCGAAGAACCAGTTCTCCCTGCAGC TGAAGTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGGAGTATAGCAACAGGTAC TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA #32 VH Amino acid: (SEQ ID NO: 34) QVQLQQSGPCLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRCLEWLCRTYYRSKWYNDYA VSVKSRITTNSDTSKNQFSLQLKSVTPEDTAVYYCARSIATGTDYWGQGTLVTVSS #69 VH DNA: (SEQ ID NO: 35) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGATGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGTAGCCGTGCTACTTGGAACTGGATCAGGGAGTCTCC AACGGGAGGCCTTGAGTGGCTGGGCAGGACATACTATAGGTCCAAGTGGTTTAATGATTATGCA GTATCTGTGAAAAGTCGAATAACTGTCAACCCAGACACATCCAAGAACCAGTTTTCCCTGCAGC TAAACTCTGTGACTCCCGAGGACAGGGGTGTCTATTACTGCGCACGCGCCAAGATGGGAGGTAT GGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA #69 VH Amino acid: (SEQ ID NO: 36) QVQLQQSGPGLMKPSQTLSLTCAISGDSVSSSRATWNWIRESPIGGLEWLGRTYYRSKWFNDYA VSVKSRITVNPDTSKNQFSLQLNSVIPEDRGVYYCARAKMGGMDVWGQGTTVTVSS A14 VH DNA: (SEQ ID NO: 37) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT CTCCCATCTCCCCCCACACTCTCTCTACCAACAGTCCTCCTTCCAACTCCATCAGCCACTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGAACACGTTGGGGTAT GGACGTCTGGGGCCAAGGGACCCTGGTCACTGTCTCCTCA A14 VH Amino acid: (SEQ ID NO: 38) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSFSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNFDTSKNQFSLQLNSVTFEDTAVYYCARGTRWGMDVWGQGTLVTVSS A21 VH DNA: (SEQ ID NO: 39) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGCGAAAGTGTACGGTGT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA A21 VH Amino acid: (SEQ ID NO: 40) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSFSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNFDTSKNQFSLQLNSVTFEDTAVYYCARAKVYGVDVWGQGTTVTVSS B103 VH DNA: (SEQ ID NO: 41) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGGCATCTCCGGGGACAGTGTCTCTAGCAAGAGTGCCACTTGGAACTGGGTCAGGCAGTCCGC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAGGTGGTTTAATGATTATGCA GTGTCTGTGAAAAGTCGAATAACCGTCAAGCCAGACACATCCAAGAACCAGTTTTCCCTGCAAT TAAATTCTGTGAGTCCCGAGGACACGGCTATCTATTACTGTGCACGCGGCAACATGGGAGCTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA B103 VH Amino acid: (SEQ ID NO: 42) QVQLQQSGPGLVKPSQTLSLTCGTSGDSVSSKSATWNWVRQSASRGLEWLGRTYYRSRWFNDYA VSVKSRITVKPDTSKNQFSLQLNSVSPEDTAIYYCARGNMGAMDVWGQGTTVTVSS B129 VH DNA: (SEQ ID NO: 43) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGCTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGGGTCTCTAGCAATAGAGCTGCTTGGAACTGGGTCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCCAGTGGTATAATGATTATGCA GTCTCTGTAAAAAGTCGAGTGACCATCAGCCCAGACGCATCCAAGAACCAAGTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGTACAGCTATGGGTGA CGCCTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA B129 VH Amino acid: (SEQ ID NO: 44) QVQLQQSGPGLLKPSQTLSLTCAISGDRVSSNRAAWNWVRQSPSRGLEWLGRTYYRSQWYNDYA VSVKSRVTTSPDASKNQVSLQLNSVTPEDTAVYYCARGTAMGDAWGQGTLVTVSS B139 VH DNA: (SEQ ID NO: 45) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCACACTCACCT GTGTCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA CTTTCTCTCAAAACTCCAATAACCATCAACCCAGACACATCCAACAACCACTTCTCCCTCCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGACAAGCCTCCAACGGTTT TGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA B139 VH Amino acid: (SEQ ID NO: 46) QVQLQQSGPGLVKPSQTLTLTCVISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYA VSLKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARQASNGFDIWGQGTMVTVSS B172 VH DNA: (SEQ ID NO: 47) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGACAGGGGACGACAGGCTT TGACTACTGGGGCCAGGGAACCACGGTCACCGTCTCCTCA B172 VH Amino acid: (SEQ ID NO: 48) QVQLQQSGPCLVKPSQTLSLTCAISCDSVSSNSAAWNWIRQSPSRCLEWLCRTYYRSKWYNDYA VSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARQGTTGFDYWGQGTTVTVSS
Antibody Sequences of Two Antibodies Derived from A14 VL-Chain Shuffled Library Selection.
[0094] Note, these antibodies have the same VH sequence as A14, therefore only VL sequences of these two antibodies were listed below.
TABLE-US-00008 #8 VL DNA (SEQ ID NO: 49) CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCT GCTCTGGAAGCAGCTCCAACATTGGGAATTATTATGTGTCCTGGTACCAGCACCTCCCAGGAAC AGCCCCCAAACTCCTCATTTATGACAATGCTAAGCGACCCTCAGGGATTCCTGACCGATTCTCT GGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACTGGGCTCCGGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAATAGCCTTAGTGGTTTGGTGTTCGGCGGAGGGACCAAGCT GACCGTCCTA #8 VL amino acid: (SEQ ID NO: 50) QSVVTQPPSVSAAPCQKVTTSCSCSSSNTCNYYVSWYQHLPCTAPKLLIYDNAKRPSCIPDRFS GSKSGTSATLGTTGLRAEDEADYYCQSYDNSLSGLVFGGGTKLTVL #20 VL DNA: (SEQ ID NO: 51) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTAAGTATGTATACTGGTACCAGCGGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAGCAGCCTGCGTGCTGTGGTTTTCGGCGGAGGGACCAAGCT GACCGTCCTA #20 VL amino acid: (SEQ ID NO: 52) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSKYVYWYQRLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL #20-m1 VL DNA: (SEQ ID NO: 53) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTTTCTATGTATACTGGTACCAGCGGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT CGCTCCAACTCTGCCACCTCACCCTCCCTCCCCATCACTCCGCTCCAGGCTCAGGATCAGCCTC ATTATTACTCCCACTCCTATCACACCACCCTCCGTCCTCTCCTTTTCCGCCCAGGCACCAACCT GACCGTCCTA #20-m1 VL amino acid: (SEQ ID NO: 54) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSFYVYWYQRLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL #20-m2 VL DNA: (SEQ ID NO: 55) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTTTCTATGTATACTGGTACCAGCAGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAGCAGCCTGCGTGCTGTGGTTTTCGGCGGAGGGACCAAGCT GACCGTCCTA #20-m2 VL amino acid: (SEQ ID NO: 56) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSFYVYWYQQLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL #20-m3 VL DNA: (SEQ ID NO: 57) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTTACTATGTATACTGGTACCAGCAGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAGCAGCCTGCGTGCTGTGGTTTTCGGCGGAGGGACCAAGCT GACCGTCCTA #20-m3 VL amino acid: (SEQ ID NO: 58) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSYYVYWYQQLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL
Sequence CWU
1
1
1481459DNAHomo sapiens 1caagttcctt tatgtgctgt ctcatcattt tggcaagaat
tcgccaccat gaaacatctg 60tggttcttcc ttctcctggt ggcagcggcc cagccggcca
tggcccagat gcagctggtg 120cagtctgggg gaggcttggt acagcctggc aggtccctga
gactctcctg tgcagcctct 180ggattcacct ttgatgatta tgccatgcac tgggtccggc
aagctccagg gaagggcctg 240gagtgggtct caggtattag ttggaatagt ggtagcatag
gctatgcgga ctctgtgaag 300ggccgattca ccatctccag agacaacgcc aagaactccc
tgtatctgca aatgaacagt 360ctgagagctg aggacacggc cttgtattac tgtgcaaaaa
cgtcctacgg gggggctttt 420gatatctggg gccaagggac aatggtcacc gtctcctca
4592330DNAHomo sapiens 2cagcctgtgc tgactcaatc
gccctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaaacacttc
caacatcgga agttattatg catactggta tcagcaactc 120ccaggaacgg cccccaaact
cctcatctat gataataatc agcggccctc ggggatccct 180gcccgattct ctggctccaa
gtctggcacc tcagcctccc tggccatcag tgggctccag 240tctgaggatg aggcagatta
ttactgtgca acatgggatg acagcctgaa tggtccggtg 300ttcggcggag ggaccaaggt
caccgtccta 3303118PRTHomo sapiens
3Gln Met Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25
30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Gly Ile
Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95Ala Lys Thr Ser Tyr Gly
Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr 100
105 110Met Val Thr Val Ser Ser 1154110PRTHomo
sapiens 4Gln Pro Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln1
5 10 15Arg Val Thr Ile
Ser Cys Ser Gly Asn Thr Ser Asn Ile Gly Ser Tyr 20
25 30Tyr Ala Tyr Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu Leu 35 40 45Ile
Tyr Asp Asn Asn Gln Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser 50
55 60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Ser Gly Leu Gln65 70 75
80Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser
Leu 85 90 95Asn Gly Pro
Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100
105 1105351DNAHomo sapiens 5caggtgcagc tggtggagtc
tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata
caccttcacc ggctactata tacattgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggacgg atcaacccta acagtggtgg cacaaactat 180gcacagaagt ttcagggcag
ggtcaccatg accagggaca cgtccatcag gacggcctac 240atggaactga gtacactgac
atctgacgac acggccgttt attactgtgc gagagaagga 300aggggcggca tggacgtctg
gggccaaggg accacggtca ccgtctcctc a 3516336DNAHomo sapiens
6gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc
180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc
240agcagagtgg aggctgagga tgttgggatt tattactgca tgcaaggtct acaacctccc
300atcaccttcg gccaggggac acgactggag attaaa
3367117PRTHomo sapiens 7Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Ile Arg Thr Ala Tyr65 70
75 80Met Glu Leu Ser Thr Leu Thr Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Gly Arg Gly Gly Met Asp Val Trp Gly Gln Gly Thr Thr
100 105 110Val Thr Val Ser Ser
1158112PRTHomo sapiens 8Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30Asn Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70
75 80Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr
Tyr Cys Met Gln Gly 85 90
95Leu Gln Pro Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 1109342DNAHomo sapiens
9gaggtgcagc tgttggagac cgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagcaa taaatactac
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagtggtgct
300tttgatatct ggggccaagg gacaatggtc accgtctctt ca
34210336DNAHomo sapiens 10gatgttgtga tgactcagtc tccactctcc ctgcccgtca
cccttggaca gccggcctcc 60atctcctgca ggtctagtca cagcctcgta tacagtgatg
gaaacaccta cttgagttgg 120tttcaccaga ggccaggcca atctccaagg cgcctaattt
ataaggtttc taatcgggac 180tttggggtcc cagacagatt cagcggcagt gggtcaggca
ctgacttcac actgaagatc 240agcagggtgg aggctgagga tgttggagtt tattactgca
tgcaaggtac acactggcct 300gggacgttcg gccaggggac caaactggat atcaaa
33611114PRTHomo sapiens 11Glu Val Gln Leu Leu Glu
Thr Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Ser Gly Ala Phe Asp Ile Trp Gly Gln Gly
Thr Met Val Thr Val 100 105
110Ser Ser12112PRTHomo sapiens 12Asp Val Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Leu Gly1 5 10
15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser His Ser Leu Val Tyr Ser
20 25 30Asp Gly Asn Thr Tyr Leu
Ser Trp Phe His Gln Arg Pro Gly Gln Ser 35 40
45Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Phe Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70
75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Gly 85 90
95Thr His Trp Pro Gly Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys
100 105 11013369DNAHomo sapiens
13caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc
60tcctgtgcca tctccgggga cagtgtctcc agcaacagtg ttgcttggaa ctggatcagg
120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat
180aatgattatg cagtctctgt gaaaagtcga ataaccatca acccagacac atccaagaac
240cagttctccc tgcagctgag ctctgtgact cccgaggaca cggctgtata ttactgtgca
300agagccgatg gttcgcgagg gggagggtat gaccagtggg gccagggaac cctggtcacc
360gtctcttca
36914339DNAHomo sapiens 14gacatcgtga tgacccagtc tccagactcc ctggctgtgt
ctctgggcga gagggccacc 60atcaaatgca agtccagtca gtctatttta tacaggtcca
acaataagaa ctacttagct 120tggtaccaac acaaaccagg acagcctcct aagctgctca
tttcctgggc atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg
ggacagattt cactctcacc 240atcaacagcc tgcaggctga agatgtggcg gtttattact
gtcagcaata ttatactact 300cctcagactt ttggccaggg gaccaaggtg gagatcaaa
33915123PRTHomo sapiens 15Gln Val Gln Leu Gln Gln
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Ser Cys Ala Ile Ser Gly Asp Ser
Val Ser Ser Asn 20 25 30Ser
Val Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser
Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln
Leu Ser Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95Tyr Tyr Cys Ala Arg Ala Asp Gly Ser Arg Gly
Gly Gly Tyr Asp Gln 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12016113PRTHomo sapiens 16Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly1 5 10
15Glu Arg Ala Thr Ile Lys Cys Lys Ser Ser Gln Ser Ile Leu Tyr Arg
20 25 30Ser Asn Asn Lys Asn Tyr Leu
Ala Trp Tyr Gln His Lys Pro Gly Gln 35 40
45Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly
Val 50 55 60Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70
75 80Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln 85 90
95Tyr Tyr Thr Thr Pro Gln Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110Lys17348DNAHomo sapiens
17caggtccagt tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct
120ccaggcaagg ggctggagca ggtggcagtt atatcatatg atggaagtaa taaatactac
180gtagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagatctaca
300tacggtatgg acgtctgggg ccaagggacc acggtcaccg tctcctca
34818333DNAHomo sapiens 18gatgttgtga tgactcagtc tccactctcc ctgcccgtca
cccttggaca gtcggcctcc 60atctcctgca ggtctagtca aagcctcgta cacagtgatg
gaaacaccta cttgaattgg 120tttcagcaga ggccaggcca atctccaagg cgcctaattt
ataaggtttc taatcgggac 180tccggggtcc cagacagatt cagcggcagt gggtcagaca
ctgatttcac actggaaatc 240agcagggtgg aggccgagga tgttgggatt tattactgca
tgcaaggtac acactggtgg 300acgttcggcc aagggaccaa gctggatatc aaa
33319116PRTHomo sapiens 19Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Gln Val 35
40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn
Lys Tyr Tyr Val Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Ser Thr Tyr Gly Met Asp Val Trp Gly
Gln Gly Thr Thr Val 100 105
110Thr Val Ser Ser 11520111PRTHomo sapiens 20Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5
10 15Gln Ser Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25
30Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser
35 40 45Pro Arg Arg Leu Ile Tyr Lys Val
Ser Asn Arg Asp Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Asp Thr Asp Phe Thr Leu Glu Ile65
70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Gly 85
90 95Thr His Trp Trp Thr Phe Gly Gln Gly Thr Lys
Leu Asp Ile Lys 100 105
11021360DNAHomo sapiens 21caggtacagc tgcagcagtc aggtccagga ctggtgaagc
cctcgcagac cctctcactc 60acctgtggca tctccgggga cagtgtctct agcaagagtg
ctgcttggaa ctggatcagg 120cagtcccctt cgagaggcct tgagtggctg ggaaggacat
actacaggtc caagtggcat 180aatgattatg cagtatctgt gaaaagtcga ataaccatca
acccagacac atccaagaac 240cagttttccc tgcagctgaa ctctgtgacc cccgaagaca
cggctgtgta ttattgtgcg 300cgcggccaga tgggagcttt ggacgtctgg ggccaaggga
ccacggtcac cgtctcctca 36022330DNAHomo sapiens 22cagtctgtgt tgacgcagcc
gccctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc
caacatcgga agttattatg tatactggta ccagcaattc 120ccaggaacgg cccccaaact
cctcatctat ggtaataatc agcggccctc aggggtccct 180gaccgattct ctggctccaa
gtctggcacc tcagcctccc tggccatcac tgggctccag 240gctgaggatg aggctgatta
ttactgtcag tcctatgaca gcagcctgag tggtgtgata 300ttcggcggag ggaccaagct
gaccgtccta 33023120PRTHomo sapiens
23Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Gly Ile Ser Gly Asp Ser Val Ser Ser Lys 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp His Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Gly
Gln Met Gly Ala Leu Asp Val Trp Gly Gln 100
105 110Gly Thr Thr Val Thr Val Ser Ser 115
12024110PRTHomo sapiens 24Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr
20 25 30Tyr Val Tyr Trp Tyr Gln
Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45Ile Tyr Gly Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65 70
75 80Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Tyr Asp Ser Ser Leu 85 90
95Ser Gly Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 11025357DNAHomo sapiens
25gaggtgcagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gaggttggtg
300gctggtcgaa gtgcttttga tatctggggc caagggacca cggtcaccgt ctcctca
35726324DNAHomo sapiens 26gaaattgtgc tgactcagtc tccagccacc ctgtctgtgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcaacttag
cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatggt gcatccacca
gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 240gaagattttg cagtttatta ctgtcagcag tataataact
ggcctccgat caccttcggc 300caagggacac gactggagat taaa
32427119PRTHomo sapiens 27Glu Val Gln Leu Val Gln
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn
Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Leu Val Ala Gly Arg Ser Ala Phe Asp
Ile Trp Gly Gln Gly 100 105
110Thr Thr Val Thr Val Ser Ser 11528108PRTHomo sapiens 28Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Asn 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Gly Ala Ser Thr
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser65 70 75 80Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95Ile Thr Phe Gly Gln Gly Thr
Arg Leu Glu Ile Lys 100 10529360DNAHomo
sapiens 29caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac
cctctcactc 60acctgtggca tctccgggga cagtgtctct agcaagagtg ttacttggaa
ctggatcagg 120gagtctccaa cgggaggcct tgagtggctg ggcaggacat actataggtc
caagtggttt 180aatgattatg cagtatctgt gaaaagtcga ataactgtca acccagacac
atccaagaac 240cagttttccc tgcagctaaa ctctgtgact cccgaggaca ggggtgtcta
ttactgcgca 300cgcgccaaga tgggaggtat ggacgtctgg ggccagggga ccacggtcac
cgtctcttca 36030120PRTHomo sapiens 30Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Gly Ile Ser Gly Asp Ser Val Ser
Ser Lys 20 25 30Ser Val Thr
Trp Asn Trp Ile Arg Glu Ser Pro Thr Gly Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp
Phe Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu Asp Arg Gly Val 85 90
95Tyr Tyr Cys Ala Arg Ala Lys Met Gly Gly Met Asp Val
Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 12031360DNAHomo
sapiens 31caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac
cctctcactc 60acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa
ctggatcagg 120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 180aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac
atccaagaac 240cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgttta
ttactgtaca 300agacagagtt ggcacggtat ggaagtctgg ggccaaggga ccacggtcac
cgtctcctca 36032120PRTHomo sapiens 32Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser
Ser Asn 20 25 30Ser Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp
Tyr Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95Tyr Tyr Cys Thr Arg Gln Ser Trp His Gly Met Glu Val
Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 12033360DNAHomo
sapiens 33caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac
cctctcactc 60acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa
ctggatcagg 120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 180aatgattatg cagtatctgt gaaaagtcga ataaccatca actcagacac
atcgaagaac 240cagttctccc tgcagctgaa gtctgtgact cccgaggaca cggctgtgta
ttactgtgca 300aggagtatag caacaggtac tgactactgg ggccagggaa ccctggtcac
cgtctcctca 36034120PRTHomo sapiens 34Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser
Ser Asn 20 25 30Ser Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp
Tyr Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Ser Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Lys
Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95Tyr Tyr Cys Ala Arg Ser Ile Ala Thr Gly Thr Asp Tyr
Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 12035360DNAHomo
sapiens 35caggtacagc tgcagcagtc aggtccagga ctgatgaagc cctcgcagac
cctctcactc 60acctgtgcca tctccgggga cagtgtctct agtagccgtg ctacttggaa
ctggatcagg 120gagtctccaa cgggaggcct tgagtggctg ggcaggacat actataggtc
caagtggttt 180aatgattatg cagtatctgt gaaaagtcga ataactgtca acccagacac
atccaagaac 240cagttttccc tgcagctaaa ctctgtgact cccgaggaca ggggtgtcta
ttactgcgca 300cgcgccaaga tgggaggtat ggacgtctgg ggccagggga ccacggtcac
cgtctcctca 36036120PRTHomo sapiens 36Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Met Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser
Ser Ser 20 25 30Arg Ala Thr
Trp Asn Trp Ile Arg Glu Ser Pro Thr Gly Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp
Phe Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu Asp Arg Gly Val 85 90
95Tyr Tyr Cys Ala Arg Ala Lys Met Gly Gly Met Asp Val
Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 12037360DNAHomo
sapiens 37caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac
cctctcactc 60acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa
ctggatcagg 120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 180aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac
atccaagaac 240cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta
ttactgtgca 300agaggaacac gttggggtat ggacgtctgg ggccaaggga ccctggtcac
tgtctcctca 36038120PRTHomo sapiens 38Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser
Ser Asn 20 25 30Ser Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp
Tyr Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95Tyr Tyr Cys Ala Arg Gly Thr Arg Trp Gly Met Asp Val
Trp Gly Gln 100 105 110Gly Thr
Leu Val Thr Val Ser Ser 115 12039360DNAHomo
sapiens 39caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac
cctctcactc 60acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa
ctggatcagg 120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caagtggtat 180aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac
atccaagaac 240cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta
ttactgtgca 300agagcgaaag tgtacggtgt ggacgtctgg ggccaaggga ccacggtcac
cgtctcctca 36040120PRTHomo sapiens 40Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser
Ser Asn 20 25 30Ser Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp
Tyr Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95Tyr Tyr Cys Ala Arg Ala Lys Val Tyr Gly Val Asp Val
Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 12041360DNAHomo
sapiens 41caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac
cctctcactc 60acctgtggca tctccgggga cagtgtctct agcaagagtg ccacttggaa
ctgggtcagg 120cagtccgcat cgagaggcct tgagtggctg ggaaggacat actacaggtc
caggtggttt 180aatgattatg cagtgtctgt gaaaagtcga ataaccgtca agccagacac
atccaagaac 240cagttttccc tgcaattaaa ttctgtgagt cccgaggaca cggctatcta
ttactgtgca 300cgcggcaaca tgggagctat ggacgtctgg ggccaaggga ccacggtcac
cgtctcttca 36042120PRTHomo sapiens 42Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Gly Ile Ser Gly Asp Ser Val Ser
Ser Lys 20 25 30Ser Ala Thr
Trp Asn Trp Val Arg Gln Ser Ala Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Arg Trp
Phe Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Ile Thr Val Lys Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn
Ser Val Ser Pro Glu Asp Thr Ala Ile 85 90
95Tyr Tyr Cys Ala Arg Gly Asn Met Gly Ala Met Asp Val
Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 12043357DNAHomo
sapiens 43caggtacagc tgcagcagtc aggtccagga ctgctgaagc cctcgcagac
cctctcactc 60acctgtgcca tctccgggga cagggtctct agcaatagag ctgcttggaa
ctgggtcagg 120cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc
ccagtggtat 180aatgattatg cagtctctgt aaaaagtcga gtgaccatca gcccagacgc
atccaagaac 240caagtctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta
ttactgtgca 300agaggtacag ctatgggtga cgcctggggc cagggaaccc tggtcaccgt
ctcttca 35744119PRTHomo sapiens 44Gln Val Gln Leu Gln Gln Ser Gly
Pro Gly Leu Leu Lys Pro Ser Gln1 5 10
15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Arg Val Ser
Ser Asn 20 25 30Arg Ala Ala
Trp Asn Trp Val Arg Gln Ser Pro Ser Arg Gly Leu Glu 35
40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gln Trp
Tyr Asn Asp Tyr Ala 50 55 60Val Ser
Val Lys Ser Arg Val Thr Ile Ser Pro Asp Ala Ser Lys Asn65
70 75 80Gln Val Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95Tyr Tyr Cys Ala Arg Gly Thr Ala Met Gly Asp Ala Trp
Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11545360DNAHomo sapiens 45caggtacagc
tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctcacactc 60acctgtgtca
tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120cagtccccat
cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180aatgattatg
cagtttctct gaaaagtcga ataaccatca acccagacac atccaagaac 240cagttctccc
tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300agacaagcct
ccaacggttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 36046120PRTHomo
sapiens 46Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln1 5 10 15Thr Leu Thr
Leu Thr Cys Val Ile Ser Gly Asp Ser Val Ser Ser Asn 20
25 30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser
Pro Ser Arg Gly Leu Glu 35 40
45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Leu Lys Ser Arg Ile Thr Ile
Asn Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val 85 90 95Tyr Tyr
Cys Ala Arg Gln Ala Ser Asn Gly Phe Asp Ile Trp Gly Gln 100
105 110Gly Thr Met Val Thr Val Ser Ser
115 12047360DNAHomo sapiens 47caggtacagc tgcagcagtc
aggtccagga ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga
cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120cagtccccat cgagaggcct
tgagtggctg ggaaggacat actacaggtc caagtggtat 180aatgattatg cagtatctgt
gaaaagtcga ataaccatca acccagacac atccaagaac 240cagttctccc tgcagctgaa
ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300agacagggga cgacaggctt
tgactactgg ggccagggaa ccacggtcac cgtctcctca 36048120PRTHomo sapiens
48Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu 35 40 45Trp Leu Gly
Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50
55 60Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Arg Gln
Gly Thr Thr Gly Phe Asp Tyr Trp Gly Gln 100
105 110Gly Thr Thr Val Thr Val Ser Ser 115
12049330DNAHomo sapiens 49cagtctgtcg tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aattattatg tgtcctggta ccagcacctc 120ccaggaacag cccccaaact cctcatttat
gacaatgcta agcgaccctc agggattcct 180gaccgattct ctggctccaa gtctggcacg
tcagccaccc tgggcatcac tgggctccgg 240gctgaggatg aggctgatta ttactgccag
tcctatgaca atagccttag tggtttggtg 300ttcggcggag ggaccaagct gaccgtccta
33050110PRTHomo sapiens 50Gln Ser Val
Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5
10 15Lys Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Asn Tyr 20 25
30Tyr Val Ser Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45Ile Tyr Asp Asn Ala Lys Arg
Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Arg65
70 75 80Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn Ser Leu 85
90 95Ser Gly Leu Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105
11051330DNAHomo sapiens 51cagtctgtgt tgacgcagcc accctcagcg tctgggaccc
ccgggcagag ggtcaccatc 60tcttgttctg gaaccagctc caacatcgga agtaagtatg
tatactggta ccagcggctc 120ccaggaacgg cccccaaact cctcatctat actaatgatc
agcggccctc aggggtccct 180gcccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcac tgggctccag 240gctgaggatg aggctgatta ttactgccag tcctatgaca
gcagcctgcg tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
33052110PRTHomo sapiens 52Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn
Ile Gly Ser Lys 20 25 30Tyr
Val Tyr Trp Tyr Gln Arg Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Thr Asn Asp Gln Arg Pro Ser
Gly Val Pro Ala Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65
70 75 80Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85
90 95Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105
11053330DNAHomo sapiens 53cagtctgtgt tgacgcagcc accctcagcg tctgggaccc
ccgggcagag ggtcaccatc 60tcttgttctg gaaccagctc caacatcgga agtttctatg
tatactggta ccagcggctc 120ccaggaacgg cccccaaact cctcatctat actaatgatc
agcggccctc aggggtccct 180gcccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcac tgggctccag 240gctgaggatg aggctgatta ttactgccag tcctatgaca
gcagcctgcg tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
33054110PRTHomo sapiens 54Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn
Ile Gly Ser Phe 20 25 30Tyr
Val Tyr Trp Tyr Gln Arg Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Thr Asn Asp Gln Arg Pro Ser
Gly Val Pro Ala Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65
70 75 80Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85
90 95Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105
11055330DNAHomo sapiens 55cagtctgtgt tgacgcagcc accctcagcg tctgggaccc
ccgggcagag ggtcaccatc 60tcttgttctg gaaccagctc caacatcgga agtttctatg
tatactggta ccagcagctc 120ccaggaacgg cccccaaact cctcatctat actaatgatc
agcggccctc aggggtccct 180gcccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcac tgggctccag 240gctgaggatg aggctgatta ttactgccag tcctatgaca
gcagcctgcg tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
33056110PRTHomo sapiens 56Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn
Ile Gly Ser Phe 20 25 30Tyr
Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Thr Asn Asp Gln Arg Pro Ser
Gly Val Pro Ala Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65
70 75 80Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85
90 95Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105
11057330DNAHomo sapiens 57cagtctgtgt tgacgcagcc accctcagcg tctgggaccc
ccgggcagag ggtcaccatc 60tcttgttctg gaaccagctc caacatcgga agttactatg
tatactggta ccagcagctc 120ccaggaacgg cccccaaact cctcatctat actaatgatc
agcggccctc aggggtccct 180gcccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcac tgggctccag 240gctgaggatg aggctgatta ttactgccag tcctatgaca
gcagcctgcg tgctgtggtt 300ttcggcggag ggaccaagct gaccgtccta
33058110PRTHomo sapiens 58Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5
10 15Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn
Ile Gly Ser Tyr 20 25 30Tyr
Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Thr Asn Asp Gln Arg Pro Ser
Gly Val Pro Ala Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln65
70 75 80Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85
90 95Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105
1105910PRTHomo sapiens 59Gly Phe Thr Phe Asp Asp Tyr Ala Met His1
5 106017PRTHomo sapiens 60Gly Ile Ser Trp Asn Ser
Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys1 5
10 15Gly6111PRTHomo sapiens 61Ala Lys Thr Ser Tyr Gly
Gly Ala Phe Asp Ile1 5 106213PRTHomo
sapiens 62Ser Gly Asn Thr Ser Asn Ile Gly Ser Tyr Tyr Ala Tyr1
5 10637PRTHomo sapiens 63Asp Asn Asn Gln Arg Pro
Ser1 56411PRTHomo sapiens 64Ala Thr Trp Asp Asp Ser Leu Asn
Gly Pro Val1 5 10659PRTHomo sapiens 65Gly
Tyr Thr Thr Gly Tyr Tyr Ile His1 56610PRTHomo sapiens 66Arg
Ile Asn Pro Asn Ser Gly Gly Thr Asn1 5
106710PRTHomo sapiens 67Ala Arg Glu Gly Arg Gly Gly Met Asp Val1
5 106814PRTHomo sapiens 68Arg Ser Ser Gln Ser Leu
Leu His Ser Asn Gly Tyr Asn Tyr1 5
10697PRTHomo sapiens 69Leu Gly Ser Asn Arg Ala Ser1
5709PRTHomo sapiens 70Met Gln Gly Leu Gln Pro Pro Ile Thr1
57110PRTHomo sapiens 71Gly Phe Thr Phe Ser Ser Tyr Ala Met His1
5 107217PRTHomo sapiens 72Val Ile Ser Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5
10 15Gly737PRTHomo sapiens 73Ala Ser Gly Ala Phe Asp
Ile1 57416PRTHomo sapiens 74Arg Ser Ser His Ser Leu Val Tyr
Ser Asp Gly Asn Thr Tyr Leu Ser1 5 10
15757PRTHomo sapiens 75Lys Val Ser Asn Arg Asp Phe1
5769PRTHomo sapiens 76Met Gln Gly Thr His Trp Pro Gly Thr1
57712PRTHomo sapiens 77Gly Asp Ser Val Ser Ser Asn Ser Val Ala Trp
Asn1 5 107818PRTHomo sapiens 78Arg Thr
Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val1 5
10 15Lys Ser7913PRTHomo sapiens 79Ala
Arg Ala Asp Gly Ser Arg Gly Gly Gly Tyr Asp Gln1 5
108017PRTHomo sapiens 80Lys Ser Ser Gln Ser Ile Leu Tyr Arg Ser
Asn Asn Lys Asn Tyr Leu1 5 10
15Ala817PRTHomo sapiens 81Trp Ala Ser Thr Arg Glu Ser1
5829PRTHomo sapiens 82Gln Gln Tyr Tyr Thr Thr Pro Gln Thr1
58310PRTHomo sapiens 83Gly Phe Thr Phe Ser Ser Tyr Ala Met His1
5 108417PRTHomo sapiens 84Val Ile Ser Tyr Asp Gly
Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys1 5
10 15Gly859PRTHomo sapiens 85Ala Arg Ser Thr Tyr Gly
Met Asp Val1 58616PRTHomo sapiens 86Arg Ser Ser Gln Ser Leu
Val His Ser Asp Gly Asn Thr Tyr Leu Asn1 5
10 15877PRTHomo sapiens 87Lys Val Ser Asn Arg Asp Ser1
5888PRTHomo sapiens 88Met Gln Gly Thr His Trp Trp Thr1
58912PRTHomo sapiens 89Gly Asp Ser Val Ser Ser Lys Ser Ala Ala
Trp Asn1 5 109015PRTHomo sapiens 90Arg
Thr Tyr Tyr Arg Ser Lys Trp His Asn Asp Tyr Ala Val Ser1 5
10 159110PRTHomo sapiens 91Ala Arg Gly
Gln Met Gly Ala Leu Asp Val1 5
109215PRTHomo sapiens 92Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr Tyr Val
Tyr Trp Tyr1 5 10
15937PRTHomo sapiens 93Gly Asn Asn Gln Arg Pro Ser1
59411PRTHomo sapiens 94Gln Ser Tyr Asp Ser Ser Leu Ser Gly Val Ile1
5 109510PRTHomo sapiens 95Gly Phe Thr Phe Ser
Ser Tyr Ala Met His1 5 109617PRTHomo
sapiens 96Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
Lys1 5 10
15Gly9712PRTHomo sapiens 97Ala Arg Leu Val Ala Gly Arg Ser Ala Phe Asp
Ile1 5 109811PRTHomo sapiens 98Arg Ala
Ser Gln Ser Val Ser Ser Asn Leu Ala1 5
10997PRTHomo sapiens 99Gly Ala Ser Thr Arg Ala Thr1
510010PRTHomo sapiens 100Gln Gln Tyr Asn Asn Trp Pro Pro Ile Thr1
5 1010112PRTHomo sapiens 101Gly Asp Ser Val Ser
Ser Lys Ser Val Thr Trp Asn1 5
1010215PRTHomo sapiens 102Arg Thr Tyr Tyr Arg Ser Lys Trp Phe Asn Asp Tyr
Ala Val Ser1 5 10
1510310PRTHomo sapiens 103Ala Arg Ala Lys Met Gly Gly Met Asp Val1
5 1010412PRTHomo sapiens 104Gly Asp Ser Val Ser
Ser Asn Ser Ala Ala Trp Asn1 5
1010515PRTHomo sapiens 105Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala Val Ser1 5 10
1510610PRTHomo sapiens 106Thr Arg Gln Ser Trp His Gly Met Glu Val1
5 1010712PRTHomo sapiens 107Gly Asp Ser Val Ser
Ser Asn Ser Ala Ala Trp Asn1 5
1010815PRTHomo sapiens 108Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala Val Ser1 5 10
1510910PRTHomo sapiens 109Ala Arg Ser Ile Ala Thr Gly Thr Asp Tyr1
5 1011012PRTHomo sapiens 110Gly Asp Ser Val Ser
Ser Ser Arg Ala Thr Trp Asn1 5
1011115PRTHomo sapiens 111Arg Thr Tyr Tyr Arg Ser Lys Trp Phe Asn Asp Tyr
Ala Val Ser1 5 10
1511210PRTHomo sapiens 112Ala Arg Ala Lys Met Gly Gly Met Asp Val1
5 1011312PRTHomo sapiens 113Gly Asp Ser Val Ser
Ser Asn Ser Ala Ala Trp Asn1 5
1011415PRTHomo sapiens 114Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala Val Ser1 5 10
1511510PRTHomo sapiens 115Ala Arg Gly Thr Arg Trp Gly Met Asp Val1
5 1011612PRTHomo sapiens 116Gly Asp Ser Val Ser
Ser Asn Ser Ala Ala Trp Asn1 5
1011715PRTHomo sapiens 117Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala Val Ser1 5 10
1511810PRTHomo sapiens 118Ala Arg Ala Lys Val Tyr Gly Val Asp Val1
5 1011912PRTHomo sapiens 119Gly Asp Ser Val Ser
Ser Lys Ser Ala Thr Trp Asn1 5
1012015PRTHomo sapiens 120Arg Thr Tyr Tyr Arg Ser Arg Trp Phe Asn Asp Tyr
Ala Val Ser1 5 10
1512110PRTHomo sapiens 121Ala Arg Gly Asn Met Gly Ala Met Asp Val1
5 1012212PRTHomo sapiens 122Gly Asp Arg Val Ser
Ser Asn Arg Ala Ala Trp Asn1 5
1012315PRTHomo sapiens 123Arg Thr Tyr Tyr Arg Ser Gln Trp Tyr Asn Asp Tyr
Ala Val Ser1 5 10
151249PRTHomo sapiens 124Ala Arg Gly Thr Ala Met Gly Asp Ala1
512512PRTHomo sapiens 125Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp
Asn1 5 1012615PRTHomo sapiens 126Arg Thr
Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser1 5
10 1512710PRTHomo sapiens 127Ala Arg Gln
Ala Ser Asn Gly Phe Asp Ile1 5
1012812PRTHomo sapiens 128Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp
Asn1 5 1012915PRTHomo sapiens 129Arg Thr
Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser1 5
10 1513010PRTHomo sapiens 130Ala Arg Gln
Gly Thr Thr Gly Phe Asp Tyr1 5
1013115PRTHomo sapiens 131Ser Gly Ser Ser Ser Asn Ile Gly Asn Tyr Tyr Val
Ser Trp Tyr1 5 10
151327PRTHomo sapiens 132Asp Asn Ala Lys Arg Pro Ser1
513311PRTHomo sapiens 133Gln Ser Tyr Asp Asn Ser Leu Ser Gly Leu Val1
5 1013415PRTHomo sapiens 134Ser Gly Thr Ser
Ser Asn Ile Gly Ser Lys Tyr Val Tyr Trp Tyr1 5
10 151357PRTHomo sapiens 135Thr Asn Asp Gln Arg Pro
Ser1 513611PRTHomo sapiens 136Gln Ser Tyr Asp Ser Ser Leu
Arg Ala Val Val1 5 1013715PRTHomo sapiens
137Ser Gly Thr Ser Ser Asn Ile Gly Ser Phe Tyr Val Tyr Trp Tyr1
5 10 151387PRTHomo sapiens 138Thr
Asn Asp Gln Arg Pro Ser1 513911PRTHomo sapiens 139Gln Ser
Tyr Asp Ser Ser Leu Arg Ala Val Val1 5
1014015PRTHomo sapiens 140Ser Gly Thr Ser Ser Asn Ile Gly Ser Phe Tyr Val
Tyr Trp Tyr1 5 10
151417PRTHomo sapiens 141Thr Asn Asp Gln Arg Pro Ser1
514211PRTHomo sapiens 142Gln Ser Tyr Asp Ser Ser Leu Arg Ala Val Val1
5 1014315PRTHomo sapiens 143Ser Gly Thr Ser
Ser Asn Ile Gly Ser Tyr Tyr Val Tyr Trp Tyr1 5
10 151447PRTHomo sapiens 144Thr Asn Asp Gln Arg Pro
Ser1 514511PRTHomo sapiens 145Gln Ser Tyr Asp Ser Ser Leu
Arg Ala Val Val1 5 1014647PRTHepatitis B
virus 146Gly Thr Asn Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His1
5 10 15Gln Leu Asp Pro
Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp Asp 20
25 30Phe Asn Pro Asn Lys Asp His Trp Pro Glu Ala
Asn Gln Val Gly 35 40
4514757PRTHepatitis B virus 147Gly Gly Trp Ser Ser Lys Pro Arg Gln Gly
Met Gly Thr Asn Leu Ser1 5 10
15Val Pro Asn Pro Leu Gly Phe Phe Pro Asp His Gln Leu Asp Pro Ala
20 25 30Phe Gly Ala Asn Ser Asn
Asn Pro Asp Trp Asp Phe Asn Pro Asn Lys 35 40
45Asp His Trp Pro Glu Ala Asn Gln Val 50
5514860PRTHomo sapiens 148Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr
Ile Asn Pro Asp1 5 10
15Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu
20 25 30Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Gly Lys Met Gly Gly Met Asp 35 40
45Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 50
55 60
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