Patent application title: METALLOPROTEINASE-BINDING PROTEINS
Inventors:
Daniel T. Dransfield (Hanson, MA, US)
Kristin Rookey (Revere, MA, US)
Robert C. Ladner (Ijamsville, MD, US)
Assignees:
DYAX CORP.
IPC8 Class: AA61K39395FI
USPC Class:
424 934
Class name: In vivo diagnosis or in vivo testing magnetic imaging agent (e.g., nmr, mri, mrs, etc.) polypeptide attached to or complexed with the agent (e.g., protein, antibody, etc.)
Publication date: 2010-11-18
Patent application number: 20100291001
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Patent application title: METALLOPROTEINASE-BINDING PROTEINS
Inventors:
DANIEL T. DRANSFIELD
KRISTIN ROOKEY
ROBERT C. LADNER
Agents:
LANDO & ANASTASI, LLP
Assignees:
Origin: CAMBRIDGE, MA US
IPC8 Class: AA61K39395FI
USPC Class:
Publication date: 11/18/2010
Patent application number: 20100291001
Abstract:
Disclosed are antibodies that interact with a matrix metalloproteases such
as MMP-26. Exemplary antibodies inhibit MMP-26 activity. These antibodies
can be used, e.g., to treat or prevent metastatic disorders,
hyperproliferative disorders, disorders which are characterized by
excessive extracellular matrix degradation, and inflammatory disorders.Claims:
1. A protein comprising a heavy chain immunoglobulin variable domain
sequence and a light chain immunoglobulin variable domain sequence,
wherein the protein binds to MMP-26 with a KD of less than
5.times.10.sup.-7 M and comprises at least one human complementarity
determining region or framework region.
2. The protein of claim 1 wherein the protein inhibits MMP-26 proteolytic activity.
3. The protein of claim 2 wherein the heavy chain variable domain sequence comprises(a) a CDR1 that comprises TABLE-US-00100 X-Y-X-M-M or (A/S/M/Y/W/F/E/Q)-Y-(A/W/F/N/Q)-M-(A/S/M/W/F;
(b) a CDR2 that comprisesR-I-X-(S/P)-S-G-G-X-T-X-Y-A-D-S-V-K-G or(G/S/V/W/R)-I-(G/S/V/Y)-(S/P)-S-G-G-(S/I/F/K/D/H)-T-(L/M/K/D/P)-Y-A-D-S- -V-K-G; and/or(c) a CDR3 that comprises F-D-I.
4. The protein of claim 2 wherein the heavy chain variable domain sequence comprises a CDR1 that comprises a sequence of which at least 3 of 5 amino acids are identical to a reference sequence from column 1, a CDR2 that comprises a sequence of which at least 13 of 16 amino acids are identical to a reference sequence from column 2, and a CDR3 of which at least 70% of the amino acids are identical to a reference sequence from column 3 of Table 7.
5. The protein of claim 4 wherein the heavy chain variable domain sequence comprises a CDR1 that comprises a sequence of which at least 3 of 5 amino acids are identical to a reference sequence from column 1 in a particular row, a CDR2 that comprises a sequence of which at least 13 of 16 amino acids are identical to a reference sequence from column 2 in the particular row, and a CDR3 of which at least 70% of the amino acids are identical to a reference sequence from column 3 in the particular row, the columns being from Table 7.
6. The protein of claim 4 wherein at least 30, 50, 60, 70, 80, 90 or 100% of the CDR amino acid residues that are not identical to residues in the reference sequences from Table 7, Table 8, Table 9, or Table 10 are identical to residues at corresponding positions in a human germline sequence.
7. The protein of claim 2 wherein the heavy chain variable domain sequence comprises a CDR1 that comprises a sequence from column 1, a CDR2 that comprises a sequence from column 2, and a CDR3 from that comprises a sequence from column 3 of Table 7, Table 8, Table 9, or Table 10.
8. The protein of claim 2 wherein the light chain variable domain sequence is a κ light chain and comprises(a) a CDR1 that comprises TABLE-US-00101 R-(A/T)-S-Q-(G/S/I)-(I/V)-(S/D/N)-(S/T/R)-Y-L- (A/N)-X, R-(A/T)-S-Q-(G/S/I/N)-(I/V)-(G/S/R/D/N)- (S/T/R/K/D/N)-(S/T/Y/W)-(L/V/Y)-(A/L/N)-A, R-A-S-Q-(G/S)-I-(S/D)-(S/T)-Y-L-(A/N)-X, R-A-S-Q-X-I-X-X-Y-L-N-X, or R-A-S-Q-(G/S/I)-(I/V)-(G/S/R/D)-(S/T/R/K/D/N)- (S/T/Y/W)-(L/V/Y)-(A/L/N)-A;
(b) a CDR2 that comprises TABLE-US-00102 (A/G)-A-S-(S/T/I/K)-L-(E/Q)-(G/S/D), (A/G/T/Q)-(A/T)-(S/T/F)-(S/T/I/K)-(L/V/R)-(A/E/Q)- (G/S/T/D/N), or A-A-S-X-L-(E/D/N/Q); and/or
(c) a CDR3 that comprises TABLE-US-00103 Q-Q-(S/T/Y)-(Y/N)-S-(S/T)-P-(G/L/P)-(T/I)-T, or Q-(R/E/Q)-(A/S/T/Y)-(G/Y/N)-(S/T/I/D)-(S/T/I/Y/F/ P)-(S/P)-(G/L/Y/F/R/P)-(T/I/F/E)-(T/V)-T.
9. The protein of claim 2 wherein the light chain variable domain sequence is a λ light chain and comprises(a) a CDR1 that comprises TABLE-US-00104 S-G-X-S-S-X-X-G-S, or T-G-T-(S/N)-S-D-(I/V)-G-(A/G)-Y-N-Y-V-S;
(b) a CDR2 that comprises TABLE-US-00105 (R/D/N/E)-(V/D/N)-(G/S/T/D/N)-(K/D/N/E/Q)-R-P-S, or (R/E)-(V/D/N)-(T/D/N)-(K/Q)-R-P-S; and/or
(c) a CDR3 that comprises TABLE-US-00106 (A/Q)-(S/T/V)-(Y/W)-(A/D)-(G/S/D)-(S/N)-(L/V/N)- (S/N)-(G/L)-P-V, or W-D-X-S-X-X-X-X-V.
10. The protein of claim 2 wherein the light chain variable domain sequence comprises a CDR1 that comprises a sequence of which at least 9 of 11 amino acids are identical to a reference sequence from column 1, a CDR2 that comprises a sequence of which at least 7 of 9 amino acids are identical to a reference sequence from column 2, and a CDR3 of which at least 70% of the amino acids are identical to a reference sequence from column 3 of Table 8.
11. The protein of claim 10 wherein the light chain variable domain sequence comprises a CDR1 that comprises a sequence of which at least 9 of 11 amino acids are identical to a reference sequence from column 1 in a particular row, a CDR2 that comprises a sequence of which at least 7 of 9 amino acids are identical to a reference sequence from column 2 in the particular row, and a CDR3 of which at least 70% of the amino acids are identical to a reference sequence from column 3 in the particular row, the columns being from Table 8.
12. The protein of claim 1 wherein the framework regions of the heavy and/or light chain variable domain are at least 70, 80, 90, 92, 95, 97, 98, or 99% identical to a corresponding framework region of a human germline sequence.
13. The protein of claim 1 wherein the heavy and/or light chain variable domain are at least 70% identical to a human germline sequence.
14. The protein of claim 1 wherein the protein inhibits MMP-26 with a Ki that is at least 20 better than its Ki for another metalloproteinases.
15. The protein of claim 1 wherein the protein reduces cell metastasis in vivo.
16. The protein of claim 1 wherein the protein comprises two independent polypeptide chains, a first chain comprising the light chain variable domain sequence and the second chain comprising the heavy chain variable domain sequence, and each chain comprising a constant immunoglobulin domain.
17. The protein of claim 1 wherein the protein comprises CL, CH1, CH2, and CH3 domains.
18. A protein comprising a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to the MMP-26; and(a) at least one of the variable domains is related to a reference antibody selected from the group consisting of a01, b04, b06, b10, c01, c08, d02, d04, d06, d08, D6-orig, a04, a11, c05, c04, c11, c12, d07, A1-orig, H6-orig, a02, a03, a05, a06, a07, a08, a09, a10, a12, b01, b02, b03, b05, b07, b08, b09, b11, b12, c02, c03, c06, c07, c09, c10, d01, d03, d05, and d09,the relationship being such that at least 80% of the amino acid residues in the variable domain are either (i) identical to a corresponding residue in the reference antibody, (ii) identical to a corresponding residue in a human germline sequence, or both; or(b) at least one of the variable domains is at least 85% identical to the corresponding variable domain of a reference antibody selected from the group consisting of a01, b04, b06, b10, c01, c08, d02, d04, d06, d08, D6-orig, a04, a11, c05, c04, c11, c12, d07, A1-orig, H6-orig, a02, a03, a05, a06, a07, a08, a09, a10, a12, b01, b02, b03, b05, b07, b08, b09, b11, b12, c02, c03, c06, c07, c09, c10, d01, d03, d05, and d09.
19. The protein of claim 18 wherein the human germline sequence is the human germline sequence with which the reference antibody is associated in an example described herein.
20. The protein of claim 18 wherein all of the amino acids residues in the variable domain are (i) identical to a corresponding residue in the reference antibody, or (ii) identical to a corresponding residue in the human germline sequence, e.g., with which the reference antibody is associated in an example described herein, or both.
21. The protein of claim 18 wherein at least 1, 2, 3, 4, or 5 of the amino acid residues in the variable domain differ from a corresponding residue in the reference antibody, but are identical to a corresponding residue in the human germline sequence.
22. The protein of claim 18 wherein, in the framework regions, at least 90% or all of the amino acid residues are identical to a corresponding residue in the human germline sequence.
23. The protein of claim 18 wherein at least 1, 2, or 3 of the amino acid residues in the CDR regions of the variable domain differ from a corresponding residue in the reference antibody, but are identical to a corresponding residue in the human germline sequence.
24. The protein of claim 18 wherein amino acid residues that are not identical are conserved substitutions relative to a corresponding residue in the reference antibody, or a human germline sequence.
25. A protein that comprises an antigen binding fragment that binds to MMP-26, wherein the protein binds to a MMP-26 epitope that overlaps with an epitope bound by an antibody selected from the group consisting of a01, b04, b06, b10, c01, c08, d02, d04, d06, d08, D6-orig, a04, a11, c05, c04, c11, c12, d07, A1-orig, H6-orig, a02, a03, a05, a06, a07, a08, a09, a10, a12, b01, b02, b03, b05, b07, b08, b09, b11, b12, c02, c03, c06, c07, c09, c10, d01, d03, d05, and d09 or the protein competes with an antibody selected from the group consisting of a01, b04, b06, b10, c01, c08, d02, d04, d06, d08, D6-orig, a04, a11, c05, c04, c11, c12, d07, A1-orig, H6-orig, a02, a03, a05, a06, a07, a08, a09, a10, a12, b01, b02, b03, b05, b07, b08, b09, b11, b12, c02, c03, c06, c07, c09, c10, d01, d03, d05, and d09, for binding to MMP-26-26.
26. An isolated nucleic acid comprising a coding sequence that encodes a polypeptide comprising a variable domain sequence of the protein of claim 1.
27. The nucleic acid of claim 26 that further comprises a second coding sequence that encodes a polypeptide comprising an immunoglobulin light chain variable domain.
28. The nucleic acid of claim 26 that further comprises a second coding sequence that encodes a polypeptide comprising an immunoglobulin heavy chain variable domain.
29. A host cell that produces the protein of claim 1, the cell comprising a first nucleic acid encoding a polypeptide comprising a heavy chain variable domain sequence of the protein and a second nucleic acid encoding a polypeptide comprising a light chain domain sequence of the protein.
30. A host cell that contains a first nucleic acid encoding a polypeptide comprising a heavy chain variable region and a second nucleic acid encoding a polypeptide comprising a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence at least 85% identical to an amino acid sequence of a heavy chain immunoglobulin variable domain sequence described herein, and the light chain variable region comprises an amino acid sequence at least 85% identical to a light chain immunoglobulin variable domain sequence described herein.
31. A method of providing a MMP-26-binding antibody, the method comprising:providing the host cell of claim 29; andexpressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen binding protein that interacts with MMP-26.
32. A method of treating or preventing a neoplastic disorder, the method comprising:administering the protein of claim 1 to a subject in an amount effective to treat or prevent a neoplastic disorder in the subject.
33. The method of claim 32 wherein the subject has, is predisposed to, or is diagnosed with a malignant cancer or metastatic disorder.
34. The method of claim 32 wherein the neoplastic disorder is associated with breast, prostate, or lung cancer.
35. A method of treating or preventing an inflammatory disorder, the method comprising:administering the protein of claim 1 to a subject in an amount effective to treat or prevent an inflammatory disorder in the subject.
36. The method of claim 35 wherein the inflammatory disorder is selected from the group consisting of: rheumatoid arthritis, lupus, restenosis, graft v. host response, or multiple sclerosis.
37. A method of treating or preventing a disorder characterized by excessive or undesired MMP-26 activity, the method comprising:administering the protein of claim 1 to a subject in an amount effective to treat or prevent a disorder characterized by excessive or undesired MMP-26 activity in the subject.
38. The method of claim 37 wherein the disorder is periodontitis, rheumatoid arthritis, or osteoarthritis.
39. A method of modulating MMP-26 activity, the method comprising:providing an MMP-26-binding protein of claim 1; andcontacting the protein to MMP-26, in vitro or in vivo, in an amount sufficient to modulate MMP-26 activity.
40. The method of claim 39 wherein the protein is contacted to MMP-26 in the vicinity of a neoplastic cell.
41. A method for detecting the presence of a MMP-26 protein, in a sample, in vitro, the method comprising:(i) contacting the sample with an MMP-26-binding protein according to claim 1, under conditions that allow interaction of the MMP-26-binding protein and the MMP-26 protein to occur; and(ii) detecting interaction between the MMP-26-binding protein, and the sample.
42. The method of claim 41 wherein at least one of the MMP-26 binding protein or the MMP-26 is immobilized.
43. A method for detecting the presence of MMP-26 in vivo, the method comprising:(i) administering to a subject an MMP-26-binding protein, under conditions that allow interaction of the MMP-26-binding protein and the MMP-26 protein to occur; and(ii) detecting location of the MMP-26-binding protein in the subject or formation of a complex between the MMP-26-binding protein and MMP-26 in the subject.
44. The method of claim 43 wherein the detecting comprises imaging the subject.
45. The method of claim 43 wherein the MMP-26-binding protein is labeled with an MRI detectable label.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of U.S. patent application Ser. No. 10/993,543, filed on Nov. 19, 2004, which claims priority to U.S. Application Ser. No. 60/523,745, filed on Nov. 19, 2003, the contents of which are hereby incorporated by reference in their entireties.
BACKGROUND
[0002]Matrix metalloproteinases (MMPs) are endopeptidases which, in their typical homeostatic role, digest unneeded matrix proteins including tissue components such as collagens, fibronectin, and proteoglycans. Matrix metalloproteinase-26 (MMP-26) is one member of this class of proteins. In certain cancers, these proteins are overexpressed, thus causing increased hydrolytic activity and digestion of extracellular matrix components. This process facilitates the metastatic spread of these cells.
SUMMARY
[0003]In one aspect, the invention features an antibody that interacts with a matrix metalloproteases such as MMP-26, e.g., human MMP-26. The antibody can include one or more human regions, e.g., one or more human complementarity determining regions (CDRs), one or more human frameworks (e.g., germline or somatically-mutated human FR), or one or more human constant regions, or effectively human regions of the same. In one embodiment, the antibody inhibits MMP-26 activity.
[0004]In certain implementations, the antibody is used to prevent metastatic spread of certain cancers by inhibiting MMP-26-induced cleavage of extracellular matrix proteins. In certain implementations, the antibody also decreases the hyperproliferative properties of a neoplastic cell by modulating the availability of a MMP-26 cleavage product, e.g., a growth factor, e.g., an Insulin-like Growth Factor (IGF) or an alpha-1-anti-trypsin (α1AT). Other disorders which are characterized by excessive extracellular matrix degradation include periodontitis, rheumatoid arthritis, and osteoarthritis. In certain implementations, the antibody also modulates (e.g., decreases) inflammation, and accordingly can be used to treat an inflammatory disorder.
[0005]In one aspect, the invention features an antibody that interacts with a matrix metalloproteases such as MMP-26, e.g., human MMP-26. The antibody can include one or more human regions, e.g., one or more human CDRs, one or more human frameworks (e.g., germline or somatically mutated human FR), or one or more human constant regions, or effectively human regions of the same. In one embodiment, the antibody inhibits MMP-26 activity.
[0006]In certain implementations, the antibody is used to prevent metastatic spread of certain cancers by inhibiting MMP-26-induced cleavage of extracellular matrix proteins. In certain implementations, the antibody also decreases the hyperproliferative properties of a neoplastic cell by modulating the availability of a MMP-26 cleavage product, e.g., a growth factor, e.g., an IGF or an α1AT. Other disorders which are characterized by excessive extracellular matrix degradation include periodontitis, rheumatoid arthritis, and osteoarthritis. In certain implementations, the antibody also modulates (e.g., decreases) inflammation, and accordingly can be used to treat an inflammatory disorder.
[0007]In another aspect, the invention features a protein including a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence. The protein binds to MMP-26. For example, the protein binds to MMP-26 with a KD of less than 1×10-7 M, 3×10-8, 1×10-8, 3×10-9, 1×10-9, 3+10-10, 1×10-10, 3×10-11, 1×10-11, or 1×10-12 M. In one embodiment, the protein includes at least one human CDR or at least one human framework.
[0008]In one embodiment, the protein inhibits MMP-26 proteolytic activity.
[0009]In one embodiment, the heavy chain variable domain sequence includes
[0010](a) a CDR1 that includes [0011]X-Y-X-M-M (SEQ ID NO:236)("X" as defined herein refers to any amino acid, for example, any of all twenty natural amino acids or any of the nineteen non-cysteine residues), [0012](A/S/M/Y/W/F/E/Q)-Y-(A/W/F/N/Q)-M-(A/S/M/W/F) (SEQ ID NO:237)(as used herein, amino acids in parentheses refer to amino acids that are used in the alternative at a particular position);
[0013](b) a CDR2 that includes [0014]R-I-X-(S/P)-S--G-G-X-T-X-Y-A-D-S-V-K-G, (SEQ ID NO:185), or (G/S/V/W/R)-I-(G/S/V/Y)-(S/P)-S-G-G-(S/I/F/K/D/H)-T-(L/M/K/D/P)-Y-A-D-SV-- K-G, (SEQ ID NO:186); and/or
[0015](c) a CDR3 that includes F-D-I.
[0016]In one embodiment, the heavy chain variable domain sequence includes a CDR1 that includes a sequence of which at least 3 of 5 amino acids are identical to a reference sequence from column 1, a CDR2 that includes a sequence of which at least 13 of 16 amino acids are identical to a reference sequence from column 2, and a CDR3 of which at least 70, 80, 85, 87, 90, 92, 94, 96, 97, or 98% of the amino acids are identical to a reference sequence from column 3 of Table 7.
[0017]In one embodiment, the heavy chain variable domain sequence includes a CDR1 that includes a sequence of which at least 3 of 5 amino acids are identical to a reference sequence from column 1 in a particular row (or two or three particular rows), a CDR2 that includes a sequence of which at least 13 of 16 amino acids are identical to a reference sequence from column 2 in the particular row (or two or three particular rows), and a CDR3 of which at least 70, 80, 85, 87, 90, 92, 94, 96, 97, or 98% of the amino acids are identical to a reference sequence from column 3 in the particular row (or two or three particular rows), the columns being from Table 7.
[0018]In one embodiment, at least 30, 50, 60, 70, 80, 90 or 100% of the CDR amino acid residues that are not identical to residues in the reference sequences from Table 7 are identical to residues at corresponding positions in a human germline sequence (e.g., a human germline sequence described herein, e.g., the human germline sequence that is associated with the respective CDR in an example described herein).
[0019]In one embodiment, the heavy chain variable domain sequence includes a CDR1 that includes a sequence from column 1, a CDR2 that includes a sequence from column 2, and a CDR3 from that includes a sequence from column 3 of Table 7.
[0020]In one embodiment, the light chain variable domain sequence is a κ light chain and includes
[0021](a) a CDR1 that includes
TABLE-US-00001 (SEQ ID NO: 187) R-(AT)-S-Q-(GSI)-(IV)-(SDN)-(STR)-Y-L-(AN)-X,; (SEQ ID NO: 188) R-(AT)-S-Q-(GSIN)-(IV)-(GSRDN)-(STRKDN)-(STYW)- (LVY)-(ALN)-A,; (SEQ ID NO: 189) R-A-S-Q-(GS)-I-(SD)-(ST)-Y-L-(AN)-X,; (SEQ ID NO: 190) R-A-S-Q-X-I-X-X-Y-L-N-X,, or (SEQ ID NO: 191) R-A-S-Q-(GSI)-(IV)-(GSRD)-(STRKDN)-(STYW)-(LVY)- (ALN)-A,;
[0022](b) a CDR2 that includes
TABLE-US-00002 (SEQ ID NO: 238) (AG)-A-S-(STIK)-L-(EQ)-(GSD),, (SEQ ID NO: 239) (AGTQ)-(AT)-(STF)-(STIK)-(LVR)-(AEQ)-(GSTDN), or (SEQ ID NO: 192) A-A-S-X-L-(EDNQ),; and/or
[0023](c) a CDR3 that includes
TABLE-US-00003 (SEQ ID NO: 193) Q-Q-(STY)-(YN)-S-(ST)-P-(GLP)-(TI)-T,; or (SEQ ID NO: 194) Q-(REQ)-(ASTY)-(GYN)-(STID)-(STIYFP)-(SP)- (GLYFRP)-(TIFE)-(TV)-T,.
[0024]In one embodiment, the light chain variable domain sequence is a X light chain and includes
[0025](a) a CDR1 that includes
TABLE-US-00004 (SEQ ID NO: 195) S-G-X-S-S-X-X-G-S,; or (SEQ ID NO: 196) T-G-T-(SN)-S-D-(IV)-G-(AG)-Y-N-Y-V-S,;
[0026](b) a CDR2 that includes
TABLE-US-00005 (SEQ ID NO: 240) (RDNE)-(VDN)-(GSTDN)-(KDNEQ)-R-P-S,, or (SEQ ID NO: 241) (RE)-(VDN)-(TDN)-(KQ)-R-P-S,;
andor
[0027](c) a CDR3 that includes
TABLE-US-00006 (SEQ ID NO: 242) (AQ)-(STV)-(YW)-(AD)-(GSD)-(SN)-(LVN)-(SN)-(GL)- P-V,, or (SEQ ID NO: 197) W-D-X-S-X-X-X-X-V,.
[0028]In one embodiment, the light chain variable domain sequence includes a CDR1 that includes a sequence of which at least 9 of 11 amino acids are identical to a reference sequence from column 1, a CDR2 that includes a sequence of which at least 7 of 9 amino acids are identical to a reference sequence from column 2, and a CDR3 of which at least 70, 80, 85, 87, 90, 92, 94, 96, 97, or 98% of the amino acids are identical to a reference sequence from column 3 of Table 8.
[0029]In one embodiment, the light chain variable domain sequence includes a CDR1 that includes a sequence of which at least 9 of 11 amino acids are identical to a reference sequence from column 1 in a particular row (or two or three particular rows), a CDR2 that includes a sequence of which at least 7 of 9 amino acids are identical to a reference sequence from column 2 in the particular row (or two or three particular rows), and a CDR3 of which at least 70, 80, 85, 87, 90, 92, 94, 96, 97, or 98% of the amino acids are identical to a reference sequence from column 3 in the particular row (or two or three particular rows), the columns being from Table 8.
[0030]In one embodiment, at least 30, 50, 60, 70, 80, 90 or 100% of the CDR amino acid residues that are not identical to residues in the reference sequences from Table 8 are identical to residues at corresponding positions in a human germline sequence (e.g., a human germline sequence described herein, e.g., the human germline sequence that is associated with the respective CDR in an example described herein).
[0031]In one embodiment, the light chain variable domain sequence includes a CDR1 that includes a sequence from column 1, a CDR2 that includes a sequence from column 2, and a CDR3 from that includes a sequence from column 3 of Table 8.
[0032]In one embodiment, CDR2 of the heavy chain variable domain sequence includes: serine at position 4, and lysine at position 10, and CDR3 of the heavy chain variable domain sequence includes: A-F-D-I (SEQ ID NO:253).
[0033]In one embodiment, CDR2 of the heavy chain variable domain sequence includes: R or S at position 1, and P at position 4, and CDR3 of the heavy chain variable domain sequence includes: F-D-Y.
[0034]In one embodiment, CDR3 of the heavy chain variable domain sequence further includes a dityrosine sequence.
[0035]In one embodiment, CDR1 of the light chain variable domain sequence includes S-G-S-S-S-N-I-G-S-X-Y-V, (SEQ ID NO:198), wherein X is any amino acid. In one embodiment, CDR2 of the light chain variable domain sequence includes R-N-X-Q-R-P-S, (SEQ ID NO:250) wherein X is any amino acid. In one embodiment, CDR3 includes W-T-D-D-S (SEQ ID NO:251).
[0036]In one embodiment, CDR2 of the heavy chain variable domain sequence includes: P at position 4 and M, F, or R at position 10, and CDR1 of the light chain variable domain sequence includes: R-(A/T)-S-Q-X-(V/I)-X-X-(Y/W)-(L/V), (SEQ ID NO:199).
[0037]In one embodiment, CDR3 of the light chain variable domain sequence includes Q-Q-X-(Y/N)-(S/T)-X-(P/S) (SEQ ID NO:254).
[0038]In one embodiment, CDR2 of the heavy chain variable domain sequence includes: Y at position 3 and F at position 10, CDR1 of the light chain variable domain sequence includes: T-G-T-(S/N)-S-D-(V/I)-G-G-Y-N-Y-V-S, (SEQ ID NO:200), and CDR2 of the light chain variable domain sequence includes: E-V-X-X-R-P-S, (SEQ ID NO:201).
[0039]The protein can also bind to MMP-26, e.g., and not substantially inhibit its enzymatic activity.
[0040]In one embodiment, the heavy chain variable domain sequence includes
[0041](a) a CDR1 that includes
TABLE-US-00007 P-Y-F-M-F,, (SEQ ID NO: 202) or (ALVEP)-Y-(SMWFD)-M-(YFKDNP),; (SEQ ID NO: 255)
and/or
[0042](b) a CDR2 that includes
TABLE-US-00008 (SEQ ID NO: 203) (SV)-I-Y-(SP)-S-G-G-X-T-X-Y-A-D-S-V-K-G,, or (SEQ ID NO: 204) (GSVY)-I-(GSVYW)-(SP)-S-G-G-(SIYFD)-T-(SLRNQ)-Y-A- D-S-V-K-G,.
[0043]In one embodiment, the heavy chain variable domain sequence includes a CDR1 that includes a sequence of which at least 3 of 5 amino acids are identical to a sequence from column 1, a CDR2 that includes a sequence of which at least 13 of 16 amino acids are identical to a sequence from column 2, and a CDR3 of which at least 70, 80, 85, 87, 90, 92, 94, 96, 97, or 98% of the amino acids are identical to a sequence from column 3 of Table 9.
[0044]In one embodiment, at least 30, 50, 60, 70, 80, 90 or 100% of the CDR amino acid residues that are not identical to residues in the reference sequences from Table 9 are identical to residues at corresponding positions in a human germline sequence (e.g., a human germline sequence described herein, e.g., the human germline sequence that is associated with the respective CDR in an example described herein).
[0045]In one embodiment, the heavy chain variable domain sequence includes a CDR1 that includes a sequence from column 1, a CDR2 that includes a sequence from column 2, and a CDR3 from that includes a sequence from column 3 of Table 9.
[0046]In one embodiment, the light chain variable domain sequence is a κ light chain and includes
[0047](a) a CDR1 that includes
TABLE-US-00009 (SEQ ID NO: 205) R-A-S-Q-S-(IV)-S-(SN)-(SY)-(LY)-(ALN)-A,; (SEQ ID NO: 206) R-A-S-Q-(GS)-(IV)-S-(STN)-(SY)-(LY)-(ALN)-A,; (SEQ ID NO: 207) R-A-S-Q-S-(IV)-S-S-Y-L,; or (SEQ ID NO: 208) R-A-S-Q,
[0048](b) a CDR2 that includes
TABLE-US-00010 (SEQ ID NO: 256) (AGDP)-(AN)-S-(STIKDN)-(LR)-(AEPQ)-(STRDN),; (SEQ ID NO: 257) (AGD)-A-S-(STN)-(LR)-(AEQ)-(ST),; (SEQ ID NO: 258) (AGD)-A-S-(STN)-(LR)-(AQ)-(ST),; (SEQ ID NO: 259) (AGD)-A-S-S-(LR)-(AQ)-(ST),; (SEQ ID NO: 244) D-(AD)-S-X-(LR)-(AP)-(ST),; (SEQ ID NO: 245) (AD)-(ADN)-S-(SKDNQ)-(LR)-(APQ)-(ST),; or (SEQ ID NO: 246) (AGRD)-(ADN)-(SYN)-(SKDNQ)-(LR)-(APQ)-(ST),.
[0049]In one embodiment, the light chain variable domain sequence is a X light chain and includes
[0050](a) a CDR1 that includes
TABLE-US-00011 (SEQ ID NO: 209) S-G-X-S-S-N-I-G-S-N-X-V-X-X,; (SEQ ID NO: 260) (GST)-G-(GSTN)-(SN)-(SI)-(GDN)-(TIV)-(GK)-(GS)- (VYN)-(YFNH)-(VY)-(VY)-S,; (SEQ ID NO: 210) (ST)-G-(GST)-S-S-(DN)-(IV)-G-(GS)-(YN)-(YFN)-(VY)- (VY)-S,; (SEQ ID NO: 234) G-G-(SN)-(SN)-(ID)-(GI)-(GT)-(SK)-(SYN)-V-H,; or (SEQ ID NO: 235) G-(GST)-(SN)-(SN)-(IDN)-(GIV)-(GST)-(SKN)-(SYN)- (VFN)-(VYH),;
[0051](b) a CDR2 that includes
TABLE-US-00012 (SEQ ID NO: 261) D-(AD)-S-X-(LR)-(AP)-(ST),; (SEQ ID NO: 262) (AD)-(ADN)-S-(SKDNQ)-(LR)-(APQ)-(ST),; (SEQ ID NO: 263) (AGRD)-(ADN)-(SYN)-(SKDNQ)-(LR)-(APQ)-(ST),; (SEQ ID NO: 211) D-(DN)-S-(DQ)-R-P-S-X,; or (SEQ ID NO: 247) (RDNE)-(VDN)-(SYN)-(KDQ)-R-P-S-X,;
[0052]and/or
[0053](c) a CDR3 that includes
TABLE-US-00013 (SEQ ID NO: 212) A-A-W-D-D-(SN)-(LV),; (SEQ ID NO: 248) (QA)-X-W-D-(SDT)-(GSN),; or (SEQ ID NO: 249) (AQ)-(ASV)-(YW)-(AD)-(GSID)-(GSN)-(STLVN)-(GSDN)- (GSLVH)-(VQ)-V,.
[0054]In one embodiment, the light chain variable domain sequence includes a CDR1 that includes a sequence of which at least 9 of 11 amino acids are identical to a sequence from column 1, a CDR2 that includes a sequence of which at least 7 of 9 amino acids are identical to a sequence from column 2, and a CDR3 of which at least 70, 80, 85, 87, 90, 92, 94, 96, 97, or 98% of the amino acids are identical to a sequence from column 3 of Table 10.
[0055]In one embodiment, at least 30, 50, 60, 70, 80, 90 or 100% of the CDR amino acid residues that are not identical to residues in the reference sequences from Table 10 are identical to residues at corresponding positions in a human germline sequence (e.g., a human germline sequence described herein, e.g., the human germline sequence that is associated with the respective CDR in an example described herein).
[0056]In one embodiment, the light chain variable domain sequence includes a CDR1 that includes a sequence from column 1, a CDR2 that includes a sequence from column 2, and a CDR3 from that includes a sequence from column 3 of Table 10.
[0057]In one embodiment, the framework regions of the heavy and/or light chain variable domain are at least 70, 80, 90, 92, 95, 97, 98, or 99% identical to a corresponding heavy or light FR sequence, e.g., of a known FR sequence or a FR sequence described herein.
[0058]In one embodiment, the framework regions of the heavy and/or light chain variable domain are at least 70, 80, 90, 92, 95, 97, 98, or 99% identical to a corresponding framework region of a human germline sequence (e.g., the human germline sequence with which the CDRs of the protein are associated herein).
[0059]In one embodiment, the heavy and/or light chain variable domain are at least 70, 80, 90, 92, 95, 97, or 98% identical to a human germline sequence (e.g., the human germline sequence with which the CDRs of the protein are associated herein).
[0060]In one embodiment, the H1 and H2 hypervariable loops have the same canonical structure as an antibody described herein. In one embodiment, the L1 and L2 hypervariable loops have the same canonical structure as an antibody described herein.
[0061]In one embodiment, the protein binds MMP-26 with a KD that is at least 2, 4, 5, 10, 20, 50, or 100 better than its KD for another metalloproteinase, e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, or MMP-14. In another embodiment, the protein binds MMP-26 with a KD that is at least 2, 4, 5, 10, 20, 50, or 100 better than its KD for MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, or MMP-14.
[0062]In one embodiment, the protein inhibits MMP-26 with a Ki of less than 1×10-7 M, 3×10-8, 1×10-8, 3×10-9, 1×10-9, 3×1010, 1×1010, 3×1011, or 1×10-12 M. In an embodiment, the protein inhibits MMP-26 with a Ki that is at least 2, 4, 5, 10, 20, 50, or 100 better than its Ki for another metalloproteinase, e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, or MMP-14.
[0063]In one embodiment, the protein reduces cell metastasis in vivo.
[0064]In one embodiment, the protein includes two independent polypeptide chains, a first chain including the light chain variable domain sequence and the second chain including the heavy chain variable domain sequence, and each chain including a constant immunoglobulin domain. In an embodiment, the protein is composed of a single polypeptide chain that includes the light chain variable domain sequence and the heavy chain variable domain sequence.
[0065]In one embodiment, the protein further includes a label, a cytotoxic or cytostatic agent, or a serum-residence prolonging moiety. The protein can include additional features described herein.
[0066]In another aspect, the invention features a protein that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to the MMP-26 catalytic domain and one of the immunoglobulin variable domains is at least 70, 75, 80, 85, 87, 90, 92, 94, 95, 96, 97, 98, 99, or 100% identical to a variable domain sequence of a variable domain described herein, e.g., a variable domain of a01, b04, b06, b10, c01, c08, d02, d04, d06, d08, D6-orig, a04, a11, c05, c04, c11, c12, d07, A1-orig, H6-orig, a02, a03, a05, a06, a07, a08, a09, a10, a12, b01, b02, b03, b05, b07, b08, b09, b11, b12, c02, c03, c06, c07, c09, c10, d01, d03, d05, or d09. The protein can include additional features described herein.
[0067]In another aspect, the invention features a protein that includes a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the protein binds to MMP-26. At least one of the variable domains is related to a reference antibody selected from the group consisting of a01, b04, b06, b10, c01, c08, d02, d04, d06, d08, D6-orig, a04, a11, c05, c04, c11, c12, d07, A1-orig, H6-orig, a02, a03, a05, a06, a07, a08, a09, a10, a12, b01, b02, b03, b05, b07, b08, b09, b11, b12, c02, c03, c06, c07, c09, c10, d01, d03, d05, and d09. The relationship is such that at least 75, 80, 82, 84, 87, 90, 92, 94, 95, 96, 97, 98, 99, or 100% of the amino acid residues in the variable domain are either (i) identical to a corresponding residue in the reference antibody, (ii) identical to a corresponding residue in a human germline sequence, or both. In one embodiment, the human germline sequence is the human germline sequence with which the reference antibody is associated in an example described herein.
[0068]In one embodiment, all of the amino acids residues in the variable domain are (i) identical to a corresponding residue in the reference antibody, or (ii) identical to a corresponding residue in the human germline sequence, e.g., with which the reference antibody is associated in an example described herein, or both.
[0069]In one embodiment, at least 1, 2, 3, 4, or 5 of the amino acid residues in the variable domain differ from a corresponding residue in the reference antibody, but are identical to a corresponding residue in the human germline sequence, e.g., with which the reference antibody is associated in an example described herein.
[0070]In one embodiment, in the framework regions, at least 90, 92, 94, 96, 97, 98, or 99% or all of the amino acid residues are identical to a corresponding residue in the human germline sequence, e.g., with which the reference antibody is associated in an example described herein.
[0071]In one embodiment, at least 1, 2, or 3 of the amino acid residues in the CDR regions of the variable domain differ from a corresponding residue in the reference antibody, but are identical to a corresponding residue in the human germline sequence, e.g., with which the reference antibody is associated in an example described herein.
[0072]In one embodiment, amino acid residues that are not identical are conserved substitutions relative to a corresponding residue in the reference antibody, or a human germline sequence with which the reference antibody is associated in an example described herein.
[0073]The protein can include additional features described herein.
[0074]In another aspect, the invention features a protein that includes an antigen binding fragment that binds to MMP-26, wherein the protein binds to a MMP-26 epitope that overlaps with an epitope bound by an antibody described herein. The protein can include additional features described herein.
[0075]In another aspect, the invention features a protein that includes an antigen binding fragment that binds to MMP-26, wherein the protein competes with an antibody described herein for binding to MMP-26. The protein can include additional features described herein.
[0076]An MMP-26-binding antibody is typically monospecific, e.g., a monoclonal antibody, or antigen-binding fragment thereof. The MMP-26-binding antibodies can be full-length (e.g., an IgG (e.g., an IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE) or can include only an antigen-binding fragment (e.g., a Fab, F(ab')2, Fv, or scFv fragment). The antibody, or antigen-binding fragment thereof, can include two heavy chain immunoglobulins and two light chain immunoglobulins, or can be a single chain antibody. The antibodies can, optionally, include a constant region chosen from a kappa, lambda, alpha, gamma, delta, epsilon or a mu constant region gene. An MMP-26-binding antibody can include a heavy and light chain constant region, e.g. a constant region substantially from a human antibody, e.g., a human IgG1, IgG2, IgG3, or IgG4, or a portion thereof.
[0077]In one embodiment, the antibody (or fragment thereof) is a recombinant or modified antibody, e.g., a chimeric, a humanized, a deimmunized, or an in vitro generated antibody. The term "recombinant" or "modified" antibody, as used herein, is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include human, humanized, CDR grafted, chimeric, deimmunized, in vitro generated antibodies, and may optionally include framework and/or constant regions derived from human germline immunoglobulin-encoding nucleic acid sequences.
[0078]In one embodiment, the antibody binds to an epitope distinct from an epitope bound by known antibodies that bind to MMP-26. In other embodiments, the antibody does not compete with known antibodies that bind to MMP-26. In still other embodiments, the antibody does not compete with an antibody described herein.
[0079]In one embodiment, the antibody binds to overlapping epitopes of, or competitively inhibits, the binding of an antibody disclosed herein to MMP-26. In one embodiment, the antibody binds to an epitope that includes an amino acid that is within at least 12, 10, 8, 6, 5, or 3 amino acids of one or more of amino acid 208, 209, 212, or 218. In one embodiment, the antibody binds to an epitope that includes an amino acid between residues 200-230, e.g., between 205-220. In one embodiment, the antibody includes an antigen binding site structure that includes one or more side chains that are positioned within 12, 10, 8, 6 or 4 Angstroms of amino acid residues 200-230, e.g., between 205-220, e.g., 208, 209, 212, or 218.
[0080]Further, any combination of MMP-26-binding antibodies is within the scope of the invention, e.g., two or more antibodies that bind to different regions of MMP-26, e.g., antibodies that bind to two different epitopes on MMP-26, e.g., a bispecific antibody.
[0081]In one embodiment, the MMP-26-binding antibody includes at least one light or heavy chain immunoglobulin (or two light chain immunoglobulins and two heavy chain immunoglobulins). Preferably, each immunoglobulin includes a light or a heavy chain variable region having at least one, two and, preferably, three CDR's substantially identical to a CDR from an anti-MMP-26 light or heavy chain variable region, respectively, e.g., from a variable region of an antibody described herein.
[0082]In one aspect, the invention features an isolated nucleic acid including a coding sequence that encodes a polypeptide including a variable domain sequence of a protein described herein, e.g., a protein described above. The nucleic acid can further include a second coding sequence that encodes a polypeptide including a second immunoglobulin variable domain, e.g., thereby providing two sequences that respectively encode a heavy and light chain variable domain.
[0083]In one aspect, the invention features a host cell that produces a protein described herein. The cell can include a first nucleic acid encoding a polypeptide including a heavy chain variable domain sequence of the protein and a second nucleic acid encoding a polypeptide including a light chain domain sequence of the protein.
[0084]For example, the host cell contains a first nucleic acid encoding a polypeptide including a heavy chain variable region and a second nucleic acid encoding a polypeptide including a light chain variable region. The heavy chain variable region includes an amino acid sequence at least 70, 80, 90, 92, 95, 97, 98, or 99% identical to an amino acid sequence of a heavy chain immunoglobulin variable domain sequence described herein, and the light chain variable region includes an amino acid sequence at least 70, 80, 90, 92, 95, 97, 98, or 99% identical to a light chain immunoglobulin variable domain sequence described herein.
[0085]In another aspect, the invention features a nucleic acid that includes a coding sequence that encodes a polypeptide comprising an immunoglobulin heavy chain variable domain that binds to MMP-26, e.g., an immunoglobulin heavy chain variable domain described herein. For example, the immunoglobulin heavy chain variable domain can include: a CDR motif or CDR described herein. The immunoglobulin heavy chain variable domain can include a framework region described herein. In one example, the variable domain is a heavy chain variable domain is at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to an amino acid sequence described herein or a variable domain sequence thereof.
[0086]In another aspect, the invention features a nucleic acid that includes a coding sequence that encodes a polypeptide comprising an immunoglobulin light chain variable domain that binds to MMP-26, e.g., an immunoglobulin light chain variable domain described herein. For example, the immunoglobulin light chain variable domain can include: a CDR motif or CDR described herein. The immunoglobulin light chain variable domain can include a framework region described herein. In one example, the variable domain is a light chain variable domain is at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to an amino acid sequence described herein or a variable domain sequence thereof.
[0087]A nucleic acid described herein can further include a promoter operably linked to the coding sequence. A nucleic acid can include a first and second coding sequence, e.g., wherein the first coding sequence encodes a polypeptide that includes an immunoglobulin heavy chain variable domain and the second coding sequence encodes a polypeptide that includes an immunoglobulin light chain variable domain.
[0088]In another aspect, the invention features a host cell that contains a first nucleic acid encoding a polypeptide comprising a heavy chain variable region and a second nucleic acid encoding a polypeptide comprising a light chain variable region. The heavy chain variable region and the light chain variable region can associate to form a MMP-26 binding protein. These variable regions can have one or more properties described herein, e.g., at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to a sequence described herein, e.g., the sequence of a variable domain from an isolated antibody described herein or a human germline sequence described herein. The invention also includes a method of providing an MMP-26-binding antibody. The method can include providing a host cell described herein; and expressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen-binding protein that interacts with MMP-26.
[0089]In another aspect, the invention features a protein ligand that includes a human or effectively human heavy chain immunoglobulin variable domain and a human or effectively human light chain immunoglobulin variable domain, wherein the protein ligand binds to human MMP-26 catalytic domain. The protein can bind to MMP-26 with a Kd of less than 1×10-7 M, 3×10-8, 1×10-8, 3×10-9, 1×10-9, 3×10-10, 1×10-10, 3×10-11, 1×10-11, or 1×10-12M. In one embodiment, the protein inhibits MMP-26 proteolytic activity. The protein can include one or more additional features described herein.
[0090]In another aspect, the invention provides compositions, e.g., pharmaceutical compositions, which include a pharmaceutically acceptable carrier, excipient or stabilizer, and at least one of the MMP-26-binding proteins (e.g., antibodies or fragments thereof) described herein. In one embodiment, the compositions, e.g., the pharmaceutical compositions, include a combination of two or more of the aforesaid MMP-26-binding proteins.
[0091]In another aspect, the invention features a kit that includes an MMP-26-binding antibody (or fragment thereof), e.g., an MMP-26-binding antibody (or fragment thereof) as described herein, for use alone or in combination with other therapeutic modalities, e.g., a cytotoxic or labeling agent, e.g., a cytotoxic or labeling agent as described herein, along with instructions on how to use the MMP-26 antibody or the combination of such agents to treat, prevent or detect a neoplastic disorder, an inflammatory disorder, or a disorder characterized by excessive MMP-26 activity.
[0092]In another aspect, the invention features a method of identifying a protein that specifically binds to MMP-26. In one embodiment, the method includes: providing a MMP-26 antigen; providing a library of proteins (e.g., a display library, e.g., a phage display library); and identifying a member that specifically binds to the MMP-26 antigen, e.g., the catalytic domain of MMP-26.
[0093]In another aspect, the invention features a method of identifying a protein that specifically binds to MMP-26. The method includes: providing an MMP-26 antigen; immunizing a mouse with the MMP-26 antigen; producing hybridoma cells from the spleen of the immunized mouse; and identifying individual hybridoma cell lines expressing an antibody that specifically binds to the MMP-26 antigen.
[0094]In one embodiment, the MMP-26 antigen is of human origin and includes, e.g., the extracellular domain of human MMP-26 or some fragment thereof, e.g., the catalytic domain of MMP-26. The MMP-26 antigen can be a recombinant polypeptide optionally fused to another polypeptide, e.g., a purification handle.
[0095]In preferred embodiments, the methods further include isolating a nucleic acid molecule from the identified phage or hybridoma, wherein the nucleic acid molecule encodes the polypeptide or antibody that specifically binds to the MMP-26 antigen. The isolated nucleic acid molecules can be used to produce therapeutic agents, as described herein.
[0096]In another aspect, the invention features nucleic acids that encode proteins identified by the methods described herein. In preferred embodiments, the nucleic acids include sequences encoding a heavy and light chain immunoglobulin or immunoglobulin fragment described herein. For example, the invention features, a first and second nucleic acid encoding a heavy and light chain variable region, respectively, of an MMP-26-binding antibody molecule as described herein. Sequences encoding a heavy and light chain that function together can be present on separate nucleic acid molecules or on the same nucleic acid molecule. In another aspect, the invention features host cells and vectors containing a nucleic acid described herein.
[0097]In yet another aspect, the invention features a method of producing an MMP-26-binding antibody, or antigen-binding fragment thereof. The method includes: providing a host cell that contains a first nucleic acid encoding a polypeptide comprising a heavy chain variable region, e.g., a heavy chain variable region as described herein; providing a second nucleic acid encoding a polypeptide comprising a light chain variable region, e.g., a light chain variable region as described herein; and expressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen binding protein that interacts with MMP-26. The first and second nucleic acids can be linked or unlinked, e.g., expressed on the same or different vector, respectively. The first and second nucleic acids can be components of the same molecule or can reside on different molecules (e.g., different chromosomes or plasmids).
[0098]The host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), Human Embryonic Kidney cells (HEK293, HEK293T), COS cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell. For example, nucleic acids encoding the antibodies described herein can be expressed in a transgenic animal. In one embodiment, the nucleic acids are placed under the control of a tissue-specific promoter (e.g., a mammary specific promoter) and the antibody is produced in the transgenic animal. For example, the antibody molecule is secreted into the milk of the transgenic animal, such as a transgenic cow, pig, horse, sheep, goat or rodent. To produce a single chain antibody, the nucleic acid is configured to encode a single polypeptide that comprises both the heavy and light chain variable domains.
[0099]In another aspect, the invention features a method that includes: providing a host cell, e.g., as described herein, that contains nucleic acids for expressing an antigen binding protein that interacts with MMP-26; and expressing said first and second nucleic acids in the host cell under conditions that allow assembly of said light and heavy chain variable regions to form an antigen binding protein that interacts with MMP-26. The protein can include additional features described herein.
[0100]In another aspect, the invention features a method of treating or preventing a neoplastic disorder. The method includes: administering an MMP-26 binding protein described herein to a subject in an amount effective to treat or prevent a neoplastic disorder in the subject. In one embodiment, the subject has, is predisposed to, or is diagnosed with a malignant cancer or metastatic disorder. For example, the neoplastic disorder is associated with epithelial carcinomas, breast cancer, prostate cancer, endometrial, esophageal squamous cell carcinoma, colon cancer, squamous cell carcinoma (SCC) of the oral cavity, verrucous carcinoma of the oral cavity, or lung cancer. For example, a patient having breast, prostate, endometrial, or esophageal, or lung cancer can be treated by administration of one or more injections of an IgG which binds and inhibits MMP-26.
[0101]In another aspect of the invention, an IgG antibody that binds to MMP-26 is administered to a patient (e.g., injected into the patient), e.g., a patient having a disease characterized by excess MMP-26 activity. In one embodiment, the antibody clears MMP-26 from the patient. The antibody can bind MMP-26 with high affinity, e.g. a Kd of less than 1×10-7 M, 3×10-8, 1×10-8, 3×10-9, 1×10-9, 3×10-10, 1×10-10, 3×10-11, 1×10-11, or 1×10-12 M. The antibody can be an antibody described herein.
[0102]The method can further include, prior to, during, or after the administering, evaluating cells of the subject for MMP-26 protein, mRNA, or activity.
[0103]The method can further include monitoring the subject for a metastatis.
[0104]In one embodiment, the administering includes administering a plurality of doses of the protein. For example, the doses are administered at regular intervals. The method can include other features described herein.
[0105]In another aspect, the invention features a method of treating or preventing an inflammatory disorder. The method includes: administering an MMP-26 binding protein described herein to a subject in an amount effective to treat or prevent an inflammatory disorder in the subject. In one embodiment, the inflammatory disorder is rheumatoid arthritis, lupus, restenosis, graft v. host response, or multiple sclerosis. In one embodiment, the administering includes administering a plurality of doses of the protein. For example, the doses are administered at regular intervals. The method can include other features described herein.
[0106]In another aspect, the invention features a method of treating or preventing a disorder characterized by excessive or undesired MMP-26 activity. The method includes: administering an MMP-26 binding protein to a subject in an amount effective to treat or prevent a disorder characterized by excessive or undesired MMP-26 activity in the subject. For example, the disorder is periodontitis, rheumatoid arthritis, or osteoarthritis. In one embodiment, the administering includes administering a plurality of doses of the protein. For example, the doses are administered at regular intervals. The method can include other features described herein.
[0107]In another aspect, the invention features a method of modulating MMP-26 activity. The method includes: providing an MMP-26-binding protein described herein; and contacting the protein to MMP-26, in an amount sufficient to modulate MMP-26 activity. In one embodiment, the modulated activity is MMP-26 proteolytic activity. For example, the contacting is in vitro or in vivo. In one embodiment, the protein is contacted to MMP-26 in the vicinity of a neoplastic cell (e.g., a cell found in laryngeal, epidermal, pulmonary, breast, renal, urothelial, colonic, prostatic, or hepatic cancer and/or metastasis). The method can include other features described herein.
[0108]In another aspect, the invention features a method for detecting the presence of a MMP-26 protein, e.g., in a sample, in vitro. The method includes: (i) contacting the sample (and optionally, a reference, e.g., control, sample) with an MMP-26-binding protein described herein, under conditions that allow interaction of the MMP-26-binding protein and the MMP-26 protein to occur; and (ii) detecting interaction between the MMP-26-binding protein, and the sample (and optionally, the reference, e.g., control, sample). In one embodiment, at least one of the MMP-26 binding protein or the MMP-26 is immobilized.
[0109]In another aspect, the invention features a method for detecting the presence of MMP-26 (e.g., activated MMP-26), e.g., in vivo. The method includes: (i) administering to a subject (and optionally a control subject) an MMP-26-binding protein, under conditions that allow interaction of the MMP-26-binding protein and the MMP-26 protein to occur; and (ii) detecting location of the MMP-26-binding protein in the subject or formation of a complex between the MMP-26-binding protein and MMP-26 in the subject. For example, the subject is a human subject. In one embodiment, the detecting includes imaging the subject. In one embodiment, the MMP-26-binding protein is labeled with an MRI detectable label.
[0110]With respect to any administration method, an MMP-26-binding protein described herein can be used alone, e.g., can be administered to a subject or used in vitro in non-derivatized or unconjugated forms. In other embodiments, the MMP-26-binding protein can be derivatized, modified or linked to another functional molecule, e.g., another polypeptide, protein, isotope, cell, or insoluble support. For example, the MMP-26-binding protein can be functionally-linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as an antibody (e.g., if the ligand is an antibody to form a bispecific or a multispecific antibody), a toxin, a radioisotope, a serum-residence prolonging moiety (e.g. PEG), a therapeutic (e.g., a cytotoxic or cytostatic) agent or moiety, among others. The method can include other features described herein.
[0111]In another aspect, the invention features a method of inhibiting metalloproteinase activity in a subject. The method includes administering an MMP-26-binding protein described herein in an amount effective to inhibit metalloproteinase activity in a subject. The reduced metalloproteinase activity can alter proteolysis, e.g., in the vicinity of a cancer cell, e.g., a metastatic cancer cell, in placental tissue, or in a tumor, e.g., a metastatic tumor. The method can include other features described herein.
[0112]In another aspect, the invention features a method of inhibiting metastasis in a subject. The method includes administering an MMP-26-binding protein described herein in an amount effective to inhibit metastasis in a subject. The protein can be delivered systemically or locally. For example, the protein can be targeted to a tumor. The protein can modulate the integrity of an extracellular matrix, e.g., by preventing degradation of an MMP-26 substrate that is an extracellular matrix component. The method can include other features described herein.
[0113]In another aspect, the invention features a method of treating or preventing a neoplastic disorder in a subject. The method includes providing an MMP-26-binding protein, e.g. a protein described herein, and contacting the subject with the protein, in an amount sufficient to modulate or prevent a neoplastic disorder. The method can include contacting a neoplastic cell, e.g., a benign or hyperplastic cell (e.g., a cell found in laryngeal, epidermal, pulmonary, breast, renal, endometrial, ovarian, urothelial, colonic, prostatic, or hepatic cancer and/or metastasis). The protein can include a cytotoxic entity. The method can include other features described herein.
[0114]In another aspect, the invention features a method of treating or preventing a an inflammatory disorder in a subject. The method includes providing an MMP-26-binding protein, e.g. a protein described herein, and contacting the subject with the protein, in an amount sufficient to modulate or prevent the inflammatory disorder. The method can include identifying a subject as having or being at risk for having an inflammatory disorder. The method can further include monitoring at least one indicator of inflammation, e.g., local temperature, swelling (e.g., as measured), redness, local or systemic white blood cell count, presence or absence of neutrophils, cytokine levels, elastase activity, and so forth. The method can include other features described herein.
[0115]In another aspect, the invention features a method of reducing MMP-26 activity in vivo. The method includes administering an MMP-26 binding protein (e.g., an MMP-26 binding protein described herein) to a subject. The protein can be administered in an amount sufficient to cause MMP-26 (mature MMP-26 or MMP-26 in a pro-form or other immature form) to be cleared from the subject. For example, in cases in which the MMP-26 binding protein is an IgG antibody, binding of the binding protein to endogenous MMP-26 can result in improved clearance of MMP-26 from the subject. In one embodiment, the subject has a disorder characterized by excess MMP-26 activity. The method can include other features described herein.
[0116]In another aspect, the invention features a method of inhibiting MMP-26 activity in vitro. The method includes contacting an MMP-26-binding protein that inhibits MMP-26 to a composition that include the MMP-26 catalytic domain.
[0117]The method can be used to treat or prevent cancerous disorders, e.g., including but are not limited to, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., renal, urothelial cells), pharynx, as well as adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, endometrial and ovarian cancers, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In particular, metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions described herein.
[0118]The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having, or at risk of, a disorder described herein, e.g., cancer).
[0119]The MMP-26-binding protein can be administered to the subject systemically (e.g., orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation), topically, or by application to mucous membranes, such as the nose, throat and bronchial tubes.
[0120]The methods can further include the step of monitoring the subject, e.g., for a reduction in one or more of: a reduction in tumor size; reduction in cancer markers (e.g., levels of cancer specific antigen or in MMP-26 protein levels or activity); reduction in the appearance of new lesions, e.g., in a bone scan; a reduction in the appearance of new disease-related symptoms; or decreased or stabilization of size of soft tissue mass; or any parameter related to improvement in clinical outcome. The subject can be monitored in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Monitoring can be used to evaluate the need for further treatment with the same MMP-26-binding protein or for additional treatment with additional agents. Generally, a decrease in one or more of the parameters described above is indicative of the improved condition of the subject. Information about the monitoring can be recorded, e.g., in electronic or digital form.
[0121]The MMP-26-binding protein can be used alone in unconjugated form to thereby inhibit adhesion, migration, or extravasation or the MMP-26-expressing cells, or ablate or kill the MMP-26-expressing cells. If the protein is an antibody, the ablation or killing can be mediated, e.g., by an antibody-dependent cell killing mechanisms such as complement-mediated cell lysis and/or effector cell-mediated cell killing. In other embodiments, the MMP-26-binding protein can be bound to a substance, e.g., a cytotoxic agent or moiety, effective to kill or ablate the MMP-26-expressing cells. For example, the MMP-26-binding protein can be coupled to a radioactive ion (e.g., an α-, γ-, or β-emitter), e.g., iodine (131I or 125I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), or bismuth (213Bi). The methods and compositions described herein can be used in combination with other therapeutic modalities. In one embodiment, the method includes administering to the subject an MMP-26-binding protein, e.g., an MMP-26-binding antibody or fragment thereof, in combination with a cytotoxic agent, in an amount effective to treat or prevent said disorder. The ligand and the cytotoxic agent can be administered simultaneously or sequentially. In other embodiments, the methods and compositions described herein are used in combination with surgical and/or radiation procedures.
[0122]The subject methods also can be used on cells in culture, e.g. in vitro or ex vivo. The cultured cells can be MMP-26-producing cells, e.g., tumor cells or placental cells. For example, the cells can be cultured in vitro in culture medium and the contacting step can be effected by adding the MMP-26-binding protein to the culture medium. The method can be performed on cells (e.g., cancerous or metastatic cells) present in a subject, as part of an in vivo (e.g., therapeutic or prophylactic) protocol.
[0123]In another aspect, the invention features methods for detecting the presence of a MMP-26 protein, in a sample, in vitro (e.g., a biological sample, a tissue biopsy, e.g., a cancerous lesion). The subject method can be used to evaluate, e.g., diagnose or stage a disorder described herein, e.g., a cancerous disorder. The method includes: (i) contacting the sample (and optionally, a reference, e.g., control, sample) with an MMP-26-binding protein, as described herein, under conditions that allow interaction of the MMP-26-binding protein and the MMP-26 protein to occur; and (ii) detecting MMP-26, e.g., by detecting formation of a complex between the MMP-26-binding protein or by detecting an interaction between the MMP-26-binding protein and MMP-26, in the sample (and optionally, the reference, e.g., control, sample). Formation of the complex can be indicative of the presence of MMP-26 protein (e.g., activated MMP-26 protein), and can indicate the suitability or need for a treatment described herein. For example, a statistically significant change in the formation of the complex in the sample relative to the reference sample, e.g., the control sample, is indicative of the presence of MMP-26 (e.g., activated MMP-26) in the sample.
[0124]In yet another aspect, the invention provides a method for detecting the presence of MMP-26 (e.g., activated MMP-26) in vivo (e.g., in vivo imaging in a subject). The subject method can be used to evaluate, e.g., diagnose, localize, or stage a disorder described herein, e.g., a cancerous disorder. The method includes: (i) administering to a subject (and optionally a control subject) an MMP-26-binding protein (e.g., an antibody or antigen binding fragment thereof), under conditions that allow interaction of the MMP-26-binding protein and the MMP-26 protein to occur; and (ii) detecting formation of a complex between the ligand and MMP-26, wherein a statistically significant change in the formation of the complex in the subject relative to the reference, e.g., the control subject or subject's baseline, is indicative of the presence of the MMP-26. The presence of activated MMP-26 in particular locations within a subject can be indicative of cancer, e.g., metastatic cancer.
[0125]In other embodiments, a method of diagnosing or staging, a disorder as described herein (e.g., an inflammatory or cancerous disorder), is provided. The method includes: (i) identifying a subject having, or at risk of having, the disorder; (ii) obtaining a sample of a tissue or cell affected with the disorder; (iii) contacting said sample or a control sample with an MMP-26-binding protein, under conditions that allow interaction of the binding agent and the MMP-26 protein to occur, and (iv) detecting formation of a complex. A statistically significant increase in the formation of the complex between the ligand with respect to a reference sample, e.g., a control sample, is indicative of the disorder or the stage of the disorder. For example, the finding of activated MMP-26 on tumor cells located in a solid tumor can indicate that the tumor is progressing into a metastatic tumor.
[0126]Preferably, the MMP-26-binding protein used in the in vivo and in vitro diagnostic methods is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound binding agent. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. In one embodiment, the MMP-26-binding protein is coupled to a radioactive ion, e.g., indium (111In), iodine (131I or 125I), yttrium (90Y), actinium (225Ac), bismuth (213Bi), sulfur (35S), carbon (14C), tritium (3H), rhodium (188Rh), or phosphorous (32P). In another embodiment, the ligand is labeled with an NMR contrast agent.
[0127]The invention also provides polypeptides and nucleic acids that encompass a range of amino acid and nucleic acid sequences. In addition, the invention features a host cell that includes a nucleic acid described herein. The cell can express a protein described herein, e.g., on its surface.
[0128]MMP-26 is an ideal target for therapeutic intervention, particularly in the several cancers in which it is upregulated. Because MMP-26 is normally expressed in very limited areas of normal adult tissues (uterus and placenta), a protein that binds MMP-26 can be used to specifically target cancer cells. The limited expression of MMP-26 in normal adults also makes this molecule useful for detecting cancer cells in a subject.
[0129]As used herein, the term "antibody" refers to a protein comprising at least one immunoglobulin variable domain. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein.
[0130]Each VH and VL is composed of three CDR's and four FR's, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The canonical structures of hypervariable loops of an immunoglobulin variable can be inferred from its sequence, as described in Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798); and Tomlinson et al. (1995) EMBO J. 14(18):4628-38.
[0131]As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may omit one, two or more N- or C-terminal amino acids, internal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form an MMP-26-binding structure.
[0132]The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.
[0133]One or more regions of an antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoeitic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. One or more of the constant regions can be human or effectively human. All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the N-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the C-terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
[0134]The term "antigen-binding fragment" refers to a protein that includes an immunoglobulin light chain variable domain sequence and an immunoglobulin heavy chain variable domain sequence, the protein being able to specifically bind to an antigen, e.g., MMP-26. In one embodiment, an antigen-binding fragment is a fragment of a full-length antibody (or simply "antibody portion," or "fragment") that retain the ability to specifically bind to MMP-26 (e.g., human MMP-26). Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated CDR that retains functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab')2, a Fd fragment, a Fv fragments, and dAb fragments) as well as complete antibodies. Antibody fragments can be obtained using any appropriate technique including conventional techniques known to those with skill in the art. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.
[0135]An "effectively human" immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human amino acid positions (e.g., framework positions and/or CDR positions) such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
[0136]A "humanized" immunoglobulin variable region is an immunoglobulin variable region that is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of "humanized" immunoglobulins include, for example, U.S. Pat. No. 6,407,213 and U.S. Pat. No. 5,693,762.
[0137]As used herein, "binding affinity" refers to the apparent association constant or Ka. The Ka is the reciprocal of the dissociation constant (Kd). A ligand may, for example, have a binding affinity of at least 10-5, 10-6, 10-7 or 10-8 M for a particular target molecule. Higher affinity binding of a ligand to a first target relative to a second target can be indicated by a higher Ka (or a smaller numerical value Kd) for binding the first target than the Ka (or numerical value Kd) for binding the second target. In such cases the ligand has specificity for the first target relative to the second target. Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 5, 10, 50, 100, or 1000-fold.
[0138]Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in PBS (phosphate buffered saline) at pH 7.2 at 30° C. These techniques can be used to measure the concentration of bound and free ligand as a function of ligand (or target) concentration. The concentration of bound ligand ([Bound]) is related to the concentration of free ligand ([Free]) and the concentration of binding sites for the ligand on the target where (N) is the number of binding sites per target molecule by the following equation:
[Bound]=N[Free]/((1/Ka)+[Free]).
[0139]It is not always necessary to make an exact determination of Ka, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to Ka, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.
[0140]An "isolated composition" refers to a composition that is removed from at least 90% of at least one component of a natural sample from which the isolated composition can be obtained. Compositions produced artificially or naturally can be "compositions of at least" a certain degree of purity if the species or population of species of interests is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-weight basis.
[0141]An "epitope" refers to the site on a target compound that is bound by a ligand, e.g., a polypeptide ligand or an antigen-binding ligand (e.g., an antibody such as a Fab or full length antibody). In the case where the target compound is a protein, the site can be entirely composed of amino acid components, entirely composed of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or composed of combinations thereof. Overlapping epitopes include at least one common amino acid residue.
[0142]Calculations of "homology" or "sequence identity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
[0143]In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. For example, the reference sequence may be the length of the immunoglobulin variable domain sequence.
[0144]The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
[0145]As used herein, the term "substantially identical" (or "substantially homologous") is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities. In the case of antibodies, the second antibody has the same specificity and has at least 50% of the affinity relative to the same antigen.
[0146]Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiment, the sequence identity can be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., highly stringent hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
[0147]As used herein, the term "homologous" is synonymous with "similarity" and means that a sequence of interest differs from a reference sequence by the presence of one or more amino acid substitutions (although modest amino acid insertions or deletions) may also be present. In addition to the GAP program described above, a variety of means of calculating degrees of homology or similarity to a reference sequence are available. One method uses the BLAST algorithms (available from the National Center of Biotechnology Information (NCBI), National Institutes of Health, Bethesda Md.), in each case, using the algorithm default or recommended parameters for determining significance of calculated sequence relatedness. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
[0148]As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified. The invention includes nucleic acids that hybridize with low, medium, high, or very high stringency to a nucleic acid described herein or to a complement thereof. The nucleic acids can be the same length or within 30, 20, or 10% of the length of the reference nucleic acid.
[0149]It is understood that a MMP-26-binding protein may have mutations relative to a ligand described herein (e.g., a conservative or non-essential amino acid substitutions), which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect desired biological properties, such as binding activity can be determined as described in Bowie, et al. (1990) Science 247:1306-1310. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is possible for many framework and CDR amino acid residues to include one or more conservative substitutions.
[0150]A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the binding agent, e.g., the antibody, without abolishing or more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change.
[0151]The terms "polypeptide" or "peptide" (which may be used interchangeably) refer to a polymer of three or more amino acids linked by a peptide bond, e.g., between 3 and 30, 12 and 60, or 30 and 300, or over 300 amino acids in length. The polypeptide may include one or more unnatural amino acids. Typically, the polypeptide includes only natural amino acids. A "protein" can include one or more polypeptide chains. Accordingly, the term "protein" encompasses polypeptides. A protein or polypeptide also can include one or more modifications, e.g., a glycosylation, amidation, phosphorylation, and so forth. The term "small peptide" can be used to describe a polypeptide that is between 3 and 30 amino acids in length, e.g., between 8 and 24 amino acids in length. The term "ligand" refers to a protein that can interact with a target molecule, e.g., MMP-26. A "specific ligand" refers to a protein that specifically interacts with the target molecule.
[0152]Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02. Particular ligands may show a difference, e.g., in specificity or binding, that are statistically significant (e.g., P value <0.05 or 0.02).
[0153]Other features and advantages of the instant invention will become more apparent from the following detailed description and claims. Embodiments of the invention can include any combination of features described herein. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.
DETAILED DESCRIPTION
[0154]The invention provides, inter alia, proteins (e.g., antibodies) that bind to matrix metalloproteinase-26 (MMP-26). In one embodiment, the proteins inhibit MMP-26. Other names for MMP-26 include Matrilysin-2 and endometase. An exemplary human MMP-26 amino acid sequence is as follows (as provided by SWISSPROT entry Q9NRE1):
TABLE-US-00014 (SEQ ID NO: 213) MQLVILRVTIFLPWCFAVPVPPAADHKGWDFVEGYFHQFFLTKKESPLLT QETQTQLLQQFHRNGTDLLDMQMHALLHQPHCGVPDGSDTSISPGRCKWN KHTLTYRIINYPHDMKPSAVKDSIYNAVSIWSNVTPLIFQQVQNGDADIK VSFWQWAHEDGWPFDGPGGILGHAFLPNSGNPGVVHFDKNEHWSASDTGY NLFLVATHEIGHSLGLQHSGNQSSIMYPTYWYHDPRTFQLSADDIQRIQH LYGEKCSSDIP
TABLE-US-00015 TABLE 1 MMP-26 Features From To Length Description 1 17 17 Signal Sequence 18 89 72 Propeptide 90 261 172 mature protein chain 82 82 potential cysteine switch (potential). 208 208 catalytic zinc - interacting resdiue 209 209 Active Site 212 212 catalytic zinc - interacting resdiue 218 218 catalytic zinc - interacting resdiue
After removal of the signal sequence and the prodomain, the amino acid sequence of the mature MMP-26 protein can be as follows:
TABLE-US-00016 (SEQ ID NO: 214) TSISPGRCKWNKHTLTYRIINYPHDMKPSAVKDSIYNAVSIWSNVTPLIF QQVQNGDADIKVSFWQWAHEDGWPFDGPGGILGHAFLPNSGNPGVVHFDK NEHWSASDTGYNLFLVATHEIGHSLGLQHSGNQSSIMYPTYWYHDPRTFQ LSADDIQRIQHLYGEKCSSDIP
[0155]Typically, MMP-26 is specifically expressed in human placenta and uterine tissue. MMP-26 is expressed by endometrial tumors and other tumors of epithelial origin, including carcinomas of the breast, lung and prostate. In addition, a variety of breast, lung and prostate cancer cell lines express this MMP. Accordingly, MMP-26 may participate in tissue remodeling events associated with tumor progression and metastasis as well as embryo implantation.
[0156]MMP-26 cleaves a variety of substrates, including type I gelatin, serpin alpha-1-anti-trypsin (α-1AT), fibronectin, vitronectin, denatured collagen and pro-gelatinase B (pro-MMP-9) (thereby activating the zymogen). MMP-26 does not substantially cleave non-denatured collagens, laminin, elastin, and plasminogen. In addition, MMP-26 cleaves insulin-like growth factor binding protein-1 (IGFBP-1). Cleavage of this binding protein can increase local concentrations of insulin-like growth factor-1 (IGF-1) and may similarly effect IGF-2. Insulin-like growth factors have been linked to proliferation of a variety of cancer cells. Accordingly, an inhibitor of MMP-26 (e.g., an inhibitory antibody described herein) can be used to reduce insulin-like growth factor activity (e.g., levels or presence of IGF-1 or IGF-2).
[0157]MMP-26-mediated α1AT hydrolysis inactivates this regulator of serine proteases resulting in enhanced protease activity and further destruction of the extracellular matrix. MMP-26 protease activity previously has been associated with invasiveness by breast cancer cells.
Identification of MMP-26 Binding Proteins
[0158]A number of methods can be used to identify proteins that bind to MMP-26. To identify antibodies, it is possible to immunize a non-human animal with the target molecule. Spleen cells can be isolated from the immunized animal and used to produce hybridoma cells using standard methods. In one embodiment, the non-human animal includes one or more human immunoglobulin genes. One method for identifying proteins that bind to MMP-26 includes: providing a library and selecting from the library one or more members that encode a protein that binds to the MMP-26 antigen. The selection can be performed in a number of ways. For example, the library can be a display library.
[0159]The MMP-26 can be tagged and recombinantly expressed. The MMP-26 is purified and attached to a support, e.g., to affinity beads, or paramagnetic beads or other magnetically responsive particles.
[0160]The MMP-26 can also be expressed on the surface of a cell. Members of the display library that specifically bind to the cell, e.g., only if the MMP-26 is activated, can be selected.
Display Libraries
[0161]In one embodiment, a display library is used to identify proteins that bind to MMP-26. A display library is a collection of entities; each entity includes an accessible protein component (e.g., a Fab or scFv) and a recoverable component (e.g., a nucleic acid) that encodes or identifies the protein component. The protein component can be of any length, e.g. from three amino acids to over 300 amino acids. In a selection, the protein component of each member of the library is probed with MMP-26 protein and if the protein component binds to MMP-26, the display library member is identified, e.g., by retention on a support. The protein component can include one or more immunoglobulin variable domains or variants of another domain. Methods using immunoglobulin domains for display are described below (see, e.g., "Antibody Display Libraries").
[0162]Retained display library members are recovered from the support and analyzed. The analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated. The analysis also can include determining the amino acid sequence of the protein component and purification of the protein component for detailed characterization.
[0163]A variety of formats can be used for display libraries. Examples include the following.
[0164]Phage Display. One format utilizes viruses, particularly bacteriophages. This format is termed "phage display." The protein component is typically covalently linked to a bacteriophage coat protein. The linkage results form translation of a nucleic acid encoding the protein component fused to the coat protein. The linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon. Phage display is described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol. 267:129-49; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.
[0165]Phage display systems have been developed for filamentous phage (phage f1, fd, and M13) as well as other bacteriophage (e.g., T7 bacteriophage and lambdoid phages; see, e.g., Santini (1998) J. Mol. Biol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6; Houshmet al. (1999) Anal Biochem 268:363-370). The filamentous phage display systems typically use fusions to a minor coat protein, such as gene III protein, and gene VIII protein, a major coat protein, but fusions to other coat proteins such as gene VI protein, gene VII protein, gene IX protein, or domains thereof also can be used (see, e.g., WO 00/71694). In one embodiment, the fusion is to a domain of the gene III protein, e.g., the anchor domain or "stump," (see, e.g., U.S. Pat. No. 5,658,727 for a description of the gene III protein anchor domain). It also is possible to physically associate the protein being displayed to the coat using a non-peptide linkage, e.g., a non-covalent bond or a non-peptide covalent bond. For example, a disulfide bond and/or c-fos and c-jun coiled-coils can be used for physical associations (see, e.g., Crameri et al. (1993) Gene 137:69 and WO 01/05950).
[0166]Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components, by infecting cells using the selected phages. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
[0167]Cell-based Display. In still another format the library is a cell-display library. Proteins are displayed on the surface of a cell, e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryotic cells include E. coli cells, B. subtilis cells, and spores (see, e.g., Lu et al. (1995) Biotechnology 13:366). Exemplary eukaryotic cells include yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hanseula, or Pichia pastoris). Yeast surface display is described, e.g., in Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557 and WO 03/029456, which describes a yeast display system that can be used to display immunoglobulin proteins such as Fab fragments and the use of mating to generate combinations of heavy and light chains.
[0168]In one embodiment, diverse nucleic acid sequences are cloned into a vector for yeast display. The cloning joins the variegated sequence with a domain (or complete) yeast cell surface protein, e.g., Aga2, Aga1, Flo1, or Gas1. A domain of these proteins can anchor the polypeptide encoded by the variegated nucleic acid sequence by a transmembrane domain (e.g., Flo1) or by covalent linkage to the phospholipid bilayer (e.g., Gas1). The vector can be configured to express two polypeptide chains on the cell surface such that one of the chains is linked to the yeast cell surface protein. For example, the two chains can be immunoglobulin chains.
[0169]In one embodiment, nucleic acids encoding immunoglobulin heavy chains that have been mutagenized based on an initial MMP-26-binding immunoglobulin are introduced into yeast cells of one cell type, and nucleic acids encoding immunoglobulin light chains that have been mutagenized based on an initial MMP-26-binding immunoglobulin are introduced into yeast cells of the other cell type. These two populations of cells can be combined to form diploid yeast that each express an immunoglobulin heavy and light chain. The yeast cells can be selected and/or screened for cells that bind to MMP-26, e.g., bind with improved affinity.
[0170]Ribosome Display. RNA and the polypeptide encoded by the RNA can be physically associated by stabilizing ribosomes that are translating the RNA and have the nascent polypeptide still attached. Typically, high divalent Mg2+ concentrations and low temperature are used. See, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35.
[0171]Polypeptide-Nucleic Acid Fusions. Another format utilizes polypeptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can be generated by the in vitro translation of mRNA that include a covalently attached puromycin group, e.g., as described in Roberts and Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302, and U.S. Pat. No. 6,207,446. The mRNA can then be reverse transcribed into DNA and crosslinked to the polypeptide.
[0172]Other Display Formats. Yet another display format is a non-biological display in which the protein component is attached to a non-nucleic acid tag that identifies the polypeptide. For example, the tag can be a chemical tag attached to a bead that displays the polypeptide or a radiofrequency tag (see, e.g., U.S. Pat. No. 5,874,214).
[0173]Epitope Specific Ligands. Display technology also can be used to obtain ligands, e.g., antibody ligands, that bind to particular epitopes of a target. Epitopes can be classified as "conformational" or "sequential". Conformational epitopes involve amino-acid residues that have a defined relative orientation in a properly folded target even though the amino acids may be substantially separated in the sequence (e.g., separated by at least one, two, four, six, eight or ten amino acids). Sequential epitopes involve short portions of the polypeptide chain that bind an antibody whatever the folding state of the protein (e.g., native or unfolded). Ligands for conformational epitopes can be identified, for example, by using competing non-target molecules that lack the particular epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating display library members that are not specific to the target. In another implementation, epitope specific ligands are identified by eluting display library members with a competing ligand that binds to the epitope of interest on the target molecule. Ligands that bind sequential epitopes can be selected, for example, using short peptides that have amino-acid sequences found in a target protein. Often ligands that bind to conformational epitopes also bind weakly to one or another peptide that contains some of the amino acids involved in the conformational epitope. Thus, one can select for binding to a peptide at very low stringency and then select for binding to the folded target protein.
[0174]Affinity Maturation. In one embodiment, a ligand that binds to a target is modified, e.g., by mutagenesis, to provide a pool of modified ligands. The modified ligands are then evaluated to identify one or more altered ligands which have altered functional properties (e.g., improved binding, improved stability, lengthened stability in vivo). In one implementation, display library technology is used to select or screen the pool of modified ligands. Higher affinity ligands are then identified from the second library, e.g., by using higher stringency or more competitive binding and washing conditions. Other screening techniques also can be used.
[0175]In one example of affinity maturation the methods described herein are used to first identify a protein ligand from a display library that binds a MMP-26 with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of less than 1 nM, 10 nM, or 100 nM. The nucleic acid sequence encoding the initial identified protein ligand are used as a template nucleic acid for the introduction of variations, e.g., to identify a second protein ligand that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial protein ligand. Alternatively, the amino-acid sequence of one or more CDRs can be used as a guide for design of a nucleic acid library that includes nucleic acids encoding the isolated sequence and many neighboring sequences. Such diversified nucleic acids can be introduced into a display vector containing the initial isolate and improved variants are selected from the library.
[0176]In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. If, for example, the identified ligands are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particular within ten, five, or three amino acids of a CDR junction. In the case of antibodies, mutagenesis also can be limited to one or a few of the CDRs, e.g., to make step-wise improvements.
[0177]Some exemplary mutagenesis techniques include: error-prone PCR (Leung et al. (1989) Technique 1:11-15), recombination (see, e.g., U.S. Ser. No. 10/279,633), DNA shuffling using random cleavage (Stemmer (1994) Nature 389-391; termed "nucleic acid shuffling"), RACHITT® (Coco et al. (2001) Nature Biotech. 19:354), site-directed mutagenesis (Zoller et al. (1987) Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) Methods Enzymol. 208:564-586) and incorporation of degenerate oligonucleotides (Griffiths et al. (1994) EMBO J 13:3245).
[0178]In one embodiment, mutagenesis is used to make an antibody more similar to one or more germline sequences. One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.
[0179]In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a CDR region. For example, the germline CDR residue can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated. Similar mutagenesis can be performed in the framework regions.
[0180]Accordingly, it is possible to isolate an antibody which has similar activity to a given antibody of interest, but is more similar to one or more germline sequences. For example, an antibody can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5% identical to a germline sequence in a region outside the CDRs (e.g., framework regions). Further an antibody can include at least 1, 2, 3, 4, or 5 germline residues in a CDR region, the germline residue being from a germline sequence of similar (e.g., most similar) to the variable region being modified. Germline sequences of primary interest are human germline sequences. The activity of the antibody (e.g., the binding activity) can be within a factor or 100, 10, 5, 2, 0.5, 0.1, and 0.001 of the original antibody.
[0181]Off-Rate Selection. Since a slow dissociation rate can be predictive of high affinity, particularly with respect to interactions between polypeptides and their targets, the methods described herein can be used to isolate ligands with a desired kinetic dissociation rate (i.e. reduced) for a binding interaction to a target.
[0182]To select for slow dissociating ligands from a display library, the library is contacted to an immobilized target. The immobilized target is then washed with a first solution that removes non-specifically or weakly bound biomolecules. Then the immobilized target is eluted with a second solution that includes a saturation amount of free target, i.e., replicates of the target that are not attached to the particle. The free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.
[0183]The second solution can have solution conditions that are substantially physiological or that are stringent. Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include biomolecules that dissociate at a slower rate from the target than biomolecules in the early fractions.
[0184]Further, it also is possible to recover display library members that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells.
[0185]Selecting and Screening for Specificity. "Selection", in the context of a display library, refers to a process in which many members of a display library are allowed to contact the target and those that bind are recovered and propagated. The selection can be from a library having numerous members, e.g., more than 1010 members. "Screening", in the context of a display library, refers to a process in which isolated members of the library are tested singly for binding to the target. Through automation, thousands of candidates may be screened in a highly parallel process. The display library selection methods described herein can include a selection process that discards display library members that bind to a non-target molecule. Examples of non-target molecules include, e.g., metalloproteinases other than MMP-26, e.g., a matrix metalloproteinase other than MMP-26, e.g., MMP-12, MMP-3 (stromelysin-1), MMP-9 (gelatinase), and so on. In one implementation, a so-called "negative selection" step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecules. The display library or a pool thereof is contacted to the non-target molecule. Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule.
[0186]In another implementation, a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to MMP-26.
[0187]The display library selection and screening methods described herein can include a selection or screening process that selects for display library members that bind to specific sites on the target molecule. For example, elution with high concentration of an antibody described herein selects for phage that bind to the epitope bound by such an antibody. One can screen for a phage that binds to a particular epitope of MMP-26 by performing ELISAs with and without a competing antibody that recognizes the epitope in the buffer.
Selection and Screening for MMP-26-Binding Antibodies:
[0188]The following provides one exemplary method for identifying antibodies that bind to MMP-26 using a phagemid Fab library. For example, three rounds of selection can be performed with decreasing amounts of target protein (e.g., 500 to 50 nM for first to third rounds, respectively). The target is immobilized on streptavidin-coated magnetic beads (Dynal). The library is depleted against streptavidin-coated magnetic beads prior to each round of selection and optionally against an unrelated protein which may include a common purification handle. For example, if the target is produced as a fusion to an Fc domain, the library can be depleted against soluble Trail-Fc (a commercially available Fc fusion protein). The depletion process removes Fc binders.
[0189]Each round of selection can include, e.g., two cycles of streptavidin magnetic bead depletion, a cycle of binding of phage to MMP-26-coated beads, ten cycles of washes, elution of bound phage, and propagation of enriched phage for the next round. Phage bound to MMP-26-coated beads after ten washes can be directly amplified or eluted before amplification. After three rounds of selection, individual clones may be grown in 96-well microtiter plates and individually screened for MMP-26 binding activity by phage ELISA. ELISAs can include evaluations of binding to MMP-26, specificity controls, and unrelated controls. Isolates can be DNA fingerprinted to determine the diversity emerging from the selection process. For example, positive isolates can be PCR amplified with the oligonucleotide primers M13-reverse and gene III-forward (see, e.g., Marks et al. (1991), J. Mol. Biol. 222:581). The products can be analyzed by BstNI fingerprinting.
[0190]An exemplary method for performing ELISA's with phage that display a ligand is as follows. Individual clones can be grown and rescued as described previously (Marks et al. (1991), J. Mol. Biol. 222:581). For ELISAs, 96-well Immulon 2 HB plates (Thermo Labsystems) are coated with 1 μg/well ImmunoPure® streptavidin (Pierce) in PBS and incubated overnight at 4° C. After three washes with PBS, 100 μL of biotinylated MMP-26 protein is allowed to bind to the immobilized streptavidin for 30-60 minutes at room temperature. Then, MMP-26-coated wells are blocked with 300 μL of 2% milk/1×PBS/0.05% Tween (2% MPBST) for two hours at 37° C. The wells are incubated with 100 μL of phage culture supernatant that had been blocked with 2% MPBST for one hour at room temperature. The wells are washed five times with 1×PBS/Tween 0.1% (PBST), and incubated with 100 μL of anti-M13-HRP secondary antibody at a 1:5,000 dilution for one hour at room temperature. The wells are washed five times with PBST before developing with TMB-solution and read at 630 nm.
[0191]For the cell ELISAs, cells are washed once in PBS and resuspended at a concentration of 1×106 to 2×106 cells/mL of PBS. A final concentration of 1-2×105 cells per well of a 96-well tissue culture plate (Falcon, VWR) can be used. The cells are fixed by adding an equal volume of 0.2% glutaraldehyde (Sigma-Aldrich) and incubating at 37° C. for 12 minutes. They are then washed three times with PBS using an automated plate washer (Bio-Tek Instruments, Inc.) and blocked with 200 μL of 2% MPBST for one hour at room temperature. The rest of the ELISA procedure can be performed as described above except that 1×PBS/Tween 0.05% is used for the washes and incubations.
Diversity
[0192]Display libraries and other libraries include variation at one or more positions in the displayed polypeptide. The variation at a given position can be synthetic or natural. For some libraries, both synthetic and natural diversity are included.
[0193]Synthetic Diversity. Libraries can include regions of diverse nucleic acid sequence that originate from artificially synthesized sequences. Typically, these are formed from degenerate oligonucleotide populations that include a distribution of nucleotides at each given position. The inclusion of a given sequence is random with respect to the distribution. One example of a degenerate source of synthetic diversity is an oligonucleotide that includes NNN wherein N is any of the four nucleotides in equal proportion.
[0194]Synthetic diversity can also be more constrained, e.g., to limit the number of codons in a nucleic acid sequence at a given trinucleotide to a distribution that is smaller than NNN. For example, such a distribution can be constructed using less than four nucleotides at some positions of the codon. In addition, trinucleotide addition technology can be used to further constrain the distribution.
[0195]So-called "trinucleotide addition technology" is described, e.g., in Wells et al. (1985) Gene 34:315-323, U.S. Pat. No. 4,760,025 and U.S. Pat. No. 5,869,644. Oligonucleotides are synthesized on a solid phase support, one codon (i.e., trinucleotide) at a time. The support includes many functional groups for synthesis such that many oligonucleotides are synthesized in parallel. The support is first exposed to a solution containing a mixture of the set of codons for the first position. The unit is protected so additional units are not added. The solution containing the first mixture is washed away and the solid support is deprotected so a second mixture containing a set of codons for a second position can be added to the attached first unit. The process is iterated to sequentially assemble multiple codons. Trinucleotide addition technology enables the synthesis of a nucleic acid that at a given position can encode a number of amino acids. The frequency of these amino acids can be regulated by the proportion of codons in the mixture. Further the choice of amino acids at the given position is not restricted to quadrants of the codon table as is the case if mixtures of single nucleotides are added during the synthesis. Synthetic oligonucleotides including randomized or spiked codons can be also be used for producing a library for an affinity maturation selection.
[0196]Natural Diversity. Libraries can include regions of diverse nucleic acid sequence that originate (or are synthesized based on) from different naturally-occurring sequences. An example of natural diversity that can be included in a display library is the sequence diversity present in immune cells (see also below). Nucleic acids are prepared from these immune cells and are manipulated into a format for polypeptide display.
Antibody Display Libraries
[0197]In one embodiment, the display library presents a diverse pool of proteins, each of which includes an immunoglobulin domain, e.g., an immunoglobulin variable domain. Display libraries are particular useful, for example, for identifying human or effectively human antibodies that recognize human antigens. Such antibodies can be used as therapeutics to treat human disorders such as cancer, e.g., metastatic cancer. Since the constant and framework regions of the antibody are human, these therapeutic antibodies may avoid themselves being recognized and targeted as antigens. The constant regions are also optimized to recruit effector functions of the human immune system. The in vitro display selection process surmounts the inability of a normal human immune system to generate antibodies against self-antigens.
[0198]A typical antibody display library displays a polypeptide that includes a VH domain and a VL domain. An "immunoglobulin domain" refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol. 6:381-405). The display library can display the antibody as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using a single polypeptide chain). Other formats can also be used.
[0199]As in the case of the Fab and other formats, the displayed antibody can include a constant region as part of a light or heavy chain. In one embodiment, each chain includes one constant region, e.g., as in the case of a Fab. In other embodiments, additional constant regions are displayed.
[0200]Antibody libraries can be constructed by a number of processes (see, e.g., de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20. and Hoogenboom et al. (2000) Immunol Today 21:371-8). Further, elements of each process can be combined with those of other processes. The processes can be used such that variation is introduced into a single immunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulin domains (e.g., VH and VL). The variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4, referring to such regions of either and both of heavy and light chain variable domains. In one embodiment, variation is introduced into all three CDRs of a given variable domain. In another preferred embodiment, the variation is introduced into CDR1 and CDR2, e.g., of a heavy chain variable domain. Any combination is feasible. In one process, antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid. The oligonucleotides can be synthesized using a variety of subunits, e.g., monomeric nucleotides or trinucleotides. For example, Knappik et al. (2000) J. Mol. Biol. 296:57-86 describe a method for constructing CDR encoding oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.
[0201]In another process, an animal, e.g., a rodent, is immunized with the MMP-26. The animal is optionally boosted with the antigen to further stimulate the response. Then spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library.
[0202]In yet another process, antibody libraries are constructed from nucleic acid amplified from naive germline immunoglobulin genes (e.g., human genes). The amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain. Sources of immunoglobulin-encoding nucleic acids are described below. Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.
[0203]Nucleic acid encoding immunoglobulin domains or fragments thereof can be obtained from the immune cells of, e.g., a human, a primate, mouse, rabbit, camel, or rodent. In one example, the cells are selected for a particular property. B cells at various stages of maturity can be selected. In another example, the B cells are naive.
[0204]In one embodiment, fluorescent-activated cell sorting (FACS) is used to sort B cells that express surface-bound IgM, IgD, or IgG molecules. Further, B cells expressing different isotypes of IgG can be isolated. In another preferred embodiment, the B or T cell is cultured in vitro. The cells can be stimulated in vitro, e.g., by culturing with feeder cells or by adding mitogens or other modulatory reagents, such as antibodies to CD40, CD40 ligand or CD20, phorbol myristate acetate, bacterial lipopolysaccharide, concanavalin A, phytohemagglutinin or pokeweed mitogen.
[0205]In still another embodiment, the cells are isolated from a subject that has an immunological disorder, e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, vasculitis, Sjogren syndrome, systemic sclerosis, or anti-phospholipid syndrome. The subject can be a human, or an animal, e.g., an animal model for the human disease, or an animal having an analogous disorder. In yet another embodiment, the cells are isolated from a transgenic non-human animal that includes a human immunoglobulin locus.
[0206]In one preferred embodiment, the cells have activated a program of somatic hypermutation. Cells can be stimulated to undergo somatic mutagenesis of immunoglobulin genes, for example, by treatment with anti-immunoglobulin, anti-CD40, and anti-CD38 antibodies (see, e.g., Bergthorsdottir et al. (2001) J Immunol. 166:2228). In another embodiment, the cells are naive.
[0207]The nucleic acid encoding an immunoglobulin variable domain can be isolated from a natural repertoire by the following exemplary method. First, RNA is isolated from the immune cell. Full length (i.e., capped) mRNAs are separated (e.g. by dephosphorylating uncapped RNAs with calf intestinal phosphatase). The cap is then removed with tobacco acid pyrophosphatase and reverse transcription is used to produce the cDNAs.
[0208]The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., de Haard et al. (1999) J. Biol. Chem 274:18218-30. The primer binding region can be constant among different immunoglobulins, e.g., in order to reverse transcribe different isotypes of immunoglobulin. The primer binding region can also be specific to a particular isotype of immunoglobulin. Typically, the primer is specific for a region that is 3' to a sequence encoding at least one CDR. In another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes).
[0209]A synthetic sequence can be ligated to the 3' end of the reverse transcribed strand. The synthetic sequence can be used as a primer binding site for binding of the forward primer during PCR amplification after reverse transcription. The use of the synthetic sequence can obviate the need to use a pool of different forward primers to fully capture the available diversity.
[0210]The variable domain-encoding gene is then amplified, e.g., using one or more rounds. If multiple rounds are used, nested primers can be used for increased fidelity. The amplified nucleic acid is then cloned into a display library vector.
[0211]Any method for amplifying nucleic acid sequences may be used for amplification. Methods that maximize and do not bias diversity are preferred. A variety of techniques can be used for nucleic acid amplification. The polymerase chain reaction (PCR; U.S. Pat. Nos. 4,683,195 and 4,683,202, Saiki, et al. (1985) Science 230, 1350-1354) utilizes cycles of varying temperature to drive rounds of nucleic acid synthesis. Transcription-based methods utilize RNA synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat. No. 6,066,457; U.S. Pat. No. 6,132,997; U.S. Pat. No. 5,716,785; Sarkar et. al., Science (1989) 244: 331-34; Stofler et al., Science (1988) 239: 491). NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517) utilizes cycles of transcription, reverse-transcription, and RNaseH-based degradation to amplify a DNA sample. Still other amplification methods include rolling circle amplification (RCA; U.S. Pat. Nos. 5,854,033 and 6,143,495) and strand displacement amplification (SDA; U.S. Pat. Nos. 5,455,166 and 5,624,825).
Exemplary MMP-26 Inhibition Assays
[0212]Methods for evaluating MMP-26 enzymatic activity are known. See, e.g., Park et al. (2002) J. Biol. Chem. 277:35168-35175. In addition, MMP-26 assay may be monitored by measuring the cleavage of MMP-26-catalyzable substrates including vitronectin and collagen with subsequent detection of the cleaved substrates by standard SDS-PAGE techniques.
[0213]Inhibitory dissociation constants can be determined using 1 μM of the peptide substrate: Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (SEQ ID NO:215). The protein being evaluated can be tested at a range of concentrations, e.g., five to ten different concentrations. Kinetic parameters can be monitored by evaluating fluorescence of 3-methoxycoumarin, by excitation at 328 nm and by monitoring emission at 393 nm. The Ki can be calculating by fitting data to an equation. Kinetic experiments can be performed in 50 mM HEPES pH 7.5, 10 mM CaCl2, 0.2 M NaCl and 0.01% Brij-35. Additional substrates include Mca-Lys-Pro-Ile-Ser(P1)-Leu-Dpa-Ala-Arg-NH2 (SEQ ID NO:216), or Mca-Pro-11e-Ser(P1)-Leu-Dpa-Ala-Arg-NH2 (SEQ ID NO:217). See, e.g., Park et al. (2002) J Biol Chem. 277(38):35168-75.
[0214]Additional assays for inhibition can monitor ability to inhibit MMP-26 mediated activation of MMP-9. Activation is monitored by MMP-9 cleavage of gelatin (Molecular Probes E-12055) or cleavage of an MMP-9 specific peptide (Calbiochem 444221). MMP-26, e.g., at a concentration of 100 nM, is incubated with the candidate inhibitors at varying concentrations for one hour. The proMMP-9, at a concentration of 10 nM, is then added to the mixture followed by fluorescently labeled gelatin or a fluorescently labeled peptide substrate. MMP-9 activity is monitored using a fluorimeter (Molecular Devices) and is observed as an increase in signal.
[0215]In one embodiment, a protein (e.g., an antibody described herein) has a Ki of less than 10-7, 10-8, 10-9, 10-10, or 10-11 M in an assay described herein.
Exemplary Biological Assays
[0216]Potential MMP-26 binding protein can also be evaluated for their activity in vivo. For example, to evaluate the activity of a protein (e.g., an antibody described herein) to reduce tumor growth through binding and/or inhibition of MMP-26, the procedures described by Jankun et al., Canc. Res., 57: 559-563 (1997) to evaluate PAI-1 can be employed. Briefly, the ATCC cell lines DU145 and LNCaP are injected into SCID mice. After tumors are established, the mice are administered the test protein. Tumor volume measurements are taken twice a week for about five weeks. A binding protein can be deemed active in this assay if an animal to which the protein was administered exhibited decreased tumor volume and/or decreased metastatic spread of the tumor, as compared to animals receiving appropriate control compounds (e.g., antibody molecules specific to other proteins, particularly proteins unrelated to tumor growth and metastatic activity, or the formulation without the protein).
[0217]To evaluate the ability of a protein (e.g., an antibody described herein) to reduce the occurrence of, or inhibit, metastasis, the procedures described by Kobayashi et al., Int. J. Canc., 57: 727-733d (1994) can be employed. Briefly, a murine xenograft selected for high lung colonization potential in injected into C57B1/6 mice i.v. (experimental metastasis) or s.c. into the abdominal wall (spontaneous metastasis). Various concentrations of the compound to be tested can be admixed with the tumor cells in MATRIGEL® basement membrane matrix prior to injection. Daily i.p. injections of the test compound are made either on days 1-6 or days 7-13 after tumor inoculation. The animals are sacrificed about three or four weeks after tumor inoculation, and the lung tumor colonies are counted. Evaluation of the resulting data permits a determination as to efficacy of the test compound, optimal dosing and route of administration.
[0218]The activity of the proteins toward decreasing tumor volume and metastasis can be evaluated in model described in Rabbani et al., Int. J. Cancer 63 : 840-845 (1995). See also Xing et al., Canc. Res., 57: 3585-3593 (1997). There, Mat LyLu tumor cells were injected into the flank of Copenhagen rats. The animals were implanted with osmotic minipumps to continuously administer various doses of test compound for up to three weeks. The tumor mass and volume of experimental and control animals were evaluated during the experiment, as were metastatic growths. Evaluation of the resulting data permits a determination as to efficacy of the test compound, optimal dosing, and route of administration. Xing et al., Canc. Res., 57: 3585-3593 (1997) describes a related protocol.
[0219]A protein (e.g., an antibody described herein) can also be evaluated in culture or in an animal for ability to modulate inflammation or an inflammatory disorder. For example, US 20030161810 provides a non-human animal model for an inflammatory disorder (including rheumatoid arthritis), the animal includes human synovial fluid. US 20030176389 describes a dextran sodium sulfate-induced mouse model of colitis. In another example, cell culture is used to monitor adhesion of leukocytes. For example, a compound can be immobilized on a solid surface and adhesion of cells expressing an adhesion molecule can be evaluated for interaction with the surface. Cells suitable for this assay include any leukocytes, such as T cells, B cells, monocytes, eosinophils, and basophils. Exemplary leukocyte cell lines include Jurkat and U937 cells.
[0220]In one embodiment, a protein (e.g., an antibody described herein) has a statistically significant effect in an assay described herein.
Secondary Screening Methods
[0221]After selecting candidate display library members that bind to a target or any candidate MMP-26-binding protein, each candidate protein can be further analyzed, e.g., to further characterize its binding properties for the target. Each candidate display library member can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, a physiological property (e.g., cytotoxicity, renal clearance, immunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.
[0222]As appropriate, the assays can use the display library member directly, a recombinant polypeptide produced from the nucleic acid encoding a displayed polypeptide, a synthetic peptide synthesized based on the sequence of a displayed polypeptide. In the case of a candidate MMP-26 binding protein from any source, the protein can be obtained, e.g., from such a source or by recombinant production. Exemplary assays for binding properties include the following.
[0223]ELISA. Proteins encoded by a display library can also be screened for a binding property using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non-specifically bound polypeptides. Then the amount of the protein bound to the plate is determined by probing the plate with an antibody that can recognize the polypeptide, e.g., a tag or constant portion of the polypeptide. The antibody is linked to an enzyme such as alkaline phosphatase, which produces a colorimetric product when appropriate substrates are provided. The protein can be purified from cells or assayed in a display library format, e.g., as a fusion to a filamentous bacteriophage coat. Alternatively, cells (e.g., live or fixed) that express the target molecule, e.g., MMP-26, can be plated in a microtitre plate and used to test the affinity of the peptides/antibodies present in the display library or obtained by selection from the display library.
[0224]In another version of the ELISA assay, each polypeptide of a diversity strand library is used to coat a different well of a microtitre plate. The ELISA then proceeds using a constant target molecule to query each well.
[0225]Homogeneous Binding Assays. The binding interaction of candidate protein with a target can be analyzed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the `acceptor` molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.
[0226]Another example of a homogenous assay is Alpha Screen (Packard Bioscience, Meriden CT). Alpha Screen uses two labeled beads. One bead generates singlet oxygen when excited by a laser. The other bead generates a light signal when singlet oxygen diffuses from the first bead and collides with it. The signal is only generated when the two beads are in proximity. One bead can be attached to the display library member, the other to the target. Signals are measured to determine the extent of binding.
[0227]The homogenous assays can be performed while the candidate protein is attached to the display library vehicle, e.g., a bacteriophage or using a candidate protein as free molecule.
[0228]Surface Plasmon Resonance (SPR). The binding interaction of a molecule isolated from a display library and a target can be analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden).
[0229]Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (Kd), and kinetic parameters, including Kon and Koff, for the binding of a biomolecule to a target. Such data can be used to compare different biomolecules. For example, proteins encoded by nucleic acid selected from a library of diversity strands can be compared to identify individuals that have high affinity for the target or that have a slow Koff. This information can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow Koff. This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.
[0230]Protein Arrays. Polypeptides identified from the display library can be immobilized on a solid support, for example, on a bead or an array. For a protein array, each of the polypeptides is immobilized at a unique address on a support. Typically, the address is a two-dimensional address. Protein arrays are described below (see, e.g., Diagnostics).
[0231]Cellular Assays. Candidate polypeptides can be selected from a library by transforming the library into a host cell; the library could have been previously identified from a display library. For example, the library can include vector nucleic acid sequences that include segments that encode the polypeptides and that direct expression, e.g., such that the polypeptides are produced within the cell, secreted from the cell, or attached to the cell surface. The cells can be screened or selected for polypeptides that bind to the MMP-26, e.g., as detected by a change in a cellular phenotype or a cell-mediated activity. For example, in the case of an antibody that binds to MMP-26, the activity may be an in vitro assay for cell invasion. In one embodiment, the antibody is contacted to an invasive mammalian cell, e.g., a carcinoma cell, e.g., JEG-3 (choriocarcinoma) cell. The ability of the cell to invade a matrix is evaluated. The matrix can be an artificial matrix, e.g., MATRIGEL® basement membrane matrix, gelatin, etc., or a natural matrix, e.g., extracellular matrix of a tissue sample, or a combination thereof. For example, the matrix can be produced in vitro by a layer of cells.
Protein Production
[0232]Standard recombinant nucleic acid methods can be used to express a MMP-26 binding protein. See, for example, the techniques described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley Interscience, N.Y. (1989). Generally, a nucleic acid sequence encoding the protein of interest is cloned into a nucleic acid expression vector. If the protein includes multiple polypeptide chains, each chain can be cloned into an expression vector, e.g., the same or different vectors, that are expressed in the same or different cells. Methods for producing antibodies also are provided below.
[0233]The expression vector for expressing the protein can include, in addition to the segment encoding the protein or fragment thereof, regulatory sequences, including for example, a promoter, operably linked to the nucleic acid(s) of interest. Vectors are typically tailored for the intended expression system. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs.
[0234]Methods well known to those skilled in the art can be used to construct vectors containing a polynucleotide encoding a ligand and appropriate regulatory signals (e.g., transcriptional/translational control signals). These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley Interscience, N.Y. (1989). Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
[0235]Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., an antibiotic resistance gene for a bacterial cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The polynucleotide is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the polynucleotide can encode a fusion protein including an identification sequence (e.g., a terminus, e.g., N- or C-terminal) imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
[0236]Prokaryotic Expression. Useful expression vectors for bacteria are constructed by inserting a polynucleotide encoding a ligand together with suitable translation initiation and termination signals, optionally in operable reading phase with a functional promoter. The vector can include one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include, e.g., E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
[0237]As a representative but nonlimiting example, useful expression vectors for bacteria can include a selectable marker (e.g., an antibiotic resistance gene) and bacterial origin of replication derived from commercially available plasmids including genetic elements of the well known cloning vector pBR322 (ATCC 37017). Exemplary prokaryotic vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega, Madison, Wis., USA), pBS, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia), as well as phage and phagemid vectors. Exemplary bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P, and trc. Vectors can be introduced into bacterial cells, e.g., by chemical transformation (e.g., the Hanahan protocol), electroporation, or bacteriophage infection.
[0238]If the protein is made in bacteria (or some yeast), it may be necessary to modify the protein produced therein, for example, by glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
[0239]It also is possible to produce host cells containing a vector that can express a ligand that binds to MMP-26. The vector can be introduced into the host cell, e.g., using known transformation, transfection or infection methods. For example, the host cells can include members of a library constructed from the diversity strand. The host cell can be a eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
[0240]Yeast Expression Systems. The host cell for producing a protein ligand (e.g., antibody) also may be a yeast (e.g., Pichia, Hanseula, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, or Candida) or other fungus. In yeast, a number of vectors containing constitutive or inducible promoters may be used. Exemplary yeast promoters include the promoters of genes encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, and heat shock proteins, the PHOS promoter, and GAL promoter, among others. A yeast vector can include a selectable marker, e.g., a drug resistance gene, or an auxotrophic marker (such as the URA3, LEU2, HIS3, and TRP1 genes). It is possible to maintain yeast vectors as extrachromosomal elements in high or low copy and to integrate the vectors into yeast chromosomes (e.g., endogenous or artificial).
[0241]For a review of yeast expression systems, see, e.g., Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Grant et al., Expression and Secretion Vectors for Yeast, inMethods in Enzymology, Ed. Wu & Grossman, Acad. Press, N.Y. 153:516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3 (1986); Bitter, Heterologous Gene Expression in Yeast, in Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y. 152:673-684 (1987); Powers et al. (2001) J Immunol Methods. 251:123-35; and The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II (1982).
[0242]Mammalian Expression. Various mammalian cell culture systems can also be employed to express a protein ligand, e.g., an antibody. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts (described by Gluzman, Cell 23:175 (1981)), the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells. Mammalian expression vectors can include an origin of replication, a suitable promoter and also any necessary ribosome-binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
[0243]Exemplary eukaryotic vectors include: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, mouse metallothionein-I, and various art-known tissue specific promoters. In one example, DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements. Introduction of the recombinant construct into a mammalian host cell can be effected, for example, by calcium phosphate transfection, DEAE, dextran mediated transfection, electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)), or viral infection.
[0244]In another embodiment, cells and tissues may be engineered to express an endogenous gene that encodes a protein ligand described herein or a protein target. The method includes using homologous recombination to replace regulatory sequences of the endogenous gene with heterologous regulatory sequences, e.g., inducible regulatory elements. Such regulatory sequences may include promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals. Messenger RNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.
[0245]Purification. Protein ligands can be purified from cells or from media surrounding cells. Cells expressing the proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Recombinant polypeptides and proteins produced in culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. In some embodiments, the protein also includes a polypeptide tag, e.g., penta- or hexa-histidine. The recombinant polypeptides can then be purified using affinity chromatography. Scopes (1994) Protein Purification: Principles and Practice, New York:Springer-Verlag provides a number of general methods for purifying recombinant (and non-recombinant) proteins. The methods include, e.g., ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, selective precipitation, dialysis, and hydrophobic interaction chromatography. These methods can be adapted for devising a purification strategy for the MMP-26-binding protein. For ligands that include an Fc domain, one type of affinity chromatography uses immobilized protein A or protein G.
[0246]Antibody Production. Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells. For example, if the Fab is encoded by sequences in a phage display vector that includes a suppressible stop codon between the display entity and a bacteriophage protein (or fragment thereof), the vector nucleic acid can be shuffled into a bacterial cell that cannot suppress a stop codon. In this case, the Fab is not fused to the gene III protein and is secreted into the media.
[0247]Antibodies can also be produced in eukaryotic cells. In one embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al. (2001) J Immunol Methods. 251:123-35), Hanseula, or Saccharomyces.
[0248]In one embodiment, antibodies are produced in mammalian cells. Preferred mammalian host cells for expressing the clone antibodies or antigen-binding fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.
[0249]In addition to the nucleic acid sequence encoding the immunoglobulin domain, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
[0250]In an exemplary system for recombinant expression of an antibody (e.g., a full length antibody or an antigen-binding portion thereof), a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
[0251]For antibodies that include an Fc domain, the antibody production system preferably synthesizes antibodies in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fcγ receptors and complement C1q (Burton and Woof (1992) Adv. Immunol. 51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76). In a preferred embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297. The Fc domain can also include other eukaryotic post-translational modifications.
[0252]Antibodies also can be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly.
[0253]It also is possible to produce antibodies that bind to MMP-26 by immunization, e.g., using an animal, e.g., with natural, human, or partially human immunoglobulin loci. Non-human antibodies also can be modified to include substitutions for human immunoglobulin sequences, e.g., consensus human amino acid residues at particular positions, e.g., at one or more of the following positions (preferably at least five, ten, twelve, or all): (in the FR of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, and/or (in the FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and/or 103H (according to the Kabat numbering). See, e.g., U.S. Pat. No. 6,407,213.
[0254]MMP-26 production. Method for producing a MMP-26 protein or a catalytic domain thereof are known in the art. See, e.g., the example below and Park, et al. (2000) J. Biol. Chem. 275, 20540-20544.
[0255]Biotinylation Methods. A variety of methods are available to biotinylate proteins, e.g., an immunoglobulin protein or a target protein. For example, the protein can be incubated with a 5-fold molar excess of sulfo-NHS-SS-biotin in 50 mM HEPES, pH 8.0, 100 mM NaCl overnight at 4° C. Free biotin is removed by buffer exchange into PBS, 0.01% Tween 20, e.g., using a Biomax device with a 10 kDa molecular weight cut-off membrane or by dialysis. The number of biotin molecules incorporated per mole of protein can be determined using the HABA assay as described by the manufacturer (Pierce).
Pharmaceutical Compositions
[0256]In another aspect, the invention provides compositions, e.g., pharmaceutically acceptable compositions, which include an MMP-26-binding protein, e.g., an antibody molecule, other polypeptide or peptide identified as binding to MMP-26, or described herein, formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutical compositions" encompass labeled ligands (e.g., for in vivo imaging) as well as therapeutic compositions.
[0257]As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., protein ligand may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
[0258]A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
[0259]The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of humans with antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the MMP-26-binding protein is administered by intravenous infusion or injection. In another preferred embodiment, the MMP-26-binding protein is administered by intramuscular or subcutaneous injection.
[0260]The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
[0261]Pharmaceutical compositions typically are sterile and stable under the conditions of manufacture and storage. A pharmaceutical composition can also be tested to insure it meets regulatory and industry standards for administration. For example, endotoxin levels in the preparation can be tested using the Limulus amebocyte lysate assay (e.g., using the kit from Bio Whittaker lot # 7L3790, sensitivity 0.125 EU/mL) according to the USP 24/NF 19 methods. Sterility of pharmaceutical compositions can be determined using thioglycollate medium according to the USP 24/NF 19 methods. For example, the preparation is used to inoculate the thioglycollate medium and incubated at 35° C. for 14 or more days. The medium is inspected periodically to detect growth of a microorganism.
[0262]The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the ligand) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[0263]An MMP-26-binding protein can be administered by a variety of methods known in the art. For many applications, the route/mode of administration is intravenous injection or infusion. For example, for therapeutic applications, the MMP-26-binding protein can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or 7 to 25 mg/m2. The route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0264]In certain embodiments, the ligand may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) also may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound described herein by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
[0265]Pharmaceutical compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a pharmaceutical composition described herein can be administered with a needle-less hypodermic injection device, such as the devices disclosed in U.S. Pat. No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Of course, many other such implants, delivery systems, and modules also are known.
[0266]In certain embodiments, the compounds described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that a therapeutic can cross the BBB (if desired), it can be formulated, for example, in a liposome. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may include one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685).
[0267]Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in compounding such an active compound for the treatment of sensitivity in individuals.
[0268]An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody described herein is 0.1-20 mg/kg, more preferably 1-10 mg/kg. The MMP-26-binding antibody can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m2 or about 5 to 30 mg/m2. For ligands smaller in molecular weight than an antibody, appropriate amounts can be proportionally less. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit.
[0269]A pharmaceutical composition may include a "therapeutically effective amount" or a "prophylactically effective amount" of an MMP-26-binding protein, e.g., a protein described herein. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably inhibits a measurable parameter, e.g., inflammation or tumor growth rate by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit a measurable parameter, e.g., cancer, can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
[0270]A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
[0271]Also within the scope of the invention are kits including the protein ligand that binds to MMP-26 and instructions for use, e.g., treatment, prophylactic, or diagnostic use. In one embodiment, the instructions for diagnostic applications include the use of the MMP-26-binding protein (e.g., antibody or antigen-binding fragment thereof, or other polypeptide or peptide) to detect MMP-26, in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient having an inflammatory disorder or a cancer or neoplastic disorder, or in vivo. In another embodiment, the instructions for therapeutic applications include suggested dosages and/or modes of administration in a patient with a cancer or neoplastic disorder. The kit can further contain a least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, and/or one or more additional MMP-26-binding proteins, formulated as appropriate, in one or more separate pharmaceutical preparations.
Stabilization and Retention
[0272]In one embodiment, an MMP-26-binding protein (e.g., a MMP-26-binding antibody described herein) is physically associated with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, or other tissues.
[0273]For example, a MMP-26-binding protein can be associated with a polymer, e.g., a substantially non-antigenic polymers, such as polyalkylene oxides or polyethylene oxides. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 50,000, e.g., 1,000 to 15,000, 2,000 to 12,500, or 10,000 to about 30,000 are usually selected for the purposes of the present invention.
[0274]For example, an MMP-26-binding protein can be conjugated to a water soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides which comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparon.
[0275]Other compounds can also be attached to the same polymer, e.g., a cytotoxin, a label, or another targeting agent, e.g., another MMP-26-binding protein or an unrelated ligand. Mono-activated, alkoxy-terminated polyalkylene oxides (PAO's), e.g., monomethoxy-terminated polyethylene glycols (mPEG's); C1-4 alkyl-terminated polymers; and bis-activated polyethylene oxides (glycols) can be used for crosslinking. See, e.g., U.S. Pat. No. 5,951,974
[0276]In one embodiment, the polymer prior to cross-linking need not be, but preferably is, water soluble. Generally, after crosslinking, the product is water soluble, e.g., exhibits a water solubility of at least about 0.01 mg/ml, and more preferably at least about 0.1 mg/ml, and still more preferably at least about 1 mg/ml. In addition, the polymer should not be highly immunogenic in the conjugate form, nor should it possess viscosity that is incompatible with intravenous infusion or injection if the conjugate is intended to be administered by such routes.
[0277]In one embodiment, the polymer contains only a single group which is reactive. This helps to avoid cross-linking of protein molecules. However, it is within the scope herein to maximize reaction conditions to reduce cross-linking, or to purify the reaction products through gel filtration or ion exchange chromatography to recover substantially homogenous derivatives. In other embodiments, the polymer contains two or more reactive groups for the purpose of linking multiple ligands to the polymer backbone. Again, gel filtration or ion exchange chromatography can be used to recover the desired derivative in substantially homogeneous form.
[0278]The molecular weight of the polymer can range up to about 500,000 D, and preferably is at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D. The molecular weight chosen can depend upon the effective size of the conjugate to be achieved, the nature (e.g. structure, such as linear or branched) of the polymer, and the degree of derivatization.
[0279]The covalent crosslink can be used to attach an MMP-26-binding protein to a polymer, for example, crosslinking to the N-terminal amino group and epsilon amino groups found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. The polymer may be covalently bonded directly to the MMP-26-binding protein without the use of a multifunctional (ordinarily bifunctional) crosslinking agent. Covalent binding to amino groups is accomplished by known chemistries based upon cyanuric chloride, carbonyl diimidazole, aldehyde reactive groups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde, activated succinimidyl esters, activated dithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activated PEG.) Carboxyl groups can be derivatized by coupling PEG-amine using carbodiimide. Sulfhydryl groups can be derivatized by coupling to maleimido-substituted PEG (e.g. alkoxy-PEG amine plus sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) WO 97/10847 or PEG-maleimide commercially available from Shearwater Polymers, Inc., Huntsville, Ala.). Alternatively, free amino groups on the ligand (e.g. epsilon amino groups on lysine residues) can be thiolated with 2-imino-thiolane (Traut's reagent) and then coupled to maleimide-containing derivatives of PEG, e.g., as described in Pedley et al., Br. J. Cancer, 70: 1126-1130 (1994).
[0280]Functionalized PEG polymers that can be attached to an MMP-26-binding protein are available, e.g., from Shearwater Polymers, Inc. (Huntsville, Ala.). Such commercially available PEG derivatives include, e.g., amino-PEG, PEG amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEG succinimidyl succinate, PEG succinimidyl propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl esters of amino acid PEGs, PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEG vinylsulfone, PEG-maleimide, PEG-orthopyridyl-disulfide, heterofunctional PEGs, PEG vinyl derivatives, PEG silanes, and PEG phospholides. The reaction conditions for coupling these PEG derivatives may vary depending on the MMP-26-binding protein, the desired degree of PEGylation, and the PEG derivative utilized. Some factors involved in the choice of PEG derivatives include: the desired point of attachment (such as lysine or cysteine R-groups), hydrolytic stability and reactivity of the derivatives, stability, toxicity and antigenicity of the linkage, suitability for analysis, etc. Specific instructions for the use of any particular derivative are available from the manufacturer.
[0281]The conjugates of an MMP-26-binding protein and a polymer can be separated from the unreacted starting materials, e.g., by gel filtration or ion exchange chromatography, e.g., HPLC. Heterologous species of the conjugates are purified from one another in the same fashion. Resolution of different species (e.g. containing one or two PEG residues) is also possible due to the difference in the ionic properties of the unreacted amino acids. See, e.g., WO 96/34015.
Treatments
[0282]Protein ligands that bind to MMP-26 (e.g., those described herein) have therapeutic and prophylactic utilities. For example, these ligands can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent, and/or diagnose a variety of disorders, such as cancers, particularly metastatic cancers, an inflammatory disorder, and other disorders associated with increased MMP-26 activity, e.g., a disorder of the endometrium or placental.
[0283]As used herein, the term "treat" or "treatment" is defined as the application or administration of an MMP-26-binding antibody, alone or in combination with, a second agent to a subject, e.g., a patient, or application or administration of the agent to an isolated tissue or cell, e.g., cell line, from a subject, e.g., a patient, who has a disorder (e.g., a disorder as described herein), a symptom of a disorder or a predisposition toward a disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. Treating a cell refers to the inhibition of growth or activity, ablation, killing of a cell in vitro or in vivo, or otherwise reducing capacity of a cell, e.g., an aberrant cell, to mediate a disorder, e.g., a disorder as described herein (e.g., a cancerous disorder).
[0284]In one embodiment, "treating a cell" or "treating a tissue" refers to a reduction in the activity and/or proliferation of a cell, e.g., a hyperproliferative cell, or a tissue, e.g., a tumor. Such reduction includes a reduction, e.g., a statistically significant reduction, in the activity of a cell or tissue (e.g., metastatic tissue) or the number of the cell or size of the tissue. An example of a reduction in activity is a reduction in migration of the cell (e.g., migration through an extracellular matrix) or a reduction in cell differentiation. Another example is an activity that, directly or indirectly, reduces inflammation or an indicator of inflammation.
[0285]As used herein, an amount of an MMP-26-binding protein effective to treat a disorder, or a "therapeutically effective amount" refers to an amount of the ligand which is effective, upon single or multiple dose administration to a subject, in treating a cell, (e.g., a MMP-26-expressing cell or cancer cell, particularly a metastatic cell thereof), or in prolonging curing, alleviating, relieving or improving a subject with a disorder as described herein beyond that expected in the absence of such treatment. As used herein, "inhibiting the growth" of the neoplasm refers to slowing, interrupting, arresting or stopping its growth and metastases and does not necessarily indicate a total elimination of the neoplastic growth.
[0286]As used herein, an amount of an MMP-26-binding protein effective to prevent a disorder, or a "a prophylactically effective amount" of the ligand refers to an amount of an MMP-26-binding protein, e.g., an MMP-26-binding antibody described herein, which is effective, upon single- or multiple-dose administration to the subject, in preventing or delaying the occurrence of the onset or recurrence of a disorder, e.g., a metastatic disorder or a cancer.
[0287]The terms "induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, e.g., which denote quantitative differences between two states, refer to a difference, e.g., a statistically significant difference, between the two states. For example, "an amount effective to inhibit the proliferation of the MMP-26-expressing hyperproliferative cells" means that the rate of growth of the cells will be different, e.g., statistically significantly different, from the untreated cells.
[0288]Exemplary subjects include human and non-human animals. Preferred human animals include a human patient having a disorder characterized by abnormal cell proliferation or cell differentiation, or an inflammatory disorder, or other disorder described herein. Exemplary non-human animals include all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, sheep, dog, cow, pig, etc.
[0289]In one embodiment, the subject is a human subject. Alternatively, the subject can be a mammal expressing a MMP-26-like antigen with which an antibody described herein cross-reacts. A protein ligand described herein can be administered to a human subject for therapeutic purposes (discussed further below). Moreover, an MMP-26-binding protein can be administered to a non-human mammal expressing the MMP-26-like antigen to which the ligand binds (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of the ligand (e.g., testing of dosages and time courses of administration).
[0290]In one embodiment, the invention provides a method of treating (e.g., ablating or killing) a cell (e.g., a non-cancerous cell, e.g., a normal, benign or hyperplastic cell, or a cancerous cell, e.g., a malignant cell, e.g., cell found in a solid tumor, a soft tissue tumor, or a metastatic lesion (e.g., a cell found in renal, urothelial, colonic, rectal, pulmonary, breast or hepatic, cancers and/or metastasis)). The method can include the steps of contacting the cell with an MMP-26-binding protein, e.g., an MMP-26-binding antibody described herein, in an amount sufficient to treat or prevent a disorder, e.g., a disorder caused by a cancerous cell, e.g., a metastatic cell.
[0291]The subject method can be used on cells in culture, e.g. in vitro or ex vivo. For example, cancerous or metastatic cells (e.g., renal, urothelial, colon, rectal, lung, breast, endometrial, ovarian, prostatic, or liver cancerous or metastatic cells) can be cultured in vitro in culture medium and the contacting step can be effected by adding the MMP-26-binding protein to the culture medium. The method can be performed on cells (e.g., cancerous or metastatic cells) present in a subject, as part of an in vivo (e.g., therapeutic or prophylactic) protocol. For in vivo embodiments, the contacting step is effected in a subject and includes administering the MMP-26-binding protein to the subject under conditions effective to permit both binding of the ligand to the cell and the treating, e.g., a disorder.
[0292]The method can be used to treat a cancer. As used herein, the terms "cancer", "hyperproliferative", "malignant", and "neoplastic" are used interchangeably, and refer to those cells an abnormal state or condition characterized by rapid proliferation or neoplasm. The terms include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth.
[0293]The common medical meaning of the term "neoplasia" refers to "new cell growth" that results as a loss of responsiveness to normal growth controls, e.g. to neoplastic cell growth. A "hyperplasia" refers to cells undergoing an abnormally high rate of growth. However, as used herein, the terms neoplasia and hyperplasia can be used interchangeably, as their context will reveal, referring generally to cells experiencing abnormal cell growth rates. Neoplasias and hyperplasias include "tumors," which may be benign, premalignant or malignant.
[0294]Examples of cancerous disorders include, but are not limited to, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract (e.g., renal, urothelial cells), pharynx, prostate, ovary as well as adenocarcinomas which include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and so forth. Metastatic lesions of the aforementioned cancers also can be treated or prevented using a method or composition described herein.
[0295]The subject method can be useful in treating malignancies of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary tract, prostate, ovary, pharynx, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. Exemplary solid tumors that can be treated include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, ovarian carcinoma, endometrial carcinoma, breast carcinoma, choriocarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
[0296]The term "carcinoma" is recognized by those skilled in the art and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include choriocarcinomas and those forming from tissue of the cervix, lung, prostate, breast, endometrium, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
[0297]The term "sarcoma" is recognized by those skilled in the art and refers to malignant tumors of mesenchymal derivation.
[0298]The subject method also can be used to inhibit the proliferation of hyperplastic/neoplastic cells of hematopoietic origin shown to express MMP-26, e.g., a B cells.
[0299]Methods of administering MMP-26-binding proteins are described in "Pharmaceutical Compositions". Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The ligands can be used as competitive agents to inhibit, reduce an undesirable interaction, e.g., between a natural or pathological agent and the MMP-26.
[0300]In one embodiment, the MMP-26-binding proteins are used to kill or ablate cancerous cells and normal, benign hyperplastic, and cancerous cells in vivo. The ligands can be used by themselves or conjugated to an agent, e.g., a cytotoxic drug, radioisotope. This method includes: administering the ligand alone or attached to a cytotoxic drug, to a subject requiring such treatment.
[0301]The terms "cytotoxic agent" and "cytostatic agent" and "anti-tumor agent" are used interchangeably herein and refer to agents that have the property of inhibiting the growth or proliferation (e.g., a cytostatic agent), or inducing the killing, of hyperproliferative cells, e.g., an aberrant cancer cell. In cancer therapeutic embodiment, the term "cytotoxic agent" is used interchangeably with the terms "anti-cancer" or "anti-tumor" to mean an agent, which inhibits the development or progression of a neoplasm, particularly a solid tumor, a soft tissue tumor, or a metastatic lesion.
[0302]Nonlimiting examples of anti-cancer agents include, e.g., antimicrotubule agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis, radiation, and antibodies against other tumor-associated antigens (including naked antibodies, immunotoxins and radioconjugates). Examples of the particular classes of anti-cancer agents are provided in detail as follows: antitubulin/antimicrotubule, e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin, taxotere; topoisomerase I inhibitors, e.g., topotecan, camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin, idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantrone hydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU), methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin, alanosine, pyrazofurin, N-Phosphoracetyl-L-Asparate=PALA, pentostatin, 5-azacitidine, 5-Aza 2'-deoxycytidine, ara-A, cladribine, 5-fluorouridine, FUDR, tiazofurin, N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-- 2-thenoyl]-L-glutamic acid; alkylating agents, e.g., cisplatin, carboplatin, mitomycin C, BCNU=Carmustine, melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen mustard, uracil mustard, pipobroman, 4-ipomeanol; agents acting via other mechanisms of action, e.g., dihydrolenperone, spiromustine, and desipeptide; biological response modifiers, e.g., to enhance anti-tumor responses, such as interferon; apoptotic agents, such as actinomycin D; and anti-hormones, for example anti-estrogens such as tamoxifen or, for example antiandrogens such as 4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluorometh- yl)propionanilide.
[0303]Since the MMP-26-binding proteins recognize MMP-26-expressing cancer cells, e.g., cancerous lung, liver, colon, breast, endometrium, ovarian, epidermal, laryngeal, and cartilage cells, and particularly metastatic cells thereof, any such cells to which the ligands bind are destroyed. Alternatively, the ligands bind to cells in the vicinity of the cancerous cells and kill them, thus indirectly attacking the cancerous cells which may rely on surrounding cells for nutrients, growth signals and so forth. Thus, the MMP-26-binding proteins (e.g., modified with a cytotoxin) can selectively kill or ablate cells in cancerous tissue (including the cancerous cells themselves).
[0304]The ligands may be used to deliver a variety of cytotoxic drugs including therapeutic drugs, a compound emitting radiation, molecules of plants, fungal, or bacterial origin, biological proteins, and mixtures thereof. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as short-range radiation emitters, including, for example, short-range, high-energy α-emitters, as described herein.
[0305]Enzymatically active toxins and fragments thereof are exemplified by diphtheria toxin A fragment, nonbinding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sacrin, certain Aleurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin, and enomycin. Procedures for preparing enzymatically active polypeptides of the immunotoxins are described in WO84/03508 and WO85/03508, and in the appended Examples below. Examples of cytotoxic moieties that can be conjugated to the antibodies include adriamycin, chlorambucil, daunomycin, methotrexate, neocarzinostatin, and platinum.
[0306]In the case of polypeptide toxins, recombinant nucleic acid techniques can be used to construct a nucleic acid that encodes the ligand (or a protein component thereof) and the cytotoxin (or a protein component thereof) as translational fusions. The recombinant nucleic acid is then expressed, e.g., in cells and the encoded fusion polypeptide isolated.
[0307]Procedures for conjugating protein ligands (e.g., antibodies) with the cytotoxic agents have been previously described. Procedures for conjugating chlorambucil with antibodies are described by Flechner (1973) European Journal of Cancer, 9:741-745; Ghose et al. (1972) British Medical Journal, 3:495-499; and Szekerke, et al. (1972) Neoplasma, 19:211-215. Procedures for conjugating daunomycin and adriamycin to antibodies are described by Hurwitz, E. et al. (1975) Cancer Research, 35:1175-1181 and Amon et al. (1982) Cancer Surveys, 1:429-449. Procedures for preparing antibody-ricin conjugates are described in U.S. Pat. No. 4,414,148 and by Osawa, T., et al. (1982) Cancer Surveys, 1:373-388 and the references cited therein. Coupling procedures as also described in EP 86309516.2.
[0308]To kill or ablate normal, benign hyperplastic, or cancerous cells, a first protein ligand is conjugated with a prodrug which is activated only when in close proximity with a prodrug activator. The prodrug activator is conjugated with a second protein ligand, preferably one which binds to a non-competing site on the target molecule. Whether two protein ligands bind to competing or non-competing binding sites can be determined by conventional competitive binding assays. Some suitable drug-prodrug pairs are described in Blakely et al., (1996) Cancer Research, 56:3287-3292.
[0309]Alternatively, the MMP-26-binding protein can be coupled to high energy radiation emitters, for example, a radioisotope, such as 131I, a γ-emitter, which, when localized at the tumor site, results in a killing of several cell diameters. See, e.g., S. E. Order, "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press 1985). Other suitable radioisotopes include α-emitters, such as 212Bi, 213Bi, and 211At, and β-emitters, such as 186Re and 90Y. Moreover, Lu117 may also be used as both an imaging and cytotoxic agent.
[0310]Radioimmunotherapy (RIT) using antibodies labeled with 131I, 90Y, and 177Lu is under intense clinical investigation. There are significant differences in the physical characteristics of these three nuclides and as a result, the choice of radionuclide is very critical in order to deliver maximum radiation dose to the tumor. The higher beta energy particles of 90Y may be good for bulky tumors. The relatively low energy beta particles of 131I are ideal, but in vivo dehalogenation of radioiodinated molecules is a major disadvantage for internalizing antibody. In contrast, 177Lu has low energy beta particle with only 0.2-0.3 mm range and delivers much lower radiation dose to bone marrow compared to 90Y. In addition, due to longer physical half-life (compared to 90Y), the tumor residence times are higher. As a result, higher activities (more mCi amounts) of 177Lu labeled agents can be administered with comparatively less radiation dose to marrow. There have been several clinical studies investigating the use of 177Lu labeled antibodies in the treatment of various cancers. (Mulligan T et al. (1995) Clin Cancer Res. 1:1447-1454; Meredith R F, et al. (1996) J Nucl Med 37:1491-1496; Alvarez R D, et al. (1997) Gynecologic Oncology 65: 94-101).
[0311]The MMP-26-binding proteins can be used directly in vivo to eliminate antigen-expressing cells via natural complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the protein includes a complement binding effector domain, such as an Fc portion (e.g., functional portion) from IgG1, -2, or -3 or corresponding portions of IgM which bind complement. In one embodiment, a population of target cells is ex vivo treated with a binding protein described herein and appropriate effector cells. The treatment can be supplemented by the addition of complement or serum containing complement. In another embodiment, target cells coated with the protein ligand which includes a complement binding effector domain are lysed by complement.
[0312]Also encompassed by the invention is a method of killing or ablating which involves using the MMP-26 binding proteins for prophylaxis. For example, these materials can be used to prevent or delay development or progression of cancers.
[0313]Use of the therapeutic methods of the invention to treat cancers has a number of benefits. Since the protein ligands specifically recognize MMP-26, other tissue is spared and high levels of the agent are delivered directly to the site where therapy is required. Treatment in accordance with the invention can be effectively monitored with clinical parameters. Alternatively, these parameters can be used to indicate when such treatment should be employed.
[0314]A MMP-26-binding protein described herein can be administered in combination with one or more of the existing modalities for treating cancers, including, but not limited to: surgery; radiation therapy, and chemotherapy.
[0315]An MMP-26-binding protein can be administered in combination with one or more of the existing modalities for treating an inflammatory disease or disorder. Exemplary inflammatory diseases or disorders include: acute and chronic immune and autoimmune pathologies, such as, but not limited to, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA), psoriasis, graft versus host disease (GVHD), scleroderma, diabetes mellitus, allergy; asthma, acute or chronic immune disease associated with an allogenic transplantation, such as, but not limited to, renal transplantation, cardiac transplantation, bone marrow transplantation, liver transplantation, pancreatic transplantation, small intestine transplantation, lung transplantation and skin transplantation; chronic inflammatory pathologies such as, but not limited to, sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology or disease; vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, Kawasaki's pathology and vasculitis syndromes, such as, but not limited to, polyarteritis nodosa, Wegener's granulomatosis, Henoch-Schonlein purpura, giant cell arthritis and microscopic vasculitis of the kidneys; chronic active hepatitis; Sjogren's syndrome; psoriatic arthritis; enteropathic arthritis; reactive arthritis and arthritis associated with inflammatory bowel disease; and uveitis.
[0316]Inflammatory bowel diseases (IBD) include generally chronic, relapsing intestinal inflammation. IBD refers to two distinct disorders, Crohn's disease and ulcerative colitis (UC). The clinical symptoms of IBD include intermittent rectal bleeding, crampy abdominal pain, weight loss and diarrhea. A clinical index can also be used to monitor IBD such as the Clinical Activity Index for Ulcerative Colitis. See also, e.g., Walmsley et al. Gut. 1998 July; 43(1):29-32 and Jowett et al. (2003) Scand J Gastroenterol. 38(2):164-71.
[0317]A MMP26-binding protein can be used to treat or prevent one of the foregoing diseases or disorders. For example, the protein can be administered (locally or systemically) in an amount effective to ameliorate at least one symptom of the respective disease or disorder. The protein may also ameliorate inflammation, e.g., an indicator of inflammation, e.g., such as local temperature, swelling (e.g., as measured), redness, local or systemic white blood cell count, presence or absence of neutrophils, cytokine levels, elastase activity, and so forth. It is possible to evaluate a subject, e.g., prior, during, or after administration of the protein, for one or more of indicators of inflammation, e.g., an aforementioned indicator.
Diagnostic Uses
[0318]Protein ligands that bind to MMP-26 (e.g., those described herein) also have in vitro and in vivo diagnostic, therapeutic and prophylactic utilities. In one aspect, the invention provides a diagnostic method for detecting the presence of a MMP-26, in vitro (e.g., a biological sample, such as tissue, biopsy, e.g., a cancerous tissue such as a tumor) or in vivo (e.g., in vivo imaging in a subject).
[0319]The method includes: (i) contacting a sample with MMP-26-binding protein; and (ii) detecting formation of a complex between the MMP-26-binding protein and the sample. The method can also include contacting a reference sample (e.g., a control sample) with the ligand, and determining the extent of formation of the complex between the ligand and the sample relative to the same for the reference sample. A change, e.g., a statistically significant change, in the formation of the complex in the sample or subject relative to the control sample or subject can be indicative of the presence of MMP-26 in the sample.
[0320]Another method includes: (i) administering the MMP-26-binding protein to a subject; and (ii) detecting formation of a complex between the MMP-26-binding protein, and the subject. The detecting can include determining location or time of formation of the complex. In one embodiment, the subject has, is suspected of having, or is at risk for a disorder described herein, e.g., a neoplastic disorder, an inflammatory disorder, or a disorder characterized by excessive MMP-26 activity.
[0321]The MMP-26-binding protein can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
[0322]Complex formation between the MMP-26-binding protein and MMP-26 can be detected by measuring or visualizing either the ligand bound to the MMP-26 or unbound ligand. Conventional detection assays can be used, e.g., an enzyme-linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. Further to labeling the MMP-26-binding protein, the presence of MMP-26 can be assayed in a sample by a competition immunoassay utilizing standards labeled with a detectable substance and an unlabeled MMP-26-binding protein. In one example of this assay, the biological sample, the labeled standards and the MMP-26 binding agent are combined and the amount of labeled standard bound to the unlabeled ligand is determined. The amount of MMP-26 in the sample is inversely proportional to the amount of labeled standard bound to the MMP-26 binding agent.
[0323]Fluorophore and chromophore labeled protein ligands can be prepared. Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer (1968) Science, 162:526 and Brand, L. et al. (1972) Annual Review of Biochemistry, 41:843-868. The protein ligands can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110. One group of fluorescers having a number of the desirable properties described above is the xanthene dyes, which include the fluoresceins and rhodamines. Another group of fluorescent compounds are the naphthylamines. Once labeled with a fluorophore or chromophore, the protein ligand can be used to detect the presence or localization of the MMP-26 in a sample, e.g., using fluorescent microscopy (such as confocal or deconvolution microscopy).
[0324]Histological Analysis. Immunohistochemistry can be performed using the protein ligands described herein. For example, in the case of an antibody, the antibody can synthesized with a label (such as a purification or epitope tag), or can be detectably labeled, e.g., by conjugating a label or label-binding group. For example, a chelator can be attached to the antibody. The antibody is then contacted to a histological preparation, e.g., a fixed section of tissue that is on a microscope slide. After an incubation for binding, the preparation is washed to remove unbound antibody. The preparation is then analyzed, e.g., using microscopy, to identify if the antibody bound to the preparation.
[0325]Of course, the antibody (or other polypeptide or peptide) can be unlabeled at the time of binding. After binding and washing, the antibody is labeled in order to render it detectable.
[0326]Protein Arrays. The MMP-26-binding protein can also be immobilized on a protein array. The protein array can be used as a diagnostic tool, e.g., to screen medical samples (such as isolated cells, blood, sera, biopsies, and the like). Of course, the protein array can also include other ligands, e.g., that bind to MMP-26 or to other target molecules, such as hyaluronic acid.
[0327]Methods of producing polypeptide arrays are described, e.g., in De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28, e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be spotted at high speed, e.g., using commercially available robotic apparati, e.g., from Genetic MicroSystems or BioRobotics. The array substrate can be, for example, nitrocellulose, plastic, glass, e.g., surface-modified glass. The array can also include a porous matrix, e.g., acrylamide, agarose, or another polymer.
[0328]For example, the array can be an array of antibodies, e.g., as described in De Wildt, supra. Cells that produce the protein ligands can be grown on a filter in an arrayed format. Polypeptide production is induced, and the expressed polypeptides are immobilized to the filter at the location of the cell.
[0329]A protein array can be contacted with a labeled target to determine the extent of binding of the target to each immobilized polypeptide from the diversity strand library. If the target is unlabeled, a sandwich method can be used, e.g., using a labeled probed, to detect binding of the unlabeled target.
[0330]Information about the extent of binding at each address of the array can be stored as a profile, e.g., in a computer database. The protein array can be produced in replicates and used to compare binding profiles, e.g., of a target and a non-target. Thus, protein arrays can be used to identify individual members of the diversity strand library that have desired binding properties with respect to one or more molecules.
[0331]An MMP-26-binding protein described herein can also be used to detecting binding of a MMP-26 to an insoluble support. For example, a sample can be immobilized on array, and MMP-26 can be detected on the array using the MMP-26-binding protein.
[0332]In Vivo Imaging. In still another embodiment, the invention provides a method for detecting the presence of a MMP-26-expressing cancerous tissues in vivo. The method includes (i) administering to a subject (e.g., a patient having a cancer or neoplastic disorder) an MMP-26-binding antibody, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to the MMP-26-expressing tissues or cells. For example, the subject is imaged, e.g., by NMR or other tomographic means.
[0333]Examples of labels useful for diagnostic imaging in accordance with the invention include radiolabels such as 131I, 111In, 123I, 99mTc, 32P, 125I, 3H, 14C, and 188Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes can also be employed. The protein ligand can be labeled with such reagents using known techniques. For example, see Wensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York for techniques relating to the radiolabeling of antibodies and D. Colcher et al. (1986) Meth. Enzymol. 121: 802-816.
[0334]A radiolabeled ligand of this invention can also be used for in vitro diagnostic tests. The specific activity of an isotopically-labeled ligand depends upon the half-life, the isotopic purity of the radioactive label, and how the label is incorporated into the antibody.
[0335]Procedures for labeling polypeptides with the radioactive isotopes (such as 14C, 3H, 35S, 125I, 32P, 131I) are generally known. For example, tritium labeling procedures are described in U.S. Pat. No. 4,302,438. Iodinating, tritium labeling, and 35S labeling procedures, e.g., as adapted for murine monoclonal antibodies, are described, e.g., by Goding, J. W. (Monoclonal antibodies: principles and practice: production and application of monoclonal antibodies in cell biology, biochemistry, and immunology 2nd ed. London; Orlando: Academic Press, 1986. pp 124-126) and the references cited therein. Other procedures for iodinating polypeptides, such as antibodies, are described by Hunter and Greenwood (1962) Nature 144:945, David et al. (1974) Biochemistry 13:1014-1021, and U.S. Pat. Nos. 3,867,517 and 4,376,110. Exemplary radio isotopes that are useful for imaging include 123I, 131I, 111In, and 99mTc. Procedures for iodinating antibodies are described by Greenwood, F. et al. (1963) Biochem. J. 89:114-123; Marchalonis, J. (1969) Biochem. J. 113:299-305; and Morrison, M. et al. (1971) Immunochemistry 289-297. Procedures for 99mTc-labeling are described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer, New York: Masson 111-123 (1982) and the references cited therein. Procedures suitable for 111In-labeling antibodies are described by Hnatowich, D. J. et al. (1983) J. Immul. Methods, 65:147-157, Hnatowich, D. et al. (1984) J. Applied Radiation, 35:554-557, and Buckley, R. G. et al. (1984) F.E.B.S. 166:202-204.
[0336]In the case of a radiolabeled ligand, the ligand is administered to the patient, is localized to the tumor bearing the antigen with which the ligand reacts, and is detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A. R. Bradwell et al., "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin et al., (eds.), pp 65-85 (Academic Press 1985). Alternatively, a positron emission transaxial tomography scanner, such as designated Pet VI located at Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., 11C, 18F, 15O, and 13N).
[0337]MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses NMR to visualize internal features of living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds for obvious benefit. Some MRI techniques are summarized in EP-A-0 502 814. Generally, the differences related to relaxation time constants T1 and T2 of water protons in different environments are used to generate an image. However, these differences can be insufficient to provide sharp high resolution images.
[0338]The differences in these relaxation time constants can be enhanced by contrast agents. Examples of such contrast agents include a number of magnetic agents paramagnetic agents (which primarily alter T1) and ferromagnetic or superparamagnetic (which primarily alter T2 response). Chelates (e.g., EDTA, DTPA and NTA chelates) can be used to attach (and reduce toxicity) of some paramagnetic substances (e.g., Fe+3, Mn+2, Gd+3). Other agents can be in the form of particles, e.g., less than 10 μm to about 10 nM in diameter). Particles can have ferromagnetic, antiferromagnetic or superparamagnetic properties. Particles can include, e.g., magnetite (Fe3O4), γ-Fe2O3, ferrites, and other magnetic mineral compounds of transition elements. Magnetic particles may include: one or more magnetic crystals with and without nonmagnetic material. The nonmagnetic material can include synthetic or natural polymers (such as sepharose, dextran, dextrin, starch and the like
[0339]The MMP-26-binding proteins can also be labeled with an indicating group containing of the NMR-active 19F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the 19F isotope and, thus, substantially all fluorine-containing compounds are NMR-active; (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body MRI is carried out using an apparatus such as one of those described by Pykett (1982) Scientific American, 246:78-88 to locate and image cancerous tissues.
[0340]Information obtained from evaluating an MMP-26-binding protein, e.g., a ligand described herein, can be recorded on machine-compatible media, e.g., computer readable or computer accessible media. The information can be stored as a computer representation, e.g., in a database (e.g., in the case of imaging using a ligand, a database of images for one or a plurality of subjects). The term "computer representation" refers to information which is in a form that can be manipulated by a computer. The act of storing a computer representation refers to the act of placing the information in a form suitable for manipulation by a computer.
Kits
[0341]Also within the scope of the invention are kits that include a composition described herein, e.g., a composition that contains a MMP-26-binding protein. In one embodiment, the kit includes (a) a composition that includes the MMP-26-binding protein, and, optionally, (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the compound for the methods described herein, e.g., a treatment, prophylactic, or diagnostic use. For example, the informational material describes methods for administering the composition to treat a disorder, e.g., a neoplastic disorder such as a metastatic disorder, an inflammatory disorder, or a disorder characterized by excessive MMP-26 activity.
[0342]In one embodiment, the informational material can include instructions to administer the compound in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions for identifying a suitable subject, e.g., a human, e.g., a human having, or at risk for a neoplastic disorder, an inflammatory disorder, or a disorder characterized by excessive MMP-26 activity. The informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. The informational material of the kits is not limited in its form. Information about the compound can include structural information, e.g., amino acid sequence, tradename, FDA approved name, antibody isotype, and so forth. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the compound and/or its use in the methods described herein. The informational material can also be provided in any combination of formats.
[0343]In addition to the composition that includes the MMP-26-binding protein, the composition itself can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or a second agent for treating a condition or disorder described herein, e.g. a neoplastic disorder (e.g., a metastatic disorder) or an inflammatory disorder. Alternatively, such other ingredients can be included in the kit, but in different compositions or containers than the composition that includes the MMP-26-binding protein. In such embodiments, the kit can include instructions for admixing the compound and the other ingredients, or for using the compound together with the other ingredients.
[0344]The composition that includes the MMP-26-binding protein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that composition be substantially pure and/or sterile. When the composition that includes the MMP-26-binding protein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When the composition that includes the MMP-26-binding protein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
[0345]The kit can include one or more containers for the composition that includes the MMP-26-binding protein. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the MMP-26-binding protein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the compound. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
[0346]Kits can be provided that include a MMP-26-binding antibody and instructions for diagnostic, e.g., the use of the MMP-26-binding ligand (e.g., antibody or antigen-binding fragment thereof, or other polypeptide or peptide) to detect MMP-26, in vitro, e.g., in a sample, e.g., a biopsy or cells from a patient having a cancer or neoplastic disorder, or in vivo, e.g., by imaging a subject. The kit can further contain a least one additional reagent, such as a label or additional diagnostic agent. For in vivo use the ligand can be formulated as a pharmaceutical composition.
[0347]The following invention is further illustrated by the following examples, which should not be construed as limiting.
EXAMPLES
[0348]In the following examples, 48 Fab's were isolated that bind to MMP-26. Of these Fabs, 20 had inhibitory. These 20 Fab's include 12 with K light chains and 8 with λ light chains. The other 28 Fab's included 14 with K light chains and 14 with λ light chains.
Cloning and Expression of MMP-26
[0349]MMP-26 is synthesized as an inactive precursor that undergoes proteolytic cleavage and release of the propeptide in order to exhibit catalytic domain-specific enzymatic activity. A clone containing the full-length MMP-26 sequence was used as template to generate a nucleic acid encoding the catalytic domain alone. A nucleic acid encoding the catalytic domain of MMP-26 was amplified using primers mat2.top.cat.NcoI and mat2.bot.HindIII:
TABLE-US-00017 Mat2.cat.top.ncoI (SEQ ID NO: 218) 5'-GCCATCCATGGCGACCTCCATCTCGCCAGG Mat2.bot.HindIII: ((SEQ ID NO: 219) 5'-GCAGCAAGCTTCATCATCATCATCATCATAGGTATGTCAGATGAACA TTTTTCTCC
[0350]The resulting PCR product was digested with HindIII and NcoI, ligated into a modified version of the pQE60 vector (Qiagen), and electroporated into XL1 Blue MRF' cells (Stratagene). A 200 mL culture of pQE60 containing XL1 Blue MRF' cells was induced with 2.5 mM IPTG and grown overnight at 37° C. The following day the bacteria were pelleted and sonicated. The insoluble material was collected and dissolved in 8M urea-containing buffer. MMP-26 was purified using nickel-coated magnetic beads and refolded by a standard dialysis procedures.
MMP-26 Activity Assessment
[0351]MMP-26 activity was determined by zymogram gel analysis and vitronectin digestion assays. For the zymogram gel analysis assays, 375 ng of MMP-26 was resolved on a 10% gelatin-containing zymogram gels (Invitrogen). Following electrophoresis, the gel was developed overnight at 37° C. according to the manufacturers recommended directions and subsequently stained with Coomassie blue. All fractions displayed gelatinase activity. For the vitronectin digestion assays, 250 ng of MMP-26 was incubated with 250 ng of vitronectin overnight at 37° C. The MMP-26 digested vitronectin was resolved by SDS-PAGE and visualized by Coomassie blue staining. MMP-26 activity is indicated if vitronectin is cleaved.
Identification of MMP-26-Binding Fab-Displaying Phage
[0352]Selections were performed using two different methods. The first method utilized three rounds of standard solution-based selections. In each round, the amount (500 nM to 50 nM) of MMP-26 catalytic domain as target protein was decreased while the input of phage was kept constant at 3×1011 pfu. The second strategy utilized was the URSA (Ultra Rapid Screening of Antigens) method (which is described, inter alia, in U.S. Ser. No. 10/313,822). Three rounds of URSA selections were performed in one day.
[0353]Briefly, the MMP-26 target protein (tagged with hexa-histidine) was contacted to a Fab-displaying phage library. The mixture was then bound to nickel magnetic beads. After three washes, XL1 Blue MRF' cells were added to the target-containing beads in order to propagate MMP-26 specific-binding phage. The XL1 Blue cells were infected by phage bound on the beads and extruded replicates of these phage. These replicate phage then bound to the MMP-26 on the beads. The XL1 Blue MRF' cells were removed. The phage-target-bead complexes were washed to remove unbound phage, and fresh XL1 Blue MRF' cells were added to initiate Round Two. This cycle was repeated one more time such that three rounds were performed overall. The antibodies encoded by the Fab-displaying phage library include HC CDR3 and light chains that are obtained from human cells. HC CDR1 and HC CDR2 are encoded by sequences based on human CDR sequences.
ELISA Screening of Output Fab-Displaying Phage
[0354]The output Fab-displaying phage from both rounds two and three from either of the selection campaigns were screened by ELISA to positively identify MMP-26 binding phage isolates. MMP-26 (1 μg/ml) was passively immobilized on Immulon 2 HB 96-well ELISA plates (Thermo Labsystems) overnight at 4° C. The plates were blocked for thirty minutes with phosphate buffered saline containing 3% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) Tween-20. Overnight bacterial growths of Fab-displaying phage were then incubated with the target for 1 hour at room temperature. Fab-displaying phage were detected with an anti-M13 HRP-conjugated antibody. Standard solution-based selections yielded a hit rate of 23% in round 2 and a 92% hit rate for round 3. The URSA method yielded a hit rate of 69% for both rounds 2 and 3.
Reformatting of Fab Clones into Whole IgGs
[0355]Sixty eight of the Fab-displaying phage positive for MMP-26 binding were reformatted as human whole IgG antibody clones. Briefly, the Fab cassette of each positive Fab-displaying phage was PCR amplified with oligos KAPPA, LAMBDA1, 2, 3, and 4, and CjliftNheRev:
TABLE-US-00018 (SEQ ID NO: 220) KAPPA: 5'-ATATATGTGCACTCTGACATCCAGATGACCCAGTC 3',; (SEQ ID NO: 221) LAMBDA1: 5'-ATATATGTGCACTCACAGAGCGTCTTGACTC 3',; (SEQ ID NO: 222) LAMBDA2: 5'-ATATATGTGCACTCACAGAGCGCTTTGACTC 3',; (SEQ ID NO: 223) LAMBDA3: 5'-ATATATGTGCACTCAAGCTACGAATTGACTC 3',; (SEQ ID NO: 224) LAMBDA4: 5'-ATATATGTGCACTCACAGAGCGAATTGACTC 3',; (SEQ ID NO: 225) CJliftNheRev: 5'-GGAGGGTGCTAGCGGGAAGACCG 3',.
[0356]PCR products were restricted using ApaLI and NheI. The digested Fab clone was ligated into a mammalian expression vector containing the human IgG4 Fc region and electroporated into XL1 BLUE® MRF' cells. The prokaryotic ribosomal binding sequence and heavy chain leader sequence were replaced with a mammalian internal ribosomal entry and heavy chain leader sequences. Reformatted antibody clones were sequenced to confirm accuracy following the cloning steps. Endotoxin-free DNA was prepared according to the manufacturer's instructions (Qiagen) and subsequently used for transient transfection studies.
Transient Transfections of MMP-26-Binding IgG4s
[0357]Reformatted MMP-26 antibodies were expressed transiently in HEK293T (GenHunter) cells using Lipofectamine 2000 (Invitrogen). Briefly, 6×106 cells in media containing 10% (v/v) ultra low bovine IgG fetal bovine serum were seeded into 100 mm tissue culture dishes. Twenty-four hours after plating, 5 mls of fresh media was added to each dish. The transfection was then carried out exactly as described by the manufacturer (Invitrogen). Seventy two hours after transfection, the media was removed, clarified and saved, and 15 mls of fresh media was added to each dish and the cells incubated for an additional 72 hours. At the conclusion of the second 72 hour period, the media was collected, dislodged cells clarified by centrifugation and the resulting supernatant was combined with that harvested after the first 72 hour period and, if required, human antibodies were purified according to standard protein A-based chromatographic procedures.
MMP-26 Bead-Based ELISA
[0358]Reformatted and expressed full-length MMP-26 binding IgG4 antibodies were tested for specificity in a bead-based ELISA. Approximately 1.25 pg of MMP-26 protein was bound to nickel-coated magnetic beads (Novagen) which had been pre-blocked with phosphate buffered saline containing 5% (w/v) nonfat dry milk and 0.05% (v/v) Tween-20. The beads were washed in phosphate buffered saline containing 0.05% (v/v) Tween-20 (PBS-T) and 100 uls of unpurified cell culture supernatant containing transiently produced MMP-26-binding antibodies was added and incubated on a rotator for sixty minutes. The beads were washed with PBS-T and MMP-26-binding antibodies were detected with an HRP-conjugated anti-human secondary antibody. Data is expressed in fold over background where background consists of beads, blocking agent, and culture supernatant containing target antibody (Table 2; in this table, Results shown as fold over background (F>B) where background consists of beads, blocking agent, and culture supernatant).
TABLE-US-00019 TABLE 2 Bead Based ELISA Clone F > B A1-orig NT D6-orig NT H6-orig NT A01 3.3 A03 3.54 A04 3.17 A05 2.48 A06 5.4 A07 8.38 A08 1.94 A09 2.5 A10 1.84 A11 1.7 A12 1.8 B01 2.3 B02 0.36 B03 0.65 B04 0.6 B05 NT B06 2.1 B07 0.5 B08 3 B10 2.7 B11 3.13 B12 2.83 C01 2.8 C02 0.9 C03 4.1 C04 2.9 C05 3.6 C06 2.2 C07 3.12 C08 3.4 C09 3.4 C10 3.41 C11 2.6 C12 1.8 D01 3 D02 3.3 D03 2.4 D04 2.6 D05 NT D06 3.5 D07 3.3 D08 3 D09 4.3
MMP Cross Reactivity ELISA
[0359]Twenty-one of the reformatted MMP-26 antibodies were tested for MMP cross reactivity by ELISA analysis. MMP-3, MMP-7, MMP-9, and MMP-12 were coated onto Immulon 2 HB 96-well plates at a concentration of 1 μg/ml for 1 hour at 37° C. The plates were blocked with PBS-T containing 5% (w/v) nonfat dry milk for thirty minutes and subsequently washed with PBS-T. Each antibody was tested for reactivity to all of the above mentioned MMPs at a concentration of 2 μg/ml with an incubation time of 1 hour. Bound antibody was detected with an HRP-conjugated anti-human secondary antibody (Table 3, NT=not tested.).
TABLE-US-00020 TABLE 3 MMP Cross Reactivity ELISA Clone MMP Cross Reactivity A1-orig MMP-26/MMP-9 D6-orig MMP-26/MMP-9 H6-orig MMP-26 A01 MMP-26 A03 MMP-26 A04 MMP-26 A05 MMP-26 A06 MMP-26 A07 MMP-26 A08 MMP-26 A09 MMP-26 A10 MMP-26 A11 MMP-26 A12 MMP-26 B01 MMP-26 B02 MMP-26 B03 MMP-26 B04 MMP-26 B05 NT B06 MMP-26 B07 MMP-26 B08 MMP-26 B10 MMP-26/MMP-3 B11 MMP-26 B12 MMP-26 C01 MMP-26 C02 MMP-26/MMP-3/MMP-9 C03 MMP-26 C04 MMP-26 C05 MMP-26 C06 MMP-26 C07 MMP-26 C08 MMP-26 C09 MMP-26 C10 MMP-26 C11 MMP-26 C12 MMP-26/MMP-3 D01 MMP-26 D02 MMP-26 D03 MMP-26 D04 MMP-26 D05 NT D06 MMP-26 D07 MMP-26 D08 MMP-26 D09 MMP-26
In Vitro Cellular Invasion Assay
[0360]Forty-one of the expressed and purified MMP-26-binding antibodies were tested for inhibition of JEG-3 (choriocarcinoma) cell invasion through MATRIGEL® basement membrane matrix-coated filters using the growth-factor reduced system from Becton Dickinson. JEG-3 cells (104) were diluted in RPMI media containing 0.1% (v/v) fetal bovine serum and added to the upper chamber of the MATRIGEL® basement membrane matrix-coated well. Six hundred microliters of spent media from cultures of 3T3 fibroblasts was placed in the lower chamber as a source of chemo attractants. MMP-26-binding antibodies were added to the upper chamber at concentrations of 5 μg/ml and 25 μg/ml. In the absence of an inhibitor, the JEG-3 cells invaded into the lower chamber. Data is expressed as percent inhibition relative to phosphate buffered saline (set at 100% invasion). The twelve most potent antibodies were tested further at concentrations of 1 μg/ml, 5 μg/ml, 25 μg/ml, and 50 μg/ml on three consecutive days. The data is shown in Table 4 (N/E=no detectable effect).
TABLE-US-00021 TABLE 4 JEG-3 cell invasion through MATRIGEL ® basement membrane matrix in vitro % Inhibition % Inhibition Clone 5 μg/ml 25 μg/ml 1 μg/ml 5 μg/ml 25 μg/ml 50 μg/ml A1-orig 40% 66% 53% 58% 64% 74% D6-orig 41% 52% 42% 55% 70% 35% H6-orig 24% 50% 49% 52% 57% 39% A01 19% 51% 30% 47% 54% 70% A04 6% 27% A11 28% 56% 51% 54% 59% 73% B04 36% 14% B06 34% 30% B10 48% 26% 6% 5% 11% 22% C01 43% 34% -2% 16% 21% 42% C04 35% 36% 1% 11% 18% 32% C05 16% 32% C08 45% 49% 34% 42% 49% 62% C11 28% 29% C12 49% 38% 36% 35% 49% 27% D02 36% 25% D04 38% 34% 47% 34% 45% 52% D06 0% 35% D07 21% 36% D08 36% 35% 56% 50% 76% 53%
Inhibition of MMP-26 Specific Activation of proMMP-9
[0361]Two lead antibodies (A1-orig and A11) were tested for their ability to inhibit MMP-26 activation of ProMMP-9. Activation was monitored by MMP-9 cleavage of gelatin (Molecular Probes E-12055) or cleavage of an MMP-9 specific peptide (Calbiochem 444221). MMP-26, at a concentration of 100 nM, was incubated with the above designated Ab inhibitors at varying concentrations for one hour. The proMMP-9, at a concentration of 10 nM, was then added to the mixture followed by fluorescently labeled gelatin or a fluorescently labeled peptide substrate. Activation of proMMP-9 was monitored using a fluorimeter (Molecular Devices) and was seen as an increase in signal. Inhibitory activity was thus assessed by a loss in signal. Neither A1-orig nor A11 inhibited MMP-9 activity directly.
[0362]Exemplary results obtained using the MMP-9 cleavage assay include that following percentage inhibition where the candidate inhibitor is at 125 nM: TIMP (89%), A1-orig (80%), A11 (79%), negative control compound (31%), no candidate compound (0%). These results were obtained by monitoring MMP-9 peptide cleavage in a reaction that included MMP-26 and pro-MMP-9.
[0363]The following Table provides inhibition data with the A1-orig antibody at various concentrations using a fluorescently labeled substrate:
TABLE-US-00022 TABLE 5 Inhibtion of MMP-26 Cleavage by A1-orig nM Inhibition 15 8% 31 19% 62 58% 125 68% 500 86% 2000 89%
Sequence Analysis of Exemplary MMP-26-Binding Antibodies
[0364]MMP-26-binding antibodies were sequenced. Both nucleic and amino acid sequence of the VL and VH regions of each sequenced antibody are as follows (Table 6).
TABLE-US-00023 TABLE 6 Sequences of Exemplary Antibodies Antibody Sequence Identifier A1-orig GGCGTGCACTCACAGAGCGTCTTGACTCAGCCACCCTC SEQ ID NO: 1 VLC AGCGTCTGGGACCCCCGGGCAGAGGGTCATCATCTCTT Nucleic GTTCTGGAAGCAGCTCCAACATCGGAAGTCATTATGTA Acid CACTGGTACCAACAGGTCCCAGGAACGGCCCCCAAACT Sequence CCTCATTTATAGGAATGGTCAGCGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTTCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGC TAATTATTACTGTGCAACATGGGATGACAGTGTCCTAT TCGGCGGAGGGACCACGCTGACCGTCCTAGGTCAGCCC AAGGCTGCCCCC A1-orig GVHSQSVLTQPPSASGTPGQRVIISCSGSSSNIGSHYV SEQ ID NO: 2 VLC HWYQQVPGTAPKLLIYRNGQRPSGVPDRFSGFKSGTSA Amino SLAISGLRSEDEANYYCATWDDSVLFGGGTTLTVLGQP Acid KAAP Sequence A1-orig GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 3 VHC GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Nucleic GATTCACTTTCTCTTATTACCGTATGTCTTGGGTTCGC Acid CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT Sequence CGGTCCTTCTGGTGGCGATACTCTTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGATCTTTCAGCA GTGGCCCGTACTACTTTGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATC GGTCTTCCCGCTAGCGCCC A1-orig EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYRMSWVR SEQ ID NO: 4 VHC QAPGKGLEWVSSIGPSGGDTLYADSVKGRFTISRDNSK Amino NTLYLQMNSLRAEDTAVYYCARSFSSGPYYFDYWGQGT Acid LVTVSSASTKGPSVFPLAP Sequence D6-orig GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCACT SEQ ID NO: 5 VLC CTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCATCA Nucleic CTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAAT Acid TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCT Sequence GATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCAT CAAGATTCAGTGGCAGTGGAACTGGGACAGATTTCACT CTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAG TTATTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCA CTTTCGGCCCTGGGACCAAGGTGGAGATCAAACGAACT GTGGCTGCACCA D6-orig GVHSDIQMTQSPLSLSASVGDRVAITCRASQSIDTYLN SEQ ID NO: 6 VLC WYQQKPGKAPKLLIYAASKLEDGVPSRFSGSGTGTDFT Amino LTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIKRT Acid VAAP Sequence D6-orig GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 7 VHC GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Nucleic GATTCACTTTCTCTATGTACTCTATGCGTTGGGTTCGC Acid CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT Sequence CTATCCTTCTGGTGGCTCTACTGAGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGAGGGCGGGG AGAACGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCT AGCGCCC D6-orig EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYSMRWVR SEQ ID NO: 8 VHC QAPGKGLEWVSSIYPSGGSTEYADSVKGRFTISRDNSK Amino NTLYLQMNSLRAEDTAVYYCAREGGENDYWGQGTLVTV Acid SSASTKGPSVFPLAP Sequence H6-orig GGCGTGCACTCACAGAGCGAATTGACTCAGCCTCCCTC SEQ ID NO: 9 VLC CGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCT Nucleic GCACTGGAACCAGCAGTGACGTTGGTGCTTATAACTAT Acid GTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAA Sequence ACTCATAATCTATGAAGTCAATAAGCGGCCCTCAGGGG TCCCTGATCGCTTCTCTGCCTCCAAGTCTGGCAACACG GCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAAGATGA GGCTGATTATTACTGCAACTCATATGCAGGCAGCAACA GTTTGATATTCGGCGGAGGGACCAAACTGACCGTCTTA GGTCAGCCCAAGGCTGCCCCC H6-orig GVHSQSELTQPPSASGSPGQSVTISCTGTSSDVGAYNY SEQ ID NO: 10 VLC VSWYQQHPGKAPKLIIYEVNKRPSGVPDRFSASKSGNT Amino ASLTVSGLQAEDEADYYCNSYAGSNSLIFGGGTKLTVL Acid GQPKAAP Sequence H6-orig GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 11 VHC GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Nucleic GATTCACTTTCTCTCAGTACTGGATGAATTGGGTTCGC Acid CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTAT Sequence CGGTCCTTCTGGTGGCATTACTTATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGTGAGGAAG ATGGCTACAATTCTGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCGCTAGCGCCC H6-orig EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYWMNWVR SEQ ID NO: 12 VHC QAPGKGLEWVSGIGPSGGITYYADSVKGRFTISRDNSK Amino NTLYLQMNSLRAEDTAVYYCARGEEDGYNSDYWGQGTL Acid VTVSSASTKGPSVFPLAP Sequence A01 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCAGG SEQ ID NO: 13 Nucleic CACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT Acid CCTGCAGGGCCAGTCAGATTGTTCGCAGCACCTACTTA Sequence GCCTGGTATCAGCAGAAACCTGGCCAGGCTCCCAGGCT CCTCATCTATGGTACATCCAGCAGGGCCACTGGCGTCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCGGACTGGAGCCTGAAGATTTTGC ACTATACTACTGTCAGCGGTATGGTGACTCACCTCCGA TCACCTTCGGCCAAGGGACACGACTGGAGATTACACGA ACTGTGGCTGCACCATCTGTC A01 VLC GVHSDIQMTQSPGTLSLSPGERATLSCRASQIVRSTYL SEQ ID NO: 14 Amino AWYQQKPGQAPRLLIYGTSSRATGVPDRFSGSGSGTDF Acid TLTISGLEPEDFALYYCQRYGDSPPITFGQGTRLEITR Sequence TVAAPSV A01 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 15 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGCTTACAATATGTTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTAT CGGTTCTTCTGGTGGCATTGCTCCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGCCGCGTACG AGGTGGAGAACTGGTTCGACCCCTGGGGCCAGGGAACC CTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATC GGTCTTCCCG A01 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYNMFWVR SEQ ID NO: 16 Amino QAPGKGLEWVSGIGSSGGIAPYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARAAYEVENWFDPWGQGT Sequence LVTVSSASTKGPSVFP A03 VLC GGCGTGCACTCACAGAGCGCTTTGACTCAGCCACCCTC SEQ ID NO: 17 Nucleic AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT Acid GTTCTGGAGGCAGCTCCAACATCGGAAGTAATTTTGTT Sequence TACTGGTACCGGCAGCTCCCAGGAACGGCCCCCAAACT CCTCATCTATAGGAATTATCAGCGGCCCTCAGGGGTCC CTGACCGATTCTCGGGTTCCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCTGTCCGAAGATGAGGC TGATTATTACTGCGCAGCATGGGATGACAACGTGGGTG GGGTCTTCGGATCTGGGACCAAGGTCACCGTCCTGGGT CAGCCCAAGGCCAACCCCACT A03 VLC GVHSQSALTQPPSASGTPGQRVTISCSGGSSNIGSNFV SEQ ID NO: 18 Amino YWYRQLPGTAPKLLIYRNYQRPSGVPDRFSGSKSGTSA Acid SLAISGLLSEDEADYYCAAWDDNVGGVFGSGTKVTVLG Sequence QPKANPT A03 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 19 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTATTTACTCTATGGATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CTATTCTTCTGGTGGCGCTACTCGTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGGTGTAGCTGGC TACAATTAGTACCGATGCACCCTTGGGGCCAGGGAACC CTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATC GGTCTTCCCG A03 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYSMDWVR SEQ ID NO: 20 Amino QAPGKGLEWVSSIYSSGGATRYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARCSWLQLVPMHPWGQGT Sequence LVTVSSASTKGPSVFP A04 VLC GGCGTGCACTCACAGAGCGCTTTGACTCAGCCACCCTC SEQ ID NO: 21 Nucleic AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT Acid GTTCTGGAGGCTACTCCAACATGGGAAGCAATTATGCA Sequence CACTGGTACCAGCAGGTCCCAGGAACGGCCCCCAAACT CCTCATCTATAACAATAATCAGAGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCC TCCCTAGCCATCAGTGGGCTCCGGTCCGAGGATGAGGC TGATTATTACTGTGCAGCATGGGATGAAAACCTGAGTG GTCCGGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA GGTCAGCCCAAGGCCAACCCCACT A04 VLC GVHSQSALTQPPSASGTPGQRVTISCSGGYSNMGSNYA SEQ ID NO: 22 Amino HWYQQVPGTAPKLLIYNNNQRPSGVPDRFSGSKSGTSA Acid SLAISGLRSEDEADYYCAAWDENLSGPVFGTGTKVTVL Sequence GQPKANPT A04 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 23 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGAGTACAATATGGCTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CGGTTCTTCTGGTGGCAAGACTAAGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGATGAAGCCC CCGACTACGGTGACGACGCGGAAGCTTTTGATATCTGG GGCCAAGGGACAATGGTCACCGTCTCAAGCGCCTCCAC CAAGGGCCCATCGGTCTTCCCG A04 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYNMAWVR SEQ ID NO: 24 Amino QAPGKGLEWVSRIGSSGGKTKYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARDEAPDYGDDAEAFDIW Sequence GQGTMVTVSSASTKGPSVFP A05 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID NO: 25 Nucleic CTCCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT Acid CCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC Sequence TGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCT CATCTATGATGCATCCAACAGGGCCACTGGCATCCCAG CCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGT TTATTACTGTCAGCAGCGTAGCAACTGGCCTCGGACTT TCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG GCTGCACCATCTGTC A05 VLC GVHSDIQMTQSPSSLSLSPGERATLSCRASQSVSSYLA SEQ ID NO: 26 Amino WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT Acid LTISSLEPEDFAVYYCQQRSNWPRTFGGGTKVEIKRTV Sequence AAPSV A05 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 27 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGTTTACTCTATGAATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATAT CGTTCCTTCTGGTGGCAATACTCCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCAAGAGATGGGGCGG CTACGGTGGACTTAGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCG A05 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYSMNWVR SEQ ID NO: 28 Amino QAPGKGLEWVSYIVPSGGNTPYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARDGAATVDLDYWGQGTL Sequence VTVSSASTKGPSVFP A06 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID NO: 29 Nucleic CTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA Acid CTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAAT Sequence TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACT CTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAC TTACTACTGTCAACAGAGTTACAGTACCCCTCCGGAGA
ACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGA ACTGTGGCTGCACCATCTGTC A06 VLC GVHSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLN SEQ ID NO: 30 Amino WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT Acid LTISSLQPEDFATYYCQQSYSTPPENTFGQGTKLEIKR Sequence TVAAPSV A06 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 31 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTCCTTACCATATGGGTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTAT CTATCCTTCTGGTGGCTGGACTAATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGATGGGTATA GCAGTGGCTGGTTCCGGTACTGGGGCCAGGGAACCCTG GTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCG A06 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYHMGWVR SEQ ID NO: 32 Amino QAPGKGLEWVSGIYPSGGWTNYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARDGYSSGWFRYWGQGTL Sequence VTVSSASTKGPSVFP A07 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCTCCCTC SEQ ID NO: 33 Nucleic CGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCT Acid GCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTAT Sequence GTCTCCTGGTATCAACAACACCCAGACAAAGCCCCCAA ACTCCTGATTTATGAGGTCACTCAGCGGCCCTCAGGGG TCCCTGATCGCTTCTCTGGCTCCAGGTCTGGCAACACG GCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGA GGCTGATTATTACTGCAGCTCATATGCAGGCAGGAACA ATCTTTATGTCTTCGGACCTGGGACCAAGGTCACCGTC CTAGGTCAGCCCAAGGCCAACCCCACT A07 VLC GVHSQSELTQPPSASGSPGQSVTISCTGTSSDVGGYNY SEQ ID NO: 34 Amino VSWYQQHPDKAPKLLIYEVTQRPSGVPDRFSGSRSGNT Acid ASLTVSGLQAEDEADYYCSSYAGRNNLYVFGPGTKVTV Sequence LGQPKANPT A07 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 35 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGAGTACAATATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CTCTCCTTCTGGTGGCGGTACTCTTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGATCTAAATA ACAGCTCGCCCCCGGATTCCAATGATGCTTTTGATATC TGGGGCCGAGGGACAATGGTCACCGTCTCAAGCGCCTC CACCAAGGGCCCATCGGTCTTCCCG A07 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYNMMWVR SEQ ID NO: 36 Amino QAPGKGLEWVSVISPSGGGTLYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARDLNNSSPPDSNDAFDI Sequence WGRGTMVTVSSASTKGPSVFP A08 VLC GGCGTGCACTCACAGAGCGTCTTGACTCAGCCACCCTC SEQ ID NO: 37 Nucleic AGTGTCTGGGACCCCCGGACAGAGGGTCACCATCTCTT Acid GTTCTGGAGGCTACCCCAACATGGGAAGCAATTATGCA Sequence CACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACT CCTCATCTATAACGATAATCAGCGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGC TGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTG GTCCGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA GGTCAGCCCAAGGCTGCCCCCTCG A08 VLC GVHSQSVLTQPPSVSGTPGQRVTISCSGGYPNMGSNYA SEQ ID NO: 38 Amino HWYQQLPGTAPKLLIYNDNQRPSGVPDRFSGSKSGTSA Acid SLAISGLRSEDEADYYCAAWDDSLSGPVFGGGTKLTVL Sequence GQPKAAPS A08 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 39 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGTTTACGATATGCCTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CTATCCTTCTGGTGGCTTTACTCGTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGGCAGATCCGACGA TACAGCTATGGGCCTACTACTACGGTATGGACGTCTGG GGCCAAGGGACCACGGTCACCGTCTCAAGCGCCTCCAC CAAGGGCCCATCGGTCTTCCCG A08 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYDMPWVR SEQ ID NO: 40 Amino QAPGKGLEWVSVIYPSGGFTRYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCAADPTIQLWAYYYGMDVW Sequence GQGTTVTVSSASTKGPSVFP A09 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCACCCTC SEQ ID NO: 41 Nucleic AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT Acid GTTCTGGAGGCAGCTCCAACATCGGAAGTAATTTTGTT Sequence TACTGGTACCGGCAGCTCCCAGGAACGGCCCCCAAACT CCTCATCTATAGGAATTATCAGCGGCCCTCAGGGGTCC CTGACCGATTCTCGGGTTCCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCTGTCCGAAGATGAGGC TGATTATTACTGCGCAGCATGGGATGACAACGTGGGTG GGGTCTTCGGATCTGGGACCAAGGTCACCGTCCTGGGT CAGCCCAAGGCCAACCCCACT A09 VLC GVHSQSELTQPPSASGTPGQRVTISCSGGSSNIGSNFV SEQ ID NO: 42 Amino YWYRQLPGTAPKLLIYRNYQRPSGVPDRFSGSKSGTSA Acid SLAISGLLSEDEADYYCAAWDDNVGGVFGSGTKVTVLG Sequence QPKANPT A09 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 43 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTTGGTACGATATGTATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CTATTCTTCTGGTGGCTATACTGCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAAAGATCGTGATC CTTGTAGTAGAACCACCTGCTATAACTGGTTCGACCCC TGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCCTC CACCAAGGGCCCATCGGTCTTCCCG A09 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYDMYWVR SEQ ID NO: 44 Amino QAPGKGLEWVSSIYSSGGYTAYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCAKDRDPCSRTTCYNWFDP Sequence WGQGTLVTVSSASTKGPSVFP A10 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCACCCTC SEQ ID NO: 45 Nucleic AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT Acid GTTCTGGAGGCAGCTCCAACATCGGAAGTAATTATGTC Sequence TCCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACT CCTCATCTATAATAATAATCAGCGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGC TGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTT CTGCTGTGTTCGGAGGAGGCACCCAGCTGACCGTCCTC GGTCAGCCCAAGGCTGCCCCCTCG A10 VLC GVHSQSELTQPPSASGTPGQRVTISCSGGSSNIGSNYV SEQ ID NO: 46 Amino SWYQQLPGTAPKLLIYNNNQRPSGVPDRFSGSKSGTSA Acid SLAISGLRSEDEADYYCAAWDDSLSSAVFGGGTQLTVL Sequence GQPKAAPS A10 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 47 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGCTTACCGTATGTTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CTGGCCTTCTGGTGGCACTACTTCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGATCGGGGCT ATGATAGTAGTGGTTATTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCG A10 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVR SEQ ID NO: 48 Amino QAPGKGLEWVSSIWPSGGTTSYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARDRGYDSSGYFDYWGQG Sequence TLVTVSSASTKGPSVFP A11 VLC GGCGTGCACTCACAGAGCGCTTTGACTCAGCCACCCTC SEQ ID NO: 49 Nucleic GGTGTCACTGGCCCCAGGACAGACGGCCAGGATTACCT Acid GTGGGGGAAACAACATTGGAACTAAAAGTGTTCACTGG Sequence TACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGT CTATGATGACAGCGACCGGCCCTCAGGGATCCCTGAGC GATTCTCTGGCTCCAATTCTGGGAACACGGCCACCCTG ACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTA TTATTGTCAGGTGTGGGATAGTGGTAGTGATCATCAGG TCTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAG CCCAAGGCTGCCCCCTCG A11 VLC GVHSQSALTQPPSVSLAPGQTARITCGGNNIGTKSVHW SEQ ID NO: 50 Amino YQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATL Acid TISRVEAGDEADYYCQVWDSGSDHQVFGGGTKLTVLGQ Sequence PKAAPS A11 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 51 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTTGGTACACTATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CTCTCCTTCTGGTGGCCATACTCTTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTNAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGACACTTGGG ACGATTACTATGATAGTAGTGGTTATTACAACGATTTT GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCAAG CGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGC CCTG A11 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYTMMWVR SEQ ID NO: 52 Amino QAPGKGLEWVSRISPSGGHTLYADSVKGRFTISRDNSz Acid NTLYLQMNSLRAEDTAVYYCARDTWDDYYDSSGYYNDF Sequence DYWGQGTLVTVSSASTKGPSVFPLAP A12 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCAGG SEQ ID NO: 53 Nucleic CACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT Acid CCTGCAGGGCCAGTCAGAGTGTTAGTCGTAGCTACTTA Sequence GGCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCT CCTCATCTATGGTGCATCCAACAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC AGTGTATTACTGTCAGCAGTACGGTATCTCACCCCTCA CCTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACT GTGGCTGCACCATCTGTC A12 VLC GVHSDIQMTQSPGTLSLSPGERATLSCRASQSVSRSYL SEQ ID NO: 54 Amino GWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDF Acid TLTISRLEPEDFAVYYCQQYGISPLTFGPGTKVDIKRT Sequence VAAPSV A12 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 55 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGCTTACTGGATGGATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CTATCCTTCTGGTGGCTCTACTAATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGAGGGGATAG CCGCAGCAGCACCAATGGACGTCTGGGGCAAAGGGACC ACGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATC GGTCTTCCCG A12 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYWMDWVR SEQ ID NO: 56 Amino QAPGKGLEWVSVIYPSGGSTNYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCAREGIAAAAPMDVWGKGT Sequence TVTVSSASTKGPSVFP B01 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCAGG SEQ ID NO: 57 Nucleic CACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT Acid CCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTT Sequence GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCT CCTCATCTATGATGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC AGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCGA TGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA CGAACTGTGGCTGCACCATCTGTC B01 VLC GVHSDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYF SEQ ID NO: 58 Amino AWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDF Acid TLTISRLEPEDFAVYYCQQYGSSPPMYTFGQGTKLEIK Sequence RTVAAPSV B01 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 59 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTACTTACGATATGCTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CTCTCCTTCTGGTGGCTCTACTTCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGCTGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGAGAAAGCGT CGGATCTTTCGGGGACTTACTCTGAGGCCCTTGACCAC TGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCCTC CACCAAGGGCCCATCGGTCTTCCCG
B01 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYDMLWVR SEQ ID NO: 60 Amino QAPGKGLEWVSSISPSGGSTSYADSVKGRFTISRDNSK Acid NTLYLQLNSLRAEDTAVYYCAREKASDLSGTYSEALDH Sequence WGQGTLVTVSSASTKGPSVFP B02 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID NO: 61 Nucleic CTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCATCA Acid CTTGCCGTGCAAGTCAGAGCATCGACACCTATTTAAAT Sequence TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCT GATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCAT CAAGATTCAGTGGCAGTGGAACTGGGACAGATTTCACT CTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAG TTATTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCA CTTTCGGCCCTGGGACCAAGGTGGAGATCAAACGAACT GTGGCTGCACCATCTGTC B02 VLC GVHSDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLN SEQ ID NO: 62 Amino WYQQKPGKAPKLLIYAASKLEDGVPSRFSGSGTGTDFT Acid LTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIKRT Sequence VAAPSV B02 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 63 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGATTACTTTATGAAGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CTATCCTTCTGGTGGCCCTACTAAGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGAGCGTAGCA GTGGCTGGTACGGTTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCAAGCGCCTC CACCAAGGGCCCATCGGTCTTCCCG B02 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYFMKWVR SEQ ID NO: 64 Amino QAPGKGLEWVSSIYPSGGPTKYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARERSSGWYGYYYYGMDV Sequence WGQGTTVTVSSASTKGPSVFP B03 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID NO: 65 Nucleic CTTCCTGTCTGCTTCTGTAGGGGACAGAGTCACCATCA Acid CTTGCCGGGCCAGTCAGGGCATTAGGGATTTTTTAGGC Sequence TGGTATCAACAAAAACCAGGGAAAGCCCCTAATCAACT GATCTATGCTGCATCCATTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACT CTCACGATCACCAGCCTGCAGCCTGAGGATTTTGCAAC TTATTTCTGTCAACAGCTTAATGGCTACCGCGCCTTCG GCCAAGGGACACGACTGGAAATAAAGCGAACTGTGGCT GCACCATCTGTC B03 VLC GVHSDIQMTQSPSFLSASVGDRVTITCRASQGIRDFLG SEQ ID NO: 66 Amino WYQQKPGKAPNQLIYAASILQSGVPSRFSGSGSGTDFT Acid LTITSLQPEDFATYFCQQLNGYRAFGQGTRLEIKRTVA Sequence APSV B03 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 67 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTCCTTACGAGATGCAGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTAT CGGTTCTTCTGGTGGTGACTCCGTTAAAGGTCGCTTCA CTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTG CAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTA CTATTGTGCGAGAGAGAGGGTAGATTGTAGTGGTGGTG GCTGCGGGAGCTACTTTGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATC GGTCTTCCCG B03 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYEMQWVR SEQ ID NO: 68 Amino QAPGKGLEWVSGIGSSGGDSVKGRFTISRDNSKNTLYL Acid QMNSLRAEDTAVYYCARERVDCSGGGCGSYFDYWGQGT Sequence LVTVSSASTKGPSVFP B04 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID NO: 69 Nucleic TTCCGTGTCTGCATCTGTAGGAGACAGAGTCACGATCA Acid CTTGTCGGGCGAGTCAGGGTATTAGCAAGAGCTTAGCC Sequence TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCT GGTCTATGGTGCATTCAGTTTGGAAAGTGGGGTCCCAT CAAGATTCAGCGGCACTGGAGCTGGGACAGATTTCATT CTCACCATCAGCAGGCTGCAGCCTGAAGACTTTGCAAC TTATTATTGTCAACAGGCTAACAGTTTCCCGCTCACTT TCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG GCTGCACCATCTGTC B04 VLC GVHSDIQMTQSPSSVSASVGDRVTITCRASQGISKSLA SEQ ID NO: 70 Amino WYQQKPGKAPKLLVYGAFSLESGVPSRFSGTGAGTDFI Acid LTISRLQPEDFATYYCQQANSFPLTFGGGTKVEIKRTV Sequence AAPSV B04 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 71 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTTTTTACTGGATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTAT CTCTTCTTCTGGTGGCTTTACTAAGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCCAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGGGAGACCAGCC GGAGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTC ACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTT CCCG B04 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYWMMWVR SEQ ID NO: 72 Amino QAPGKGLEWVSGISSSGGFTKYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARETSRRAFDIWGQGTMV Sequence TVSSASTKGPSVFP B05 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCAGG SEQ ID NO: 73 Nucleic CACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT Acid CCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTA Sequence GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCT CCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC AGTGTATTACTGTCAGCAGTATGGTAGCTCACCTGAGA TCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGA ACTGTGGCTGCACCATCTGTC B05 VLC GVHSDIQMTQSPGTLSLSPGERATLSCRASQSVSSSYL SEQ ID NO: 74 Amino AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDF Acid TLTISRLEPEDFAVYYCQQYGSSPEITFGQGTRLEIKR Sequence TVAAPSV B05 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 75 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTGAGTACTGGATGCCTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CTATCCTTCTGGTGGCGTTACTACTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGGGGGGATT ACGATTTTTGGAGTGTACAATACTACTACTACTACATG GACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCAAG CGCCTCCACCAAGGGCCCATCGGTCTTCCCG B05 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYWMPWVR SEQ ID NO: 76 Amino QAPGKGLEWVSRIYPSGGVTTYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARGGDYDFWSVQYYYYYM Sequence DVWGKGTTVTVSSASTKGPSVFP B06 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID NO: 77 Nucleic CTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA Acid CTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCC Sequence TGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGCGGCAGTGGATCTGGAACAGATTTCACT CTCACCATCAGCAGTCTGGAACCTGAAGATTTTGCAAC TTACTACTGTCAAGAGAGTTACAGTACCCCCTTCTTTA CTTTCGGCCCTGGGACCAAAGTGGATATCAGACGAACT GTGGCTGCACCATCTGTC B06 VLC GVHSDIQMTQSPSFLSASVGDRVTITCRASQGISSYLA SEQ ID NO: 78 Amino WYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFT Acid LTISSLEPEDFATYYCQESYSTPFFTFGPGTKVDIRRT Sequence VAAPSV B06 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 79 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTCAGTACTTTATGAAGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CTCTCCTTCTGGTGGCCTTACTCAGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGTGGTATAG AAGCACCTGGGTCCCCCTCTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCG B06 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYFMKWVR SEQ ID NO: 80 Amino QAPGKGLEWVSSISPSGGLTQYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARGGIEAPGSPSDYWGQG Sequence TLVTVSSASTKGPSVFP B07 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCAGC SEQ ID NO: 81 Nucleic CACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT Acid CCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCC Sequence TGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCT CATCTATGATGCATCCAACAGGGCCACTGGCATCCCAG CCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGT TTATTACTGTCAGCAGCGTAGCAACTGGCCTCGGACTT TCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTG GCTGCACCATCTGTC B07 VLC GVHSDIQMTQSPATLSLSPGERATLSCRASQSVSSYLA SEQ ID NO: 82 Amino WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT Acid LTISSLEPEDFAVYYCQQRSNWPRTFGGGTKVEIKRTV Sequence AAPSV B07 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 83 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTCAGTACCAGATGATTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CGTTCCTTCTGGTGGCATTACTAATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGTGGGGTAG AGGCAGTGGATAGTTCGTCGCCTGACTACTGGGGCCAG GGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGG CCCATCGGTCTTCCCG B07 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYQMIWVR SEQ ID NO: 84 Amino QAPGKGLEWVSVIVPSGGITNYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARGGVEAVDSSSPDYWGQ Sequence GTLVTVSSASTKGPSVFP B08 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCCCACTC SEQ ID NO: 85 Nucleic TGTGTCGGGGTCTCCGGGGAAGACGGTAACCATCTCCT Acid GCACCCGCAGCAGTGGCAGCATTGCCGGCAACTATGTG Sequence CAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCAC TGTGATCTATGAGGATAACAAAAGACCCTCTGGGGTCC CTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAAC TCTGCCTCCCTCATCATCTCTGGACTGAAGACTGAGGA CGAGGCTGACTACTACTGTCATTCTTATGATACCAGCA ATCAGGTATTCGGCGGAGGGACCAAACTGACCGTCCTA GGTCAGCCCAAGGCTGCCCCCTCG B08 VLC GVHSQSELTQPHSVSGSPGKTVTISCTRSSGSIAGNYV SEQ ID NO: 86 Amino QWYQQRPGSSPTTVIYEDNKRPSGVPDRFSGSIDSSSN Acid SASLIISGLKTEDEADYYCHSYDTSNQVFGGGTKLTVL Sequence GQPKAAPS B08 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 87 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTCGTTACATGATGAATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CTGGTCTTCTGGTGGCAAGACTCTTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAAGAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGGGGTGGTTACA ACAACTACTACTACTCTATGGACGTCTGGGGCCAAGGG ACCACGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCG B08 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYMMNWVR SEQ ID NO: 88 Amino QAPGKGLEWVSVIWSSGGKTLYADSVKGRFTISRDNSK Acid NTLYLQMKSLRAEDTAVYYCARGGYNNYYYSMDVWGQG Sequence TTVTVSSASTKGPSVFP B10 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCTTC SEQ ID NO: 89 Nucleic CTCCCTGTCTGCCTCTGTAGGAGACAGAGTCACCATCG Acid CGTGCCGGACAAGTCAGAACGTTAATAGGTACCTGAAT Sequence TGGTATCAACATAAACTCGGCCAGGCCCCTAAACTCCT GATCTACGGTGCAACCATTTTGCAGAGTGGGGTCCCAT CAAGGTTCCGTGGCAGTGGATCTGGGACAGATTTCATC CTCACCATCACCAATCTGCAACCTGAAGATTTTGCAGT TTACTACTGTCAACAGACTTACAGTCCCCCACTGACGT TCGGCCAAGGGACCAAGGCGGAATTTAAAGGAACTGTG GCTGCACCATCTGTC B10 VLC GVHSDIQMTQSPSSLSASVGDRVTIACRTSQNVNRYLN SEQ ID NO: 90 Amino WYQHKLGQAPKLLIYGATILQSGVPSRFRGSGSGTDFI Acid LTITNLQPEDFAVYYCQQTYSPPLTFGQGTKAEFKGTV
Sequence AAPSV B10 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 91 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTTCTTACGCTATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTGGAT CGTTCCTTCTGGTGGCACTACTTTTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGCCTGTACC GGTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCC TCCACCAAGGGCCCATCGGTCTTCCCG B10 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMMWVR SEQ ID NO: 92 Amino QAPGKGLEWVSWIVPSGGTTFYADSVKGRFTISRDNSK Acid NTLYLQMNSLRAEDTAVYYCARGLYRWGQGTLVTVSSA Sequence STKGPSVFP B11 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCACCCTC SEQ ID NO: 93 Nucleic GGTGTCACTGGCCCCAGGACAGACGGCCAGGATTACCT Acid GTGGGGGAAACAACATTGGAACTAAAAGTGTTCACTGG Sequence TACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGT CTATGATGACAGCGACCGGCCCTCAGGGATCCCTGAGC GATTCTCTGGCTCCAATTCTGGGAACACGGCCACCCTG ACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTA TTATTGTCAGGTGTGGGATAGTGGTAGTGATCATCAGG TCTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAG CCCAAGGTTGCCCCCTCG B11 VLC GVHSQSELTQPPSVSLAPGQTARITCGGNNIGTKSVHW SEQ ID NO: 94 Amino YQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATL Acid TISRVEAGDEADYYCQVWDSGSDHQVFGGGTKLTVLGQ Sequence PKVAPS B11 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 95 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTCCTTACTTTATGTTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CGGTTCTTCTGGTGGCGATACTTCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTNAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGCCTGTACC GGTGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCC TCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCC B11 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYFMFWVR SEQ ID NO: 96 Amino QAPGKGLEWVSSIGSSGGDTSYADSVKGRFTISRDNSz Acid NTLYLQMNSLRAEDTAVYYCARGLYRWGQGTLVTVSSA Sequence STKGPSVFPLAP B12 VLC GGCGTGCACTCACAGAGCGCTTTGACTCAGCCACCCTC SEQ ID NO: 97 Nucleic GGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTTCCT Acid GTGGGGGCGACAACATTGACACTAAAAATGTACAGTGG Sequence TACCAGCAGAGGCCAGGCCAGGCCCCTGTGCTGGTCGT CTATGATAATAGCGACCGGCCCTCAGCGATCCCTGAGC GATTCTCTGGCTCCAACTCTGGGACCACGGCCACCCTG ACCATCAGCAGGGTCGAGGCCGGGGATGAGGCCGACTA TTACTGTCAGGTGTTTGATGGTAGGAGTGATCATCCGG TGTTCGGCGGAGGGACCAAGCTGACCGTTCCTGGGTCA GCCCAAGGCTGCCCCCTC B12 VLC GVHSQSALTQPPSVSVAPGQTARISCGGDNIDTKNVQW SEQ ID NO: 98 Amino YQQRPGQAPVLVVYDNSDRPSAIPERFSGSNSGTTATL Acid TISRVEAGDEADYYCQVFDGRSDHPVFGGGTKLTVPGS Sequence AQGCPL B12 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID NO: 99 Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG Acid GATTCACTTTCTCTCTTTACGTTATGTATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATAT CTCTTCTTCTGGTGGCATTACTCATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGCTCTATTG TAGTAGTACCAGCTGCTATACGGAGCAACAACTGGTTC GACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCAAG CGCCTCCACCAAGGGCCCATCGGTCTTCCCG B12 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSLYVMYWVR SEQ ID Amino QAPGKGLEWVSYISSSGGITHYADSVKGRFTISRDNSK NO: 100 Acid NTLYLQMNSLRAEDTAVYYCARGSIVVVPAAIRSNNWF Sequence DPWGQGTLVTVSSASTKGPSVFP C01 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCTTC SEQ ID Nucleic CACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA NO: 101 Acid CTTGCCGGGCCAGTCAGAGTATTGGAAACTGGTTGGCC Sequence TGGTATCAGCAGAAACCAGGGGAAGCCCCTCACCTCCT GATCTATCAGGCGTCTAGTTTAGAAGGTGGGGTCCCAT CAAGGTTCAGCGGCAGTGGGTCTGGGACAAAATTCACT CTCAACATCAGCAGCCTGCAGCCTGATGACTTTGCAAC TTATTACTGCCAACAGTATAATTCTTATTCGTACACTT TTGGCCAGGGGACCAAGCTGGACATCAAACGAACTGTG GCTGCACCATCTGTC C01 VLC GVHSDIQMTQSPSTLSASVGDRVTITCRASQSIGNWLA SEQ ID Amino WYQQKPGEAPHLLIYQASSLEGGVPSRFSGSGSGTKFT NO: 102 Acid LNISSLQPDDFATYYCQQYNSYSYTFGQGTKLDIKRTV Sequence AAPSV C01 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 103 Acid GATTCACTTTCTCTAATTACGGTATGTCTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CGGTCCTTCTGGTGGCATTACTATGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGACCGGGTCTAGCA GTGGCTGGTACCCTAACTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCG C01 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVR SEQ ID Amino QAPGKGLEWVSVIGPSGGITMYADSVKGRFTISRDNSK NO: 104 Acid NTLYLQMNSLRAEDTAVYYCATGSSSGWYPNFDYWGQG Sequence TLVTVSSASTKGPSVFP C02 VLC TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCA SEQ ID Nucleic GTCTCCATCCTCCCTGTCTGCATCTGTAGGAGATAGAG NO: 105 Acid TCACCATCACTTGCCGGGCAAGTCAGACCATTAGCACC Sequence TATTTAGTTTGGTATCAGCAGAAACCCGAGAAAGCCCC TACGCTCCTGATCTCCGGTGCATCCACTTTGCAAAGTG GGGTCCCAAACAGGTTCAGAGGCAGTGGATCTGGGACA GACTTCACTCTCGCCATCTCCAGTCTTCAACCTGAAGA TTTTGCAACTTACTACTGTCAACAGAGTTACACTTCCC CTAGAACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA CGAACTGTGGCTGCACCATCTGTC C02 VLC FYSHSAQDIQMTQSPSSLSASVGDRVTITCRASQTIST SEQ ID Amino YLVWYQQKPEKAPTLLISGASTLQSGVPNRFRGSGSGT NO: 106 Acid DFTLAISSLQPEDFATYYCQQSYTSPRTFGQGTKVEIK Sequence RTVAAPSV C02 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 107 Acid GATTCACTTTCTCTCATTACTCTATGCGTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATAT CGTTCCTTCTGGTGGCTTTACTCAGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGCACGCACC TCCCGGGGGTTGACTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTT CCCG C02 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYSMRWVR SEQ ID Amino QAPGKGLEWVSYIVPSGGFTQYADSVKGRFTISRDNSK NO: 108 Acid NTLYLQMNSLRAEDTAVYYCARGTHLPGVDYWGQGTLV Sequence TVSSASTKGPSVFP C03 VLC GGCGTGCACTCACAGAGCGCTTTGACTCAGCCACCCTC SEQ ID Nucleic AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT NO: 109 Acid GTTCTGGAAGCAACTCCAACATCGGAGGTAATATTGTA Sequence ATCTGGCTCCAGCAGCTCCCAGGAACGGCCCCCAAACT CATGATTTATGATGTCAGTGATCGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGC CGATTATTATTGTGCAGCCTGGGATGACAGCCTGAATG GTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA AGTCAGCCCAAGGCTGCCCCCTCG C03 VLC GVHSQSALTQPPSASGTPGQRVTISCSGSNSNIGGNIV SEQ ID Amino IWLQQLPGTAPKLMIYDVSDRPSGVPDRFSGSKSGTSA NO: 110 Acid SLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVL Sequence SQPKAAPS C03 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 111 Acid GATTCACTTTCTCTCTTTACATGATGAAGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CTCTTCTTCTGGTGGCTATACTCAGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAACTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGGGTGGGACG TCTGGGGCAAAGGGACCACGGTCACCGTCTCAAGCGCC TCCACCAAGGGCCCATCGGTCTTCCCG C03 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSLYMMKWVR SEQ ID Amino QAPGKGLEWVSVISSSGGYTQYADSVKGRFTISRDNSK NO: 112 Acid NTLYLQMNNLRAEDTAVYYCARGWDVWGKGTTVTVSSA Sequence STKGPSVFP C04 VLC GGCGTGCACTCACAGAGCGCTTTGACTCAGCCACCCTC SEQ ID Nucleic AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCT NO: 113 Acid GTTCTGGAACCAGCTCCAACATCGGAAGTCATTATGTA Sequence TTCTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACT CCTCATCCATAGGAATGATGAGCGGCCCTCAGGGGTCC CTGACCGCTTCTCTGGCTCCAAGTCTGGCACCTCCGCC TCCCTGGCCATCAGTGGCCTCCAGTCTGAGGATGAGGC TGATTATTACTGTGCTACGTGGGATGACAACCTAAATG GTCCGGTATTCGGCGGAGGGACCAAGCTGACCGGCCCT GGGTCAGCCCAAGGCTGCCCCCTC C04 VLC GVHSQSALTQPPSASGTPGQRVTISCSGTSSNIGSHYV SEQ ID Amino FWYQQLPGTAPKLLIHRNDERPSGVPDRFSGSKSGTSA NO: 114 Acid SLAISGLQSEDEADYYCATWDDNLNGPVFGGGTKLTGP Sequence GSAQGCPL C04 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 115 Acid GATTCACTTTCTCTATGTACTTTATGGTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTGGAT CGGTTCTTCTGGTGGCGAGACTCCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCAAGAGGGTACAGCA GTGGCTGGTATGTAATGGGAGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCG C04 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYFMVWVR SEQ ID Amino QAPGKGLEWVSWIGSSGGETPYADSVKGRFTISRDNSK NO: 116 Acid NTLYLQMNSLRAEDTAVYYCARGYSSGWYVMGDYWGQG Sequence TLVTVSSASTKGPSVFP C05 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCACCCTC SEQ ID Nucleic AGTGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT NO: 117 Acid GTTCTGGAAGCAGTTCCAACATCGGAAGTGAGTATGTG Sequence TACTGGTTCCAGCAGCTCCCAGGAACGGCCCCCAGACT CCTCATCTATAGGAATGATCAGCGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGAC TGATTATTACTGTACAACATGGGATGACAGCCTGAGTG GTCCGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA GGTCAGCCCAAGGCTGCCCCCTCG C05 VLC GVHSQSELTQPPSVSGTPGQRVTISCSGSSSNIGSEYV SEQ ID Amino YWFQQLPGTAPRLLIYRNDQRPSGVPDRFSGSKSGTSA NO: 118 Acid SLAISGLRSEDETDYYCTTWDDSLSGPVFGGGTKLTVL Sequence GQPKAAPS C05 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 119 Acid GATTCACTTTCTCTTCTTACCAGATGGATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CGTTCCTTCTGGTGGCGATACTACTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGACATGTCTACT ATGATAGTAGTGATTATTTCCCCAACCCGTTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCGCCTC CACCAAGGGCCCATCGGTCTTCCCG C05 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYQMDWVR SEQ ID Amino QAPGKGLEWVSRIVPSGGDTTYADSVKGRFTISRDNSK NO: 120 Acid NTLYLQMNSLRAEDTAVYYCARHVYYDSSDYFPNPFDY Sequence WGQGTLVTVSSASTKGPSVFP C06 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID Nucleic CTCCCTGTCTGCGTCTGTAGGAGACAGAGTCACCATCA NO: 121 Acid CTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCC Sequence TGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCT
GATCTATCCTGCATCCACTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACT CTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAAC TTATTATTGTCAACAGGCTGACAGTTTCCCGCCCACCT TCGGCGGAGGGACCACGGTGGAGATCAGACGAACTGTG GCTGCACCATCTGTC C06 VLC GVHSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLA SEQ ID Amino WYQQKPGKVPKLLIYPASTLQSGVPSRFSGSGSGTDFT NO: 122 Acid LTISSLQPEDFATYYCQQADSFPPTFGGGTTVEIRRTV Sequence AAPSV C06 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 123 Acid GATTCACTTTCTCTTTTTACTTTATGTTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATAT CGGTCCTTCTGGTGGCCCTACTAATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGACATTACCCCA GGGAGTACCAGCTGCCCGGGTCGTTCGACCCCTGGGGC CAGGGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAA GGGCCCATCGGTCTTCCCG C06 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYFMFWVR SEQ ID Amino QAPGKGLEWVSYIGPSGGPTNYADSVKGRFTISRDNSK NO: 124 Acid NTLYLQMNSLRAEDTAVYYCARHYPREYQLPGSFDPWG Sequence QGTLVTVSSASTKGPSVFP C07 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID Nucleic TTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA NO: 125 Acid CTTGCCGGGCAAGTCAGAGTATTAGTAACTATTTAAAT Sequence TGGTATCAGCAGAGACCAGGGAAGGCCCCTAAGCTCCT GATCTATGCTGCATCCAGTTTGGAAAGAGGGGTCCCAT CAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACT CTCACCATCAGCAGTCTGCAATCTGAAGATTTTGCAAC TTACTACTGTCAACAGAGTTACAGTCCCCCTCCTCTCA CTTTCGGCGGAGGGACCAAACTAGAGATCAAACGAACT GTGGCTGCACCATCTGTC C07 VLC GVHSDIQMTQSPSSLSASVGDRVTITCRASQSISNYLN SEQ ID Amino WYQQRPGKAPKLLIYAASSLERGVPSRFSGSGSGTDFT NO: 126 Acid LTISSLQSEDFATYYCQQSYSPPPLTFGGGTKLEIKRT Sequence VAAPSV C07 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 127 Acid GATTCACTTTCTCTTATTACGTTATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CCGTCCTTCTGGTGGCATTACTACTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGACGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAAAATCGACTACG GTGGTAACTCGTTCTACTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCG C07 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYVMMWVR SEQ ID Amino QAPGKGLEWVSVIRPSGGITTYADSVKGRFTISRDNSK NO: 128 Acid NTLYLQTNSLRAEDTAVYYCAKIDYGGNSFYFDYWGQG Sequence TLVTVSSASTKGPSVFP C08 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCACC SEQ ID Nucleic CTCCCTGTCTGCATTAGTAGGGGACAGAGTCACCATCA NO: 129 Acid CTTGCCGGGCAAGTCAGAGCATAAGCAGATATGTGAAT Sequence TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCT GATCTATGCTGCATCCATAGTAGAAAATGGGGTCCCAT CTAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCAGT CTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAC TTACTACTGTCAACAAACTTACAGTACTCCGCTCACTT TCGGCGGAGGGACCAAGCTGGCGATCAAACGAACTGTG GCTGCACCATCTGTC C08 VLC GVHSDIQMTQSPPSLSALVGDRVTITCRASQSISRYVN SEQ ID Amino WYQQKPGKAPKVLIYAASIVENGVPSRFSGSGSGTDFS NO: 130 Acid LTISSLQPEDFATYYCQQTYSTPLTFGGGTKLAIKRTV Sequence AAPSV C08 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 131 Acid GATTCACTTTCTCTTATTACGAGATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTAT CTCTCCTTCTGGTGGCCCTACTATGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGGAGTCTACTATTGTGCGAGAAAGATGGGGC GTGTAGGATATTGTAGTAGTACCAGCTGCTATCGGGAT GACTACTACGGTATGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGG TCTTCCCG C08 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYEMMWVR SEQ ID Amino QAPGKGLEWVSSISPSGGPTMYADSVKGRFTISRDNSK NO: 132 Acid NTLYLQMNSLRAEDTGVYYCARKMGRVGYCSSTSCYRD Sequence DYYGMDVWGQGTTVTVSSASTKGPSVFP C09 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCAGG SEQ ID Nucleic CACCCTGTCTTTGTCTCCAGGGGAAAGAGCAACCCTCT NO: 133 Acid CCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTATTTA Sequence GCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCT CCTCATCTCTGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGC AGTGTATTACTGTCAGCAGTATGGTAGCTCACCGTACA CTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACT GTGGCTGCACCATCTGTC C09 VLC GVHSDIQMTQSPGTLSLSPGERATLSCRASQSVSSTYL SEQ ID Amino AWYQQKPGQAPRLLISGASSRATGIPDRFSGSGSGTDF NO: 134 Acid TLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKLEIKRT Sequence VAAPSV C09 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 135 Acid GATTCACTTTCTCTCAGTACTTTATGAATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATAT TTCTGGTGGCCGTACTCCTTATGCTGACTCCGTTAAAG GTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACT CTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACAC TGCAGTCTACTATTGTGCGATCCTTCTGGGACCGAGCA GCTCCAATCACCCTTTCCTGGGGCCCTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCGCTAGCGCCC C09 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYFMNWVR SEQ ID Amino QAPGKGLEWVSYISGGRTPYADSVKGRFTISRDNSKNT NO: 136 Acid LYLQMNSLRAEDTAVYYCAILLGPSSSNHPFLGPWGQG Sequence TLVTVSSASTKGPSVFPLAP C10 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCTGCCTC SEQ ID Nucleic CGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCT NO: 137 Acid GCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTT Sequence GTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAA ACTCATGATTTATGAGGGCAGTAAGCGGCCCTCAGGGG TTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACG GCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGA GGCTGATTATTACTGCTGCTCATATGCAGGTAGTAGCA CTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA GGTCAGCCCAAGGCCAACCCCACT C10 VLC GVHSQSELTQPASVSGSPGQSITISCTGTSSDVGSYNL SEQ ID Amino VSWYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSGNT NO: 138 Acid ASLTISGLQAEDEADYYCCSYAGSSTYVFGTGTKVTVL Sequence GQPKANPT C10 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 139 Acid GATTCACTTTCTCTGTTTACGTTATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTAT CGTTCCTTCTGGTGGCAAGACTCATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGACCGGACTACG GTGGTAATTCGCGCCCCCTTGAGTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCG C10 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSVYVMMWVR SEQ ID Amino QAPGKGLEWVSGIVPSGGKTHYADSVKGRFTISRDNSK NO: 140 Acid NTLYLQMNSLRAEDTAVYYCARPDYGGNSRPLEYWGQG Sequence TLVTVSSASTKGPSVFP C11 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCTCCCTC SEQ ID Nucleic CGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCT NO: 141 Acid GCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTAT Sequence GTCTCCTGGTATCAACAACACCCAGACAAAGCCCCCAA ACTCCTGATTTATGAGGTCACTCAGCGGCCCTCAGGGG TCCCTGATCGCTTCTCTGGCTCCAGGTCTGGCAACACG GCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGA GGCTGATTATTACTGCAGCTCATATGCAGGCAGGAACA ATCTTTATGTCTTCGGACCTGGGACCAAGGTCACCGTC CTAGGTCAGCCCAAGGCCAACCCCACT C11 VLC GVHSQSELTQPPSASGSPGQSVTISCTGTSSDVGGYNY SEQ ID Amino VSWYQQHPDKAPKLLIYEVTQRPSGVPDRFSGSRSGNT NO: 142 Acid ASLTVSGLQAEDEADYYCSSYAGRNNLYVFGPGTKVTV Sequence LGQPKANPT C11 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 143 Acid GATTCACTTTCTCTGAGTACCCTATGTGGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTGGAT CTATCCTTCTGGTGGCAATACTGATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGATTCCCTATTGTA GTAGTTCCAGCTGCCCCCTACACTGGGGCCAGGGAACC CTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATC GGTCTTCCCG C11 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYPMWWVR SEQ ID Amino QAPGKGLEWVSWIYPSGGNTDYADSVKGRFTISRDNSK NO: 144 Acid NTLYLQMNSLRAEDTAVYYCAIPYCSSSSCPLHWGQGT Sequence LVTVSSASTKGPSVFP C12 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCACCCTC SEQ ID Nucleic AGTGTCCGTGTCCCCAGCACAGACAGCCAGCATCACCT NO: 145 Acid GCTCTGGAGATAAATTGGGGGATAAATATGCTTGCTGG Sequence TATCAGCAGAAGCCAGGCCAGTCCCCTGTACTGGTCAT CTATGAAGATACCAAGCGGCCCTCAGGGATCCCTGAGC GATTCTCTGGCTCCAATTCTGGGAACACAGCCACTCTG ACCATCAGCGGGACCCAGGTTATGGATGAGGCTGACTA TTACTGTCAGGTGTGGGACAGCAGCACTGCGGTATTCG GCGGAGGGACCAAGCTGACCGTCCTGGGTCAGCCCAAG GCTGCCCCCTCG C12 VLC GVHSQSELTQPPSVSVSPAQTASITCSGDKLGDKYACW SEQ ID Amino YQQKPGQSPVLVIYEDTKRPSGIPERFSGSNSGNTATL NO: 146 Acid TISGTQVMDEADYYCQVWDSSTAVFGGGTKLTVLGQPK Sequence AAPS C12 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 147 Acid GATTCACTTTCTCTGCTTACAATATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CTATCCTTCTGGTGGCTATACTCTTTATGCTGACTCGG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGACAAAAACTTA TGATTCGGGCAGTTCGCCCGTTTGACTACTGGGGCCAG GGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGG CCCATCGGTCTTCCCG C12 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYNMMWVR SEQ ID Amino QAPGKGLEWVSRIYPSGGYTLYADSVKGRFTISRDNSK NO: 148 Acid NTLYLQMNSLRAEDTAVYYCARQKLMIRAVRPFDYWGQ Sequence GTLVTVSSASTKGPSVFP D01 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCACCGTC SEQ ID Nucleic AGCGTCCGGGACCCCCGGGCAGAGGATCACCATCTCTT NO: 149 Acid GTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTA Sequence TACTGGTACCAACAGTTCCCAGAGACGGCCCCCAAACT CCTCATCTCTAGAAATGATCAGCGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCC TCCCTGGCCATCAGTGGGCTCCGGTCCGAAGATGAGGC TGATTATTACTGTGCATCATGGGATGACAGCCTGAGTG GTGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA GGTCAGCCCAAGGCTGCCCCCTCG D01 VLC GVHSQSELTQPPSASGTPGQRITISCSGSSSNIGSNYV SEQ ID Amino YWYQQFPETAPKLLISRNDQRPSGVPDRFSGSKSGTSA NO: 150 Acid SLAISGLRSEDEADYYCASWDDSLSGVVFGGGTKLTVL Sequence GQPKAAPS D01 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 151 Acid GATTCACTTTCTCTATGTACCTTATGTTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CTCTTCTTCTGGTGGCGAGACTTCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA
GGACACTGCAGTCTACTATTGTGCGAGACAGGTCAGTG ACTGGACGCGCCTCTACTCCTTTGACTACTGGGGCCAG GGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGG CCCATCGGTCTTCCCG D01 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYLMFWVR SEQ ID Amino QAPGKGLEWVSVISSSGGETSYADSVKGRFTISRDNSK NO: 152 Acid NTLYLQMNSLRAEDTAVYYCARQVSDWTRLYSFDYWGQ Sequence GTLVTVSSASTKGPSVFP D02 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID Nucleic CTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA NO: 153 Acid CTTGCCGGGCCAGTCAGGGCATTAGCACTTATTTAGCC Sequence TGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGGTCCT CATCTATACTGCATCCACTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACT CTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAAC TTACTACTGTCAACAGAGTTACATTACCCCTCCGGAGG TCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGA ACTGTGGCTGCACCATCTGTC D02 VLC GVHSDIQMTQSPSFLSASVGDRVTITCRASQGISTYLA SEQ ID Amino WYQQKPGKAPKVLIYTASTLQSGVPSRFSGSGSGTEFT NO: 154 Acid LTISSLQPEDFATYYCQQSYITPPEVTFGPGTKVDIKR Sequence TVAAPSV D02 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 155 Acid GATTCACTTTCTCTTGGTACGATATGGCTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CGTTCCTTCTGGTGGCCATACTTCTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTTTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAAGGGCAAGTC GTCCTGAGTTTTTTGACTACTGGGGCCAGGGAGCCCTG GTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCG D02 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYDMAWVR SEQ ID Amino QAPGKGLEWVSRIVPSGGHTSYADSVKGRFTISRDNFK NO: 156 Acid NTLYLQMNSLRAEDTAVYYCARRASRPEFFDYWGQGAL Sequence VTVSSASTKGPSVFP D03 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID Nucleic TGCCATGTCTGCATCTGTCGGAGACAGAGTCACCATCA NO: 157 Acid CTTGTCGGGCGAGTCAGGTCATGATCAATTATATAGCC Sequence TGGTTTCGGCAGAAACCAGGGAAAGTCCCTGAGCGCCT GATCTATGCAGCATCCACTCTGCAAAATGGGGTCCCAT CAAGGTTCAGCGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGACTAGAACCTGAGGATTTTGCAGT TTATTACTGTCAGCACCGTATCACCTGGCCTCCGGCGC TCACTTTCGGCGGAGGGACCACGGTGGAGATCAAACGA ACTGTGGCTGCACCATCTGTC D03 VLC GVHSDIQMTQSPSAMSASVGDRVTITCRASQVMINYIA SEQ ID Amino WFRQKPGKVPERLIYAASTLQNGVPSRFSGSGSGTDFT NO: 158 Acid LTISRLEPEDFAVYYCQHRITWPPALTFGGGTTVEIKR Sequence TVAAPSV D03 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 159 Acid GATTCACTTTCTCTTGGTACCGTATGGATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CGGTTCTTCTGGTGGCATGACTTATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGACGGGTAGTCG GGGGCGCCGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCG D03 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYRMDWVR SEQ ID Amino QAPGKGLEWVSVIGSSGGMTYYADSVKGRFTISRDNSK NO: 160 Acid NTLYLQMNSLRAEDTAVYYCARRVVGGAGMDVWGQGTT Sequence VTVSSASTKGPSVFP D04 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID Nucleic CTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCATCA NO: 161 Acid CTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAAT Sequence TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCT GATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCAT CAAGATTCAGTGGCAGTGGAACTGGGACAGATTTCACT CTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAG TTATTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCA CTTTCGGCCCTGGGACCAAGGTGGAGATCAAACGAACT GTGGCTGCACCATCTGTC D04 VLC GVHSDIQMTQSPSSLSASVGDRVAITCRASQSIDTYLN SEQ ID Amino WYQQKPGKAPKLLIYAASKLEDGVPSRFSGSGTGTDFT NO: 162 Acid LTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIKRT Sequence VAAPSV D04 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 163 Acid GATTCACTTTCTCTGATTACCAGATGATGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CTCTCCTTCTGGTGGCATGACTCGTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCCGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGATCGGGGCCGT ACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACC GTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCC G D04 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYQMMWVR SEQ ID Amino QAPGKGLEWVSRISPSGGMTRYADSVKGRFTISRDNSK NO: 164 Acid NTLYLPMNSLRAEDTAVYYCARSGPYYFDYWGQGTLVT Sequence VSSASTKGPSVFP D05 VLC GGCGTGCACTCACAGAGCGTCTTGACTCAGCCTGACTC SEQ ID Nucleic CGTGTCTGGGTCTCCTGGGCAGTCGATCACCATCTCCT NO: 165 Acid GCACTGGCAGCAGTCATGACATTGGTTCCTATGACTAT Sequence GTCTCCTGGTATCAGCACCACCCAGGGAAAGCCCCCAA ATTCATACTTTATGATGTCTATAATCGGCCCTCAGGTG TTTCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACG GCCTCCCTGACTATCTCTGGGCTCCAGCCTGACGACGA GGCTGACTATTTTTGTATGTCCTATACAATCACAACGC TTCTCTTCGGAACTGGGACCAGGGTCACCGTCCTGAGT CAGCCCAAGGCCAACCCCACT D05 VLC GVHSQSVLTQPDSVSGSPGQSITISCTGSSHDIGSYDY SEQ ID Amino VSWYQHHPGKAPKFILYDVYNRPSGVSDRFSGSKSGNT NO: 166 Acid ASLTISGLQPDDEADYFCMSYTITTLLFGTGTRVTVLS Sequence QPKANPT D05 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 167 Acid GATTCACTTTCTCTCATTACAATATGGCTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CCGTTCTTCTGGTGGCCTTACTGTTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGTGGCTGGCC CTGGGTACTGGGGCCAGGGAACCCTGGTCACCGTCTCA AGCGCCTCCACCAAGGGCCCATCGGTCTTCCCG D05 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYNMAWVR SEQ ID Amino QAPGKGLEWVSRIRSSGGLTVYADSVKGRFTISRDNSK NO: 168 Acid NTLYLQMNSLRAEDTAVYYCARVAGPGYWGQGTLVTVS Sequence SASTKGPSVFP D06 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID Nucleic CTCCCTGTCTGCATCTGTAGGAGACAGAGTCACTATCA NO: 169 Acid CTTGCCGGACAAGTCAAATCATTAACACCTATTTAAAT Sequence TGGTATCAACAAAAACCGGGAAAAGCCCCTAAACTCCT GATCTATGCTGCCTCCACTTTACAGGGTGGGGTCCCGT CAAGATTCAGTGGCAGTGGATCCGGGACAGACTTCACT CTCACCATCAAGAGTCTGCAACCTGACGACTTTGCAAC TTACTATTGTCAACAGAGTTATACTTCCCCGCGAACAT TCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTG GCTGCACCATCTGTC D06 VLC GVHSDIQMTQSPSSLSASVGDRVTITCRTSQIINTYLN SEQ ID Amino WYQQKPGKAPKLLIYAASTLQGGVPSRFSGSGSGTDFT NO: 170 Acid LTIKSLQPDDFATYYCQQSYTSPRTFGQGTKVEIKRTV Sequence AAPSV D06 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 171 Acid GATTCACTTTCTCTGGTTACATTATGGAGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTAT CGTTTCTTCTGGTGGCTTTACTATGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGTTGGGGATT CCAAGGGCGGGTACTACCTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCC ATCGGTCTTCCCGCTAGCGCCC D06 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYIMEWVR SEQ ID Amino QAPGKGLEWVSVIVSSGGFTMYADSVKGRFTISRDNSK NO: 172 Acid NTLYLQMNSLRAEDTAVYYCARVGDSKGGYYLDYWGQG Sequence TLVTVSSASTKGPSVFPLAP D07 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCTGCCTC SEQ ID Nucleic CGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCT NO: 173 Acid GCACTGGAACCAACAGTGACATTGGTGGTTATAATTAT Sequence GTCTCCTGGTACCAACAACACCCGGGCAAAGTCCCCAA ACTCTTGATTTTTGAGGTCAATAATCGGCCCTCAGGGG TTTCTAGTCGCTTCTCTGGCTCCAAGTCTGGCGACACG GCCTCCCTGACCATCTCTGGGCTCCAACCTGAGGACGA GGCTGTTTATTACTGCGGCTCATTTACAGTCAGCGTCA CCTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTG GGTCAGCCCAAGGCCAACCCCACT D07 VLC GVHSQSELTQPASVSGSPGQSITISCTGTNSDIGGYNY SEQ ID Amino VSWYQQHPGKVPKLLIFEVNNRPSGVSSRFSGSKSGDT NO: 174 Acid ASLTISGLQPEDEAVYYCGSFTVSVTYVFGTGTKVTVL Sequence GQPKANPT D07 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 175 Acid GATTCACTTTCTCTGAGTACAATATGTTTTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATAT CTATTCTTCTGGTGGCTCTACTGATTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGTAGGTATAG CAGCTCGTCCGTTCGACCCCTGGGGCCAGGGAACCCTG GTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCGCTAGCGCCCTG D07 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYNMFWVR SEQ ID Amino QAPGKGLEWVSYIYSSGGSTDYADSVKGRFTISRDNSK NO: 176 Acid NTLYLQMNSLRAEDTAVYYCARVGIAARPFDPWGQGTL Sequence VTVSSASTKGPSVFPLAP D08 VLC GGCGTGCACTCTGACATCCAGATGACCCAGTCTCCATC SEQ ID Nucleic CTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA NO: 177 Acid CTTGCCGGGCAAGTCAGAGCATTAGCGACTATTTAAAT Sequence TGGTATCAGCAGAAACCAGGGAAAGCCCCTGACCTCCT GATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACT CTCACCGTCAGCAGTCTGCAACCTGAAGATTTTGCAAC TTACTTCTGTCAACAGAGTTACTCTATTCCTCTCACTT TCGGCGGCGGGACCAAGGTTGAGATCACTCGAACTGTG GCTGCACCATCTGTC D08 VLC GVHSDIQMTQSPSSLSASVGDRVTITCRASQSISDYLN SEQ ID Amino WYQQKPGKAPDLLIYAASSLQSGVPSRFSGSGSGTDFT NO: 178 Acid LTVSSLQPEDFATYFCQQSYSIPLTFGGGTKVEITRTV Sequence AAPSV D08 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 179 Acid GATTCACTTTCTCTTTTTACGCTATGTGGTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTCGTAT CTATTCTTCTGGTGGCAAGACTTGGTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCTACTATTGTGCGAGAGTGGGGATGT CCACCTATGCTTTTGATATCTGGGGCCAAGGGACAATG GTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGT CTTCCCG D08 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYAMWWVR SEQ ID Amino QAPGKGLEWVSRIYSSGGKTWYADSVKGRFTISRDNSK NO: 180 Acid NTLYLQMNSLRAEDTAVYYCARVGMSTYAFDIWGQGTM Sequence VTVSSASTKGPSVFP D09 VLC GGCGTGCACTCACAGAGCGAATTGACTCAGCCACCCTC SEQ ID Nucleic AGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACCT NO: 181 Acid GCTCTGGAGATAAATTGGGGGATAAATATGCTTGCTGG Sequence TATCAGCAGAAGCCAGGCCAGTCCCCTGTGCTGGTCAT CTATCAAGATAGCAAGCGGCCCTCAGGGATCCCTGAGC GATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTG ACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTA TTACTGTCAGGCGTGGGACAGCAGCGCTGTGGTATTCG GCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAG GCTGCCCCCTCG
D09 VLC GVHSQSELTQPPSVSVSPGQTASITCSGDKLGDKYACW SEQ ID Amino YQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATL NO: 182 Acid TISGTQAMDEADYYCQAWDSSAVVFGGGTKLTVLGQPK Sequence AAPS D09 VHC GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCA SEQ ID Nucleic GCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCG NO: 183 Acid GATTCACTTTCTCTCATTACAATATGCATTGGGTTCGC Sequence CAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGGTAT CGTTTCTTCTGGTGGCAATACTGGTTATGCTGACTCCG TTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAG AATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGA GGACACTGCAGTCGACTATTGTGCGAGAGTGGTACGGT ATAGCAGTGGCTGGTACTACTGGTTCGACCCCTGGGGC CAGGGAACCCTGGTCACCGTCTCAAGCGCCTCCACCAA GGGCCCATCGGTCTTCCCG D09 VHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYNMHWVR SEQ ID Amino QAPGKGLEWVSGIVSSGGNTGYADSVKGRFTISRDNSK NO: 184 Acid NTLYLQMNSLRAEDTAVDYCARVVRYSSGWYYWFDPWG Sequence QGTLVTVSSASTKGPSVFP
[0365]A sequence analysis of the CDRs of the light and heavy chain variable regions has identified a number of consensus motifs amongst the MMP-26-binding antibodies. We have separated these clones into two groups: Group I ("Inhibitory Fab's"): Fab's that bind MMP-26 and inhibit invasion of JEG-3 cells through MATRIGEL® basement membrane matrix; and Group II ("Non-inhibitory Fab's"): Fab's that bind MMP-26 but do not substantially inhibit invasion of JEG-3 cells through MATRIGEL® basement membrane matrix.
TABLE-US-00024 TABLE 7 Group I (Inhibitors) HC HCs Column 1 Column 2 Column 3 4 Name CDR1 CDR2 CDR3 Type a01 (SEQ ID NO: 16) AYNMF GIGSSGGIAPYADSVKG AAYEVENWFDP H a04 (SEQ ID NO: 24) EYNMA RIGSSGGKTKYADSVKG DEAPDYGDDAEAFDI H a11 (SEQ ID NO: 52) WYTMM RISPSGGHTLYADSVKG DTWDDYYDSSGYYNDFDY H b04 (SEQ ID NO: 74) FYWMM GISSSGGFTKYADSVKG ETSRRAFDI H b06 (SEQ ID NO: 80) QYFMK SISPSGGLTQYADSVKG GGIEAPGSPSDY H b10 (SEQ ID NO: 92) SYAMM WIVPSGGTTFYADSVKG GLYR H c01 (SEQ ID NO: 104) NYGMS VIGPSGGITMYADSVKG GSSSGWYPNFDY H c04 (SEQ ID NO: 116) MYFMV WIGSSGGETPYADSVKG GYSSGWYVMGDY H c05 (SEQ ID NO: 120) SYQMD RIVPSGGDTTYADSVKG HVYYDSSDYFPNPFDY H c08 (SEQ ID NO: 132) YYEMM SISPSGGPTMYADSVKG KMGRVGYCSSTSCYRDDYYGMDV H c11 (SEQ ID NO: 144) EYPMW WIYPSGGNTDYADSVKG PYCSSSSCPLH H c12 (SEQ ID NO: 148) AYNMM RIYPSGGYTLYADSVKG QKLMIRAVRPFDY H d02 (SEQ ID NO: 156) WYDMA RIVPSGGHTSYADSVKG RASRPEFFDY H d04 (SEQ ID NO: 164) DYQMM RISPSGGMTRYADSVKG SGPYYFDY H d06 (SEQ ID NO: 172) GYIME VIVSSGGFTMYADSVKG VGDSKGGYYLDY H d07 (SEQ ID NO: 176) EYNMF YIYSSGGSTDYADSVKG VGIAARPFDP H d08 (SEQ ID NO: 180) FYAMW RIYSSGGKTWYADSVKG VGMSTYAFDI H A1-orig (SEQ ID YYRMS SIGPSGGDTLYADSVKG SFSSGPYYFDY H NO: 4) D6-orig (SEQ ID MYSMR SIYPSGGSTEYADSVKG EGGENDY H NO: 8) H6-orig (SEQ ID QYWMN GIGPSGGITYYADSVKG GEEDGYNSDY H NO: 12)
[0366]At least some members of Group I HC can include the following sequence in the region of CDR1:
TABLE-US-00025 X1-Y-X3-M-M, (SEQ ID NO: 236) or (ASMYWFEQ)-Y-(AWFNQ)-M-(ASMWF), (SEQ ID NO: 237)
where "(ASMYWFEQ)" means that, in one embodiment, X1 could be any of the amino-acid types given; "(AWFNQ)" means that X3 could be any of the types listed; and "(ASMWF)" means that the final position of CDR1 could be any of Ala, Ser, Met, Trp, or Phe.
[0367]At least some members of Group I HC can include one of the following sequences in the region of CDR2:
TABLE-US-00026 (SEQ ID NO: 264) R-I-X-(SP)-S-G-G-X-T, (SEQ ID NO: 185) R-I-X-(SP)-S-G-G-X-T-X-Y-A-D-S-V-K-G, (SEQ ID NO: 265) (GSVWR)-I-(GSVY)-(SP)-S-G-G-(SIFKDH)-T-(LMKDP), or (SEQ ID NO: 186) (GSVWR)-I-(GSVY)-(SP)-S-G-G-(SIFKDH)-T-(LMKDP)-Y- A-D-S-V-K-G.
[0368]At least some members of Group I HC can include the following sequence in the region of CDR3: F-D-I, e.g., A-F-D-I (SEQ ID NO:253). The CDR3 region can also include a di tyrosine, e.g., in addition to the tripeptide F-D-I.
TABLE-US-00027 TABLE 8 Group I (Inhibitors) LC CDR1 CDR2 CDR3 Name (column 1) (column 2) (column 3) Type a01 (SEQ ID NO: 14) RASQIVRSTYLA GTSSRAT QRYGDSPPIT K b04 (SEQ ID NO: 22) RASQGISKSLA GAFSLES QQANSFPLT K b06 (SEQ ID NO: 78) RASQGISSYLA AASTLQS QESYSTPFFT K b10 (SEQ ID NO: 90) RTSQNVNRYLN GATILQS QQTYSPPLT K c01 (SEQ ID NO: 102) RASQSIGNWLA QASSLEG QQYNSYSYT K c08 (SEQ ID NO: 130) RASQSISRYVN AASIVEN QQTYSTPLT K d02 (SEQ ID NO: 154) RASQGISTYLA TASTLQS QQSYITPPEVT K d04 (SEQ ID NO: 162) RASQSIDTYLN AASKLED QQSYSSPGIT K d06 (SEQ ID NO: 170) RTSQIINTYLN AASTLQG QQSYTSPRT K d08 (SEQ ID NO: 178) RASQSISDYLN AASSLQS QQSYSIPLT K D6-orig (SEQ ID NO: 6) RASQSIDTYLN AASKLED QQSYSSPGIT K a04 (SEQ ID NO: 22) SGGYSNMGSNYAH NNNQRPS AAWDENLSGPV L a11 (SEQ ID NO: 50) GGNNIGTKSVH DDSDRPS QVWDSGSDHQV L c05 (SEQ ID NO: 118) SGSSSNIGSEYVY RNDQRPS TTWDDSLSGPV L c04 (SEQ ID NO: 114) SGTSSNIGSHYVF RNDERPS ATWDDNLNGPV K c11 (SEQ ID NO: 142) TGTSSDVGGYNYVS EVTQRPS SSYAGRNNLYV L c12 (SEQ ID NO: 146) SGDKLGDKYAC EDTKRPS QVWDSSTAV L d07 (SEQ ID NO: 174) TGTNSDIGGYNYVS EVNNRPS GSFTVSVTYV L A1-orig (SEQ ID NO: 2) SGSSSNIGSHYVH RNGQRPS ATWDDSVL L H6-orig (SEQ ID NO: 10) TGTSSDVGAYNYVS EVNKRPS NSYAGSNSLI L
(CDRs sequences are from corresponding sequences for the variable domain provided with sequence identifiers in Table 6 above)
[0369]At least some members of Group I LC κ sequences can include one of the following sequences in the region of CDR1:
TABLE-US-00028 (SEQ ID NO: 187) R-(AT)-S-Q-(GSI)-(IV)-(SDN)-(STR)-Y-L-(AN)-X, (SEQ ID NO: 188) R-(AT)-S-Q-(GSIN)-(IV)-(GSRDN)-(STRKDN)-(STYW)- (LVY)-(ALN)-A, (SEQ ID NO: 189) R-A-S-Q-(GS)-I-(SD)-(ST)-Y-L-(AN)-X, (SEQ ID NO: 190) R-A-S-Q-X-I-X-X-Y-L-N-X, or (SEQ ID NO: 191) R-A-S-Q-(GSI)-(IV)-(GSRD)-(STRKDN)-(STYW)-(LVY)- (ALN)-A,
[0370]At least some members of Group I LC κ sequences can include one of the following sequences in the region of CDR2:
TABLE-US-00029 (SEQ ID NO: 238) (AG)-A-S-(STIK)-L-(EQ)-(GSD), (SEQ ID NO: 239) (AGTQ)-(AT)-(STF)-(STIK)-(LVR)-(AEQ)-(GSTDN), or (SEQ ID NO: 192) A-A-S-X-L-(EDNQ).
[0371]At least some members of Group I LC κ sequences can include one of the following sequences in the region of CDR3:
TABLE-US-00030 (SEQ ID NO: 193) Q-Q-(STY)-(YN)-S-(ST)-P-(GLP)-(TI)-T, or (SEQ ID NO: 194) Q-(REQ)-(ASTY)-(GYN)-(STID)-(STIYFP)-(SP)- (GLYFRP)-(TIFE)-(TV)-T.
[0372]At least some members of Group I LC λ sequences can include one of the following sequences in the region of CDR1:
TABLE-US-00031 (SEQ ID NO: 195) S-G-X-S-S-X-X-G-S, or (SEQ ID NO: 196) T-G-T-(SN)-S-D-(IV)-G-(AG)-Y-N-Y-V-S
[0373]At least some members of Group I LC λ sequences can include one of the following sequences in the region of CDR2:
TABLE-US-00032 (SEQ ID NO: 240) (RDNE)-(VDN)-(GSTDN)-(KDNEQ)-R-P-S, or (SEQ ID NO: 241) (RE)-(VDN)-(TDN)-(KQ)-R-P-S.
[0374]At least some members of Group I LC λ sequences can include one of the following sequences in the region of CDR3:
TABLE-US-00033 (SEQ ID NO: 242) (AQ)-(STV)-(YW)-(AD)-(GSD)-(SN)-(LVN)-(SN)-(GL)- P-V, or (SEQ ID NO: 197) W-D-X-S-X-X-X-X-V.
TABLE-US-00034 TABLE 9 Group II HC HCs 1 2 3 4 Name CDR1 CDR2 CDR3 Type a02 (SEQ ID NO: 296, PYFMF SIGSSGGDTSYADSVKG GLYR H 297) a03 (SEQ ID NO: 20) IYSMD SIYSSGGATRYADSVKG CSWLQLVPMHP H a05 (SEQ ID NO: 28) VYSMN YIVPSGGNTPYADSVKG DGAATVDLDY H a06 (SEQ ID NO: 32) PYHMG GIYPSGGWTNYADSVKG DGYSSGWFRY H a07 (SEQ ID NO: 36) EYNMM VISPSGGGTLYADSVKG DLNNSSPPDSNDAFDI H a08 (SEQ ID NO: 40) VYDMP VIYPSGGFTRYADSVKG DPTIQLWAYYYGMDV H a09 (SEQ ID NO: 44) WYDMY SIYSSGGYTAYADSVKG DRDPCSRTTCYNWFDP H a10 (SEQ ID NO: 48) AYRMF SIWPSGGTTSYADSVKG DRGYDSSGYFDY H a12 (SEQ ID NO: 56) AYWMD VIYPSGGSTNYADSVKG EGIAAAAPMDV H b01 (SEQ ID NO: 60) TYDML SISPSGGSTSYADSVKG EKASDLSGTYSEALDH H b02 (SEQ ID NO: 64) DYFMK SIYPSGGPTKYADSVKG ERSSGWYGYYYYGMDV H b03 (SEQ ID NO: 68) PYEMQ GIGSSGGDSVKG ERVDCSGGGCGSYFDY H b05 (SEQ ID NO: 76) EYWMP RIYPSGGVTTYADSVKG GGDYDFWSVQYYYYYMDV H b07 (SEQ ID NO: 84) QYQMI VIVPSGGITNYADSVKG GGVEAVDSSSPDY H b08 (SEQ ID NO: 88) RYMMN VIWSSGGKTLYADSVKG GGYNNYYYSMDV H b09 (SEQ ID NO: 298, PYFMF SIGSSGGDTSYADSVKG GLYR H 297) b11 (SEQ ID NO: 96) PYFMF SIGSSGGDTSYADSVKG GLYR H b12 (SEQ ID NO: 100) LYVMY YISSSGGITHYADSVKG GSIVVVPAAIRSNNWFDP H c02 (SEQ ID NO: 108) HYSMR YIVPSGGFTQYADSVKG GTHLPGVDY H c03 (SEQ ID NO: 112) LYMMK VISSSGGYTQYADSVKG GWDV H c06 (SEQ ID NO: 123) FYFMF YIGPSGGPTNYADSVKG HYPREYQLPGSFDP H c07 (SEQ ID NO: 128) YYVMM VIRPSGGITTYADSVKG IDYGGNSFYFDY H c09 (SEQ ID NO: 136) QYFMN YISGGRTPYADSVKG LLGPSSSNHPFLGP H c10 (SEQ ID NO: 140) VYVMM GIVPSGGKTHYADSVKG PDYGGNSRPLEY H d01 (SEQ ID NO: 152) MYLMF VISSSGGETSYADSVKG QVSDWTRLYSFDY H d03 (SEQ ID NO: 160) WYRMD VIGSSGGMTYYADSVKG RVVGGAGMDV H d05 (SEQ ID NO: 168) HYNMA RIRSSGGLTVYADSVKG VAGPGY H d09 (SEQ ID NO: 184) HYNMH GIVSSGGNTGYADSVKG VVRYSSGWYYWFDP H
(CDRs sequences are from corresponding sequences for the variable domain provided with sequence identifiers in Table 6 above)
[0375]At least some members of Group II HC sequences can include one of the following sequences in the region of CDR1:
TABLE-US-00035 P-Y-F-M-F, (SEQ ID NO: 202) or (ALVEP)-Y-(SMWFD)-M-(YFKDNP). (SEQ ID NO: 243)
[0376]At least some members of Group II HC sequences can include one of the following sequences in the region of CDR2:
TABLE-US-00036 (SEQ ID NO: 203) (SV)-I-Y-(SP)-S-G-G-X-T-X-Y-A-D-S-V-K-G, or (SEQ ID NO: 204) (GSVY)-I-(GSVYW)-(SP)-S-G-G-(SIYFD)-T-(SLRNQ)-Y-A- D-S-V-K-G.
TABLE-US-00037 TABLE 10 Group II (LC) Kappas 1 2 3 4 Name CDR1 CDR2 CDR3 type a05 (SEQ ID NO: 26) RASQSVSSYLA DASNRAT QQRSNWPRT K a06 (SEQ ID NO: 30) RASQSISSYLN AASSLQS QQSYSTPPENT K a12 (SEQ ID NO: 54) RASQSVSRSYLG GASNRAT QQYGISPLT K b01 (SEQ ID NO: 58) RASQSVSSSYFA DASSRAT QQYGSSPPMYT K b02 (SEQ ID NO: 62) RASQSIDTYLN AASKLED QQSYSSPGIT K b03 (SEQ ID NO: 66) RASQGIRDFLG AASILQS QQLNGYRA K b05 (SEQ ID NO: 74) RASQSVSSSYLA GASSRAT QQYGSSPEIT K b07 (SEQ ID NO: 82) RASQSVSSYLA DASNRAT QQRSNWPRT K b12 (SEQ ID NO: 98) GGDNIDTKNVQ DNSDRPS QVFDGRSDHPV K c02 (SEQ ID NO: 106) RASQTISTYLV GASTLQS QQSYTSPRT K c06 (SEQ ID NO: 122) RASQGISNYLA PASTLQS QQADSFPPT K c07 (SEQ ID NO: 126) RASQSISNYLN AASSLER QQSYSPPPLT K c09 (SEQ ID NO: 134) RASQSVSSTYLA GASSRAT QQYGSSPYT K d03 (SEQ ID NO: 158) RASQVMINYIA AASTLQN QHRITWPPALT K a02 (SEQ ID NO: 295) GGNNIGTKSVH DDSDRPS QVWDSGSDHQV L a03 (SEQ ID NO: 18) SGGSSNIGSNFVY RNYQRPS AAWDDNVGGV L a07 (SEQ ID NO: 34) TGTSSDVGGYNYVS EVTQRPS SSYAGRNNLYV L a08 (SEQ ID NO: 38) SGGYPNMGSNYAH NDNQRPS AAWDDSLSGPV L a09 (SEQ ID NO: 42) SGGSSNIGSNFVY RNYQRPS AAWDDNVGGV L a10 (SEQ ID NO: 46) SGGSSNIGSNYVS NNNQRPS AAWDDSLSSAV L b08 (SEQ ID NO: 86) TRSSGSIAGNYVQ EDNKRPS HSYDTSNQV L b09 (SEQ ID NO: 299) GGNNIGTKSVH DDSDRPS QVWDSGSDHQV L b11 (SEQ ID NO: 94) GGNNIGTKSVH DDSDRPS QVWDSGSDHQV L c03 (SEQ ID NO: 110) SGSNSNIGGNIVI DVSDRPS AAWDDSLNGWV L c10 (SEQ ID NO: 138) TGTSSDVGSYNLVS EGSKRPS CSYAGSSTYV L d01 (SEQ ID NO: 150) SGSSSNIGSNYVY RNDQRPS ASWDDSLSGVV L d05 (SEQ ID NO: 166) TGSSHDIGSYDYVS YDVYNRPS MSYTITTLL L d09 (SEQ ID NO: 182) SGDKLGDKYAC QDSKRPS QAWDSSAVV L
(CDRs sequences are from corresponding sequences for the variable domain provided with sequence identifiers in Table 6 above)
[0377]At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR1:
TABLE-US-00038 (SEQ ID NO: 205) R-A-S-Q-S-(IV)-S-(SN)-(SY)-(LY)-(ALN)-A,; (SEQ ID NO: 206) R-A-S-Q-(GS)-(IV)-S-(STN)-(SY)-(LY)-(ALN)-A,; (SEQ ID NO: 207) R-A-S-Q-S-(IV)-S-S-Y-L,; or (SEQ ID NO: 208) R-A-S-Q,.
[0378]At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR2:
TABLE-US-00039 (SEQ ID NO: 256) (AGDP)-(AN)-S-(STIKDN)-(LR)-(AEPQ)-(STRDN), (SEQ ID NO: 257) (AGD)-A-S-(STN)-(LR)-(AEQ)-(ST), (SEQ ID NO: 258) (AGD)-A-S-(STN)-(LR)-(AQ)-(ST), or (SEQ ID NO: 259) (AGD)-A-S-S-(LR)-(AQ)-(ST).
[0379]At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR3:
TABLE-US-00040 (SEQ ID NO: 266) Q-Q-X-X-X-X-P, (SEQ ID NO: 267) Q-Q-(SYR)-(GSYD)-(GSTN)-(SW)-P-(RP)-(TI)-T-T, (SEQ ID NO: 268) Q-Q-(SYR)-(GSY)-(SN)-(SW)-P-(RP)-(TI)-T-T, or (SEQ ID NO: 269) Q-Q-(ASLYR)-(GSYDN)-(GSTIN)-(STYWFP)-(RP)- (AGLYREP)-(TILME)-(TYN)-T.
[0380]At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR1:
TABLE-US-00041 (SEQ ID NO: 209) S-G-X-S-S-N-I-G-S-N-X-V-X-X,; (SEQ ID NO: 233) (GST)-G-(GSTN)-(SN)-(SI)-(GDN)-(TIV)-(GK)-(GS)- (VYN)-(YFNH)-(VY)-(VY)-S; (SEQ ID NO: 210) (ST)-G-(GST)-S-S-(DN)-(IV)-G-(GS)-(YN)-(YFN)-(VY)- (VY)-S,; (SEQ ID NO: 234) G-G-(SN)-(SN)-(ID)-(GI)-(GT)-(SK)-(SYN)-V-H,; or (SEQ ID NO: 235) G-(GST)-(SN)-(SN)-(IDN)-(GIV)-(GST)-(SKN)-(SYN)- (VFN)-(VYH).
[0381]At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR2:
TABLE-US-00042 D-(DN)-S-(DQ)-R-P-S-X,; (SEQ ID NO: 233) or (RDNE)-(VDN)-(SYN)-(KDQ)-R-P-S-X. (SEQ ID NO: 247)
[0382]Group II LC (e.g., κ or λ) can include one or more of the following sequences in the region of CDR2:
TABLE-US-00043 (SEQ ID NO: 230) D-(AD)-S-X-(LR)-(AP)-(ST), (SEQ ID NO: 231) (AD)-(ADN)-S-(SKDNQ)-(LR)-(APQ)-(ST), or (SEQ ID NO: 232) (AGRD)-(ADN)-(SYN)-(SKDNQ)-(LR)-(APQ)-(ST)
[0383]At least some members of Group II LC λ sequences can include one of the following sequences in the region of CDR3:
TABLE-US-00044 (SEQ ID NO: 234) A-A-W-D-D-(SN)-(LV),; (SEQ ID NO: 248) (QA)-X-W-D-(SDT)-(GSN); or (SEQ ID NO: 249) (AQ)-(ASV)-(YW)-(AD)-(GSID)-(GSN)-(STLVN)-(GSDN)- (GSLVH)-(VQ)-V.
[0384]Fab's from Group I can be classified as described below:
[0385]Set 1: a04(lambda) and b04(kappa).
[0386]Set 2 includes a11, c05, A1-orig, d02, and d04.
[0387]Set 3 includes b10, c01, c08, d04, and d06.
[0388]Set 4 includes c11 and d07
[0389]Some features that may be present in inhibitors include:
TABLE-US-00045 (SEQ ID NO: 294) Cons: I-G-P-S-G-G-I-T-X-Y-A-D-S-V-K-G
[0390]Where two or more antibodies share the property of inhibiting MMP-26 and have recognizable similarity in amino-acid sequence, particularly where the similarity occurs in more than one CDR, it is likely that the two antibodies bind related epitopes, e.g., they bind at essentially the same site. In this case it can be useful to recombine the sequences within a set.
[0391]Since a04 and b04 appear to have similar HCs, antibodies that include features of HC-a04/LC-b04 and HC-b04/LC-a04 are likely to also be inhibitors of MMP-26 and may have useful functional properties (e.g., match or surpass either or both parents in binding ability). Since a11, c05, A1-orig, d02, and d04 have similarities, combinations involving one of these HCs with of these LCs, or combinations of features thereof, are likely to be inhibitors of MMP-26. Since a11, c05, and A1-orig all have lambda LCs, new light chains having the common lambda framework and all recombinations of the lambda CDRs coupled to each of the HCs are likely to give additional MMP-26 inhibitory antibodies, some of which may have useful functional properties. For example, a light chain comprising FR1(a11)-CDR1(a11)-FR2(a11)-CDR2(c05)-FR3(a11)-CDR3(A1-orig)-FR4(a11) could be combined with all of the HC of Set 2. Similarly, d02 and d04 show similarity in HC CDR2, HC CDR3, and kappa CDR1, new LCs having the kappa CDRs mixed between d02 and d04 when combined with any of the HC of Set 2 are likely to give antibodies that inhibit MMP-26. One can also exchange the HC CDRs to make additional antibodies that are likely to inhibit MMP-26.
[0392]Set 3 comprises b10, c01, c08, d04, and d06, all having kappa LCs. Combining each of these kappa chains with each of the HCs in set 3 will give new antibodies that are likely to inhibit MMP-26, some of which may have useful functional properties. One can make additional antibodies by exchanging the CDRs of the LC and HC within set 3.
[0393]Set 4 comprises c11 and d07 which have highly similar lambda chains. Exchanging LC and HCs gives two new antibodies that are likely to inhibit MMP-26. Exchanging CDRs gives rise to a further set of 120 antibodies which are likely to inhibit MMP-26.
Example
[0394]With respect to each example below, there are related antibody variable domains whose amino acid sequence is encoded by a germline gene (e.g., the germline-encoded amino acid sequence aligned in the example or a related germline-encoded amino acid sequence) that can include one or more of the above mutations (e.g., at least 30, 50, 70, 75, 80, 85, 90, or 95% of the above mutations), e.g., one or more of the above mutations (e.g., at least 30, 50, 70, 75, 80, 85, 90, or 95%) of the mutations that are located in the CDRs.
[0395]The germline-encoded amino acid sequences referred to below are provided as:
TABLE-US-00046 Name SEQ ID NO: A27 270 V2-A14 271 V2-A17 272 L6 273 O2 274 V1-2 275 L5 276 V1-22 277 L12 278 V1-12 279 V1-16 280 A20 281 V1-17 282 L8 283 L14 284 V1-4 285 V2-1 286 V3-23 287
Name: A27 is related to VKIII
TABLE-US-00047 Mutation Location E1D, V3Q, L4M FR1 S28I, S30R, S31aT CDR1 A51T CDR2 I58V, R77G, V85L FR3 Q90R, S93D CDR3 K107T FR4
This is relative to the A27 VKIII germline sequence (SEQ ID NO:270).Name: A02 (Same as All) is related to V2-14 (VL2).The sequence of the A02 light chain variable domain includes:
TABLE-US-00048 (SEQ ID NO: 295) GVHSQSELTQ PPSVSLAPGQ TARITCGGNN IGTKSVHWYQ QKPGQAPVLV VYDDSDRPSG IPERFSGSNS GNTATLTISR VEAGDEADYY CQVWDSGSDH QVFGGGTKLT VLGQPKAAPS
TABLE-US-00049 Mutation Location S1Q, Y2S, V3A, V13L FR1 S31aT CDR1 FR2 CDR2 FR3 S94G CDR3 FR4 GLG for Q96 could be P or V
Name: A03 is related to V1-17 (VL1).
TABLE-US-00050 Mutation Location V3A FR1 S26G, Y32F CDR1 Q37R FR2 S50N, N52Y CDR2 R79L FR3 S95aG CDR3 T100S FR4 G96 is GLG if from VL, but Y96G if from JL1 V97 is GLG if from JL1, but P97V if from VL
Name: A04 is related to V1-17.
TABLE-US-00051 Mutation Location V3A FR1 S26G, S27Y, I30M, V33A, Y34H CDR1 L39V FR2 S50N CDR2 FR3 D93E, S94N CDR3 FR4 P96 is GLG if from V1-17, but Y96P if from JL1.
Name: A05 is related to L6 (VKIII)
TABLE-US-00052 Mutation Location E1D, V3Q, L4M, A9S, T10S FR1 CDR1 FR2 CDR2 FR3 CDR3 L96R FR4
Name: A06 is related to O2 (VKI)
TABLE-US-00053 Mutation Location FR1 CDR1 FR2 CDR2 FR3 CDR3 Y96N FR4
Name: A07 is related to V1-2 (VL1).
TABLE-US-00054 Mutation Location A3E FR1 CDR1 G41D, M47L FR2 S52T, K53Q CDR2 K66R FR3 S94R CDR3 T100P FR4
Name: A08 is related to V1-17 (VL1).
TABLE-US-00055 Mutation Location A11V FR1 S26G, S27Y, S28P, I30M, V33A, Y34H CDR1 FR2 S50N, N51D CDR2 FR3 CDR3 FR4 P96 is GLG if from V1-17, P96 is V96P if from JL2
Name: A09 is related to V1-17 (VL1).
TABLE-US-00056 Mutation Location V3E FR1 S26G, Y32F CDR1 Q37R FR2 S50R, N51aY CDR2 R79L FR3 S95aG CDR3 T100S FR4 G96 is GLG if from V1-17, but G96 is Y96G if from JL1.
Name: A10 is related to V1-17 (VL1).
TABLE-US-00057 Mutation Location V3E FR1 S26G, Y34S CDR1 S50N CDR2 G95bS CDR3 P95cA is a mutation only if this came from VL, but A is GLG if this came from JL7.
Name: A11 is related to V2-14 (VL2).
TABLE-US-00058 Mutation Location S1Q, Y2S, V3A, V13L FR1 S31aT CDR1 FR2 CDR2 FR3 S94G CDR3 FR4 GLG for Q96 could be P or V.
Name: A12 is related to A27 (VKIII)
TABLE-US-00059 Mutation Location E1D, V3Q, L4M FR1 S31R, A34G CDR1 FR2 S53N CDR2 FR3 S93I CDR3 F96L FR4
Name: A1-O (same as a1-orig) is related to V1-17.
TABLE-US-00060 Mutation Location T20I FR1 N31bH CDR1 Y34H L39V FR2 S50R CDR2 N52G S65F FR3 D85N A90T CDR3 L95aV S95bL S95c S95d V96 FR4 V97
Name: B01 is related to A27.
TABLE-US-00061 Mutation Location E1D FR1 V3Q L4M L33F CDR1 FR2 G50D CDR2 FR3 CDR3 FR4
Name: B10 is related to O2.
TABLE-US-00062 Mutation Location T22A FR1 A25T CDR1 S28N I29V S30N S31R Q38H FR2 P40L K42Q A50G CDR2 S52T S53I S63R FR3 T72I S76T S77N T85V S91T CDR3 T94P -95aP -95bL W96- FR4 V104A I106F
Name: B11 is related to V2-14.
TABLE-US-00063 Mutation Location 1SQ FR1 Y2S V3E V13L S31aT CDR1 FR2 CDR2 FR3 CDR3 S94G P95cQ V96- FR4
Name: B12 is related to V2-14.
TABLE-US-00064 Mutation Location S1Q FR1 Y2S V3A T22S N28D CDR1 G31D S31aT S32N H33Q K39R FR2 I48V D51aN CDR2 G57A FR3 W91F CDR3 S93G S94R V96P FR4
Name: B02 is related to O2.
TABLE-US-00065 Mutation Location T20A FR1 S30D S31T CDR1 FR2 S53K CDR2 Q55E S56D S67T FR3 S76R T85S Y87F T94S CDR3 .95aG F96I FR4 D105E
Name: B03 is related to L8.
TABLE-US-00066 Mutation Location L4M FR1 S30R CDR1 S31D Y32F A34G K45N FR2 L46Q T53I CDR2 E70D FR3 S76T Y87F S93G CDR3 P95R .95aA I96. FR4 T97.
Name: B04 is related to L5.
TABLE-US-00067 Mutation Location FR1 S31K CDR1 W32S I48V FR2 A50G CDR2 S52F Q55E S65T FR3 S67A T72I S77R CDR3 FR4
Name: B05 is related to A27.
TABLE-US-00068 Mutation Location E1D FR1 V3Q L4M CDR1 FR2 CDR2 FR3 .95aE CDR3 FR4
Name: B06 is related to L8.
TABLE-US-00069 Mutation Location L4M FR1 CDR1 FR2 CDR2 E70D FR3 Q79E Q90E CDR3 L91S N92Y Y94T .95aF K107R FR4
Name: B07 is related to L6.
TABLE-US-00070 Mutation Location E1D FR1 V3Q L4M CDR1 FR2 CDR2 FR3 CDR3 L96. FR4
Name: B08 is related to V1-22.
TABLE-US-00071 Mutation Location N1Q FR1 F2S M3E E13G S31aG CDR1 FR2 Q53KT CDR2 T74I FR3 Q89H CDR3 V96Q FR4
Name: B09 is related to V2-14.The B09 light chain variable domain can include:
TABLE-US-00072 (SEQ ID NO: 299) GVHSQSELTQ PPSVSLAPGQ TARITCGGNN IGTKSVHWYQ QKPGQAPVLV VYDDSDRPSG IPERFSGSNS GNTATLTISR VEAGDEADYY CQVWDSGSDH QVFGGGTKLT VLGQPKAAPS
TABLE-US-00073 Mutation Location S1Q FR1 Y2S V3E V13L S31aT CDR1 FR2 CDR2 FR3 S94G CDR3 P95cQ V96. FR4
Name: C01 is related to L12.
TABLE-US-00074 Mutation Location FR1 S30G CDR1 S31N K42E FR2 K45H D50Q CDR2 S56G E70K FR3 T74N CDR3 E105D FR4
Name: C10 is related to V1-7.
TABLE-US-00075 Mutation Location A3E FR1 CDR1 FR2 CDR2 FR3 F95b. CDR3 FR4
Name: C11 is related to V1-2.
TABLE-US-00076 Mutation Location A3E FR1 CDR1 G41D FR2 M47L S52T CDR2 K53Q K66R FR3 S94R CDR3 F95bL T100P FR4
Name: C12 is related to V2-1.
TABLE-US-00077 Mutation Location S1Q FR1 Y2S G16A CDR1 FR2 Q50E CDR2 S52T A80V FR3 A90V CDR3 V96. FR4
Name: C03 is related to V1-16.
TABLE-US-00078 Mutation Location V3A FR1 S27N CDR1 S31aG T32I N34I Y36L FR2 L47M S50D CDR2 N51V N52S Q53D FR3 P96cW CDR3 V96. FR4
Name: C04 is related to V1-17.
TABLE-US-00079 Mutation Location V3A FR1 S26T CDR1 N31bH Y34F Y49H FR2 S50R CDR2 N52D Q53E R79Q FR3 A90T CDR3 S94N S95aN V96. FR4 V106G L107P
Name: C05 is related to V1-17.
TABLE-US-00080 Mutation Location V3E FR1 A11V N31bE CDR1 Y36F FR2 K45R S50R CDR2 N52D A84T FR3 A89T CDR3 A90T V96. FR4
Name: C06 is related to A20.
TABLE-US-00081 Mutation Location FR1 CDR1 FR2 A50P CDR2 V83F FR3 K90Q CDR3 Y91A N92D A94F .95aP L96. FR4 K107R
Name: C07 is related to O2.
TABLE-US-00082 Mutation Location FR1 S31N CDR1 K39R FR2 Q55E CDR2 S56R P80S FR3 T94P CDR3 .95aP FR4
Name: C08 is related to O2.
TABLE-US-00083 Mutation Location S9P FR1 S14L S31R CDR1 L33N L46V FR2 S53I CDR2 L54V Q55E S56N T72S FR3 S91T CDR3 E105A FR4
Name: C09 is related to A27.
TABLE-US-00084 Mutation Location E1D FR1 V3Q L4M S31aT CDR1 Y49S FR2 CDR2 FR3 CDR3 FR4
Name: D01 is related to V1-17.
TABLE-US-00085 Mutation Location V3E FR1 V19I CDR1 L39F FR2 G41E Y49S S50R CDR2 N52D FR3 A90S CDR3 P95a. FR4
Name: D02 is related to L8.
TABLE-US-00086 Mutation Location L4M FR1 S31T CDR1 L46V FR2 A50T CDR2 FR3 L91S CDR3 N92Y S93I Y94T .95aP .95bE F96V FR4
Name: D03 is related to L14.
TABLE-US-00087 Mutation Location N1D FR1 R26S CDR1 G28V I29M S30I L33I Q37R FR2 K45E H46R S53T CDR2 S56N E70D FR3 S77R Q79E T85V L89Q CDR3 Q90H H91R N92I S93T Y94W .95aP .95bA K103T FR4
Name: D04 is related to O2.
TABLE-US-00088 Mutation Location T20A FR1 S30D CDR1 S31T FR2 S53K CDR2 Q55E S56D S67T FR3 S76R T85S Y87F T94S .95aG CDR3 F96I D105E FR4
Name: D05 is related to V1-4.
TABLE-US-00089 Mutation Location A3V FR1 A9D T26S CDR1 S28H V30I G31aS N31cD Q38H FR2 L46F M47I I48L E50D CDR2 S52Y N60D FR3 A81P E82D Y87F S89M CDR3 S93I S94T S95T T95aL Y96. FR4 V97L K103R
Name: D06 is related to O2.
TABLE-US-00090 Mutation Location FR1 A25T CDR1 S28I S30N S31T FR2 S53T CDR2 S56G S76K FR3 E81D S93T CDR3 T94S W96R FR4
Name: D6-O is related to O2.
TABLE-US-00091 Mutation Location S9L FR1 T20A S30D CDR1 S31T FR2 S53K CDR2 Q55E S56D S67T FR3 S76R T85S Y87F T94S CDR3 .95aG F96I FR4 D105E
Name: D07 is related to V1-4.
TABLE-US-00092 Mutation Location A3E FR1 S27N CDR1 V30I A43V FR2 M47L Y49F S52N CDR2 N60D FR3 A80P D85V S89G CDR3 Y91F S93V S95V L95b. FR4
Name: D07 is related to V1-4.
TABLE-US-00093 Mutation Location FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
Name: D08 is related to O2.
TABLE-US-00094 Mutation Location FR1 S31D CDR1 K45D FR2 CDR2 Y87F FR3 T94I CDR3 K107T FR4
Name: D09 is related to V2-1.
TABLE-US-00095 Mutation Location S1Q FR1 Y2S CDR1 FR2 CDR2 FR3 T95A CDR3 A95a. FR4
Name: H6-O is related to V1-2.
TABLE-US-00096 Mutation Location A3E FR1 G29A CDR1 M47I FR2 S51aN CDR2 G64A FR3 CDR3 N97S, F98L FR4
Example
[0396]This example includes an analysis of HC germline differences In each example the isolate name is written under the GLG.
[0397]The AA sequence encoded by the synthetic DNA up to Cys-92. The germ-line gene (GLG) for 3-23 is shown in SEQ ID NO:287. The AA sequences of the six human JH segments are shown below:
TABLE-US-00097 AA sequences of human JHs JH1 ---AEYFQHWGQGTLVTVSS (SEQ ID NO: 288) JH2 ---YWYFDLWGRGTLVTVSS (SEQ ID NO: 289) JH3 -----AFDIWGQGTMVTVSS (SEQ ID NO: 290) JH4 -----YFDYWGQGTLVTVSS (SEQ ID NO: 291) JH5 ----NWFDPWGQGTLVTVSS (SEQ ID NO: 292) JH6 YYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 293)
[0398]The following analysis of the 48 isolates. For each isolate the entry shows: the name, 2) and the mutations written as if from GLG (SEQ ID NO:287) to the isolate. With respect to each example, there are related antibody variable domains whose amino acid sequence is encoded by a germline gene (e.g., the germline-encoded amino acid sequence aligned in the example or a related germline-encoded amino acid sequence) that can include one or more of the above mutations (e.g., at least 30, 50, 70, 75, 80, 85, 90, or 95% of the above mutations), e.g., one or more of the above mutations (e.g., at least 30, 50, 70, 75, 80, 85, 90, or 95%) of the mutations that are located in the CDRs.
[0399]Below the listing of muations is a description of which JH matched best and an alignment of the best JH with all the isolate AA sequence after C92.
Isolate: A01
S31A, A33N, S35F, A500, S51aG, G52S, S56I, T57A, Y58P
JH is 5
Isolate: A02
S31P, A33F, S35F, A50S, S51aG, G52S, S56D, Y58S
JH is 4
[0400]The A02 heavy chain variable domain can include:
TABLE-US-00098 (SEQ ID NO: 296) EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYFMFWVRQA PGKGLEWVSS IGSSGGDTSY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYC
and a CDR3 that includes GLYR (SEQ ID NO:297).
Isolate: A03
S31I, A33S, S35D, A50S, S51aY, G52S, S56A, Y58R
[0401]JH is 4
Isolate: a04
S31E, A33N, S35A, A50R, S51aG, G52S, S56K, Y58K
[0402]JH is 3
Isolate: a05
S31V, A33S, S35N, A50Y, S51aV, G52P, S56N, Y58P
[0403]JH is 4
Isolate: a06
S31P, A33H, S35G, A50G, S51aY, G52P, S56W, Y58N
JH is 4
Isolate: a07
S31E, A33N, S35M, A50V, G52P, S56G, Y58L
[0404]JH is 3
Isolate: a08
S31V, A33D, S35P, A50V, S51aY, G52P, S56F, Y58R
[0405]JH is 6
Isolate: a09
S31W, A33D, S35Y, A50S, S51aY, G52S, S56Y, Y58A
[0406]JH is 5
Isolate: a10
S31A, A33R, S35F, A50S, S51aW, G52P, S56T, Y58S
[0407]JH is 4
Isolate: a11
S31W, A33T, S35M, A50R, G52P, S56H, Y58L, K75z
[0408]JH is 4
Isolate: a12
S31A, A33W, S35D, A50V, S51aY, G52P, Y58N
[0409]JH is 3
Isolate: b01
S31T, A33D, S35L, A50S, G52P, Y58S, M82L
[0410]JH is 3
Isolate: b02
S31D, A33F, S35K, A50S, S51aY, G52P, S56P, Y58K
[0411]JH is 6
Isolate: b03
S31P, A33E, S35Q, A500, S51aG, G52S, S56-, T57-, Y58-,
[0412]Y59-, A60-N.B. 5 AA deletion in CDR2.
[0413]JH is 4
Isolate: b04
S31F, A33W, S35M, A50G, G52S, S56F, Y58K
[0414]JH is 3
Isolate: b05
S31E, A33W, S35P, A50R, S51aY, G52P, S56V, Y58T
[0415]JH is 6
Isolate: b06
S31Q, A33F, S35K, A50S, G52P, S56L, Y58Q
[0416]JH is 4
Isolate: b07
S31Q, A33Q, S35I, A50V, S51aV, G52P, S56I, Y58N
[0417]JH is 4
Isolate: b08
S31R, A33M, S35N, A50V, S51aW, G52S, S56K, Y58L, N82aK
[0418]JH is 6
Isolate: b09
S31P, A33F, S35F, A50S, S51aG, G52S, S56D, Y58S
[0419]JH is 4
The b09 heavy chain variable domain can include:
TABLE-US-00099 (SEQ ID NO: 298) EVQLLESGGG LVQPGGSLRL SCAASGFTFS PYFMFWVRQA PGKGLEWVSS IGSSGGDTSY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYC
and a CDR3 that includes GLYR (SEQ ID NO:297).
Isolate: b10
S35M, A50W, S51aV, G52P, S56T, Y58F
[0420]JH is 4
Isolate: b11
S31P, A33F, S35F, A50S, S51aG, G52S, S56D, Y58S, K75z
[0421]JH is 4
Isolate: b12
S31L, A33V, S35Y, A50Y, G52S, S56I, Y58H
[0422]JH is 5
Isolate: c01
S31N, A33G, A50V, S51aG, G52P, S56I, Y58M
[0423]JH is 4
Isolate: c02
S31H, A33S, S35R, A50Y, S51aV, G52P, S56F, Y58Q
[0424]JH is 4
Isolate: c03
S31L, A33M, S35K, A50V, G52S, S56Y, Y58Q, S82bN
[0425]JH is 3
Isolate: c04
S31M, A33F, S35V, A50W, S51aG, G52S, S56E, Y58P
[0426]JH is 4
Isolate: c05
A33Q, S35D, A50R, S51aV, G52P, S56D, Y58T
[0427]JH is 4
Isolate: c06
S31F, A33F, S35F, A50Y, S51aG, G52P, S56P, Y58N
[0428]JH is 4
Isolate: c07
S31Y, A33V, S35M, A50V, S51aR, G52P, S56I, Y58T, M82T
[0429]JH is 4
Isolate: c08
S31Y, A33E, S35M, A50S, G52P, S56P, Y58M, A88G
[0430]JH is 6
Isolate: c09
[0431]S31Q, A33F, S35N, A50Y, G52-, S53-, S56R, Y58P N.B. 2 AA deletion in CDR2.
[0432]JH is 4
Isolate: c10
S31V, A33V, S35M, A500, S51aV, G52P, S56K, Y58H
[0433]JH is 4
Isolate: c11
S31E, A33P, S35W, A50W, S51aY, G52P, S56N, Y58D
[0434]JH is 4
Isolate: c12
S31A, A33N, S35M, A50R, S51aY, G52P, S56Y, Y58L
[0435]JH is 4
Isolate: d01
S31M, A33L, S35F, A50V, G52S, S56E, Y58S
[0436]JH is 4
Isolate: d02
S31W, A33D, S35A, A50R, S51aV, G52P, S56H, Y58S, S74F
[0437]JH is 4
Isolate: d03
S31W, A33R, S35D, A50V, S51aG, G52S, S56M
[0438]JH is 3
Isolate: d04
S31D, A33Q, S35M, A50R, G52P, S56M, Y58R, Q81P
[0439]JH is 4
Isolate: d05
S31H, A33N, S35A, A50R, S51aR, G52S, S56L, Y58V
[0440]JH is 4
Isolate: d06
S31G, A33I, S35E, A50V, S51aV, G52S, S56F, Y58M
[0441]JH is 4
Isolate: d07
S31E, A33N, S35F, A50Y, S51aY, G52S, Y58D
[0442]JH is 4
Isolate: d08
S31F, S35W, A50R, S51aY, G52S, S56K, Y58W
[0443]JH is 3
Isolate: d09
S31H, A33N, S35H, A50G, S51aV, G52S, S56N, Y58G, Y90D
[0444]JH is 5
A1-orig
S31Y, A33R, A50S, S51aG, G52P, S56D, Y58L
[0445]JH is 4
D6-orig
S31M, A33S, S35R, A50S, S51aY, G52P, Y58E
[0446]JH is 4
H6-orig
S31Q, A33W, S35N, A500, S51aG, G52P, S56I
[0447]JH is 4
[0448]Other embodiments of the invention are within the following claims.
Sequence CWU
1
2991354DNAHomo sapiens 1ggcgtgcact cacagagcgt cttgactcag ccaccctcag
cgtctgggac ccccgggcag 60agggtcatca tctcttgttc tggaagcagc tccaacatcg
gaagtcatta tgtacactgg 120taccaacagg tcccaggaac ggcccccaaa ctcctcattt
ataggaatgg tcagcggccc 180tcaggggtcc ctgaccgatt ctctggcttc aagtctggca
cctcagcctc cctggccatc 240agtgggctcc ggtccgagga tgaggctaat tattactgtg
caacatggga tgacagtgtc 300ctattcggcg gagggaccac gctgaccgtc ctaggtcagc
ccaaggctgc cccc 3542118PRTHomo sapiens 2Gly Val His Ser Gln Ser
Val Leu Thr Gln Pro Pro Ser Ala Ser Gly1 5
10 15Thr Pro Gly Gln Arg Val Ile Ile Ser Cys Ser Gly
Ser Ser Ser Asn 20 25 30Ile
Gly Ser His Tyr Val His Trp Tyr Gln Gln Val Pro Gly Thr Ala 35
40 45Pro Lys Leu Leu Ile Tyr Arg Asn Gly
Gln Arg Pro Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Phe Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65
70 75 80Ser Gly Leu Arg Ser
Glu Asp Glu Ala Asn Tyr Tyr Cys Ala Thr Trp 85
90 95Asp Asp Ser Val Leu Phe Gly Gly Gly Thr Thr
Leu Thr Val Leu Gly 100 105
110Gln Pro Lys Ala Ala Pro 1153399DNAHomo sapiens 3gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg
cttccggatt cactttctct tattaccgta tgtcttgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttcttct atcggtcctt ctggtggcga tactctttat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga
acagcttaag ggctgaggac actgcagtct actattgtgc gagatctttc 300agcagtggcc
cgtactactt tgactactgg ggccagggaa ccctggtcac cgtctcaagc 360gcctccacca
agggcccatc ggtcttcccg ctagcgccc 3994133PRTHomo
sapiens 4Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20
25 30Arg Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser
Ser Ile Gly Pro Ser Gly Gly Asp Thr Leu 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 Ser
Phe Ser Ser Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln 100
105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro 1305354DNAHomo sapiens 5ggcgtgcact ctgacatcca
gatgacccag tctccactct ccctgtctgc atctgtggga 60gacagagtcg ccatcacttg
ccgcgcaagt cagagcatcg acacctattt aaattggtat 120cagcagaaac cagggaaagc
ccctaaactc ctgatctatg ctgcatccaa gttggaagac 180ggggtcccat caagattcag
tggcagtgga actgggacag atttcactct caccatcaga 240agtctgcaac ctgaagattt
tgcaagttat ttctgtcaac agagctactc tagtccaggg 300atcactttcg gccctgggac
caaggtggag atcaaacgaa ctgtggctgc acca 3546118PRTHomo sapiens
6Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Ser1
5 10 15Ala Ser Val Gly Asp Arg
Val Ala Ile Thr Cys Arg Ala Ser Gln Ser 20 25
30Ile Asp Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45Lys Leu Leu
Ile Tyr Ala Ala Ser Lys Leu Glu Asp Gly Val Pro Ser 50
55 60Arg Phe Ser Gly Ser Gly Thr Gly Thr Asp Phe Thr
Leu Thr Ile Arg65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Ser Tyr
85 90 95Ser Ser Pro Gly Ile Thr
Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100
105 110Arg Thr Val Ala Ala Pro 1157387DNAHomo
sapiens 7gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct atgtactcta tgcgttgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttcttct atctatcctt ctggtggctc
tactgagtat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc
gagagagggc 300ggggagaacg actactgggg ccagggaacc ctggtcaccg tctcaagcgc
ctccaccaag 360ggcccatcgg tcttcccgct agcgccc
3878129PRTHomo sapiens 8Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met
Tyr 20 25 30Ser Met Arg Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ser Ile Tyr Pro Ser Gly Gly Ser Thr Glu 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 Glu Gly Gly Glu Asn Asp Tyr Trp Gly Gln Gly Thr
Leu Val 100 105 110Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115
120 125Pro9363DNAHomo sapiens 9ggcgtgcact cacagagcga
attgactcag cctccctccg cgtccgggtc tcctggacag 60tcagtcacca tctcctgcac
tggaaccagc agtgacgttg gtgcttataa ctatgtctcc 120tggtaccaac aacacccagg
caaagccccc aaactcataa tctatgaagt caataagcgg 180ccctcagggg tccctgatcg
cttctctgcc tccaagtctg gcaacacggc ctccctgacc 240gtctctgggc tccaggctga
agatgaggct gattattact gcaactcata tgcaggcagc 300aacagtttga tattcggcgg
agggaccaaa ctgaccgtct taggtcagcc caaggctgcc 360ccc
36310121PRTHomo sapiens
10Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly1
5 10 15Ser Pro Gly Gln Ser Val
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp 20 25
30Val Gly Ala Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His
Pro Gly Lys 35 40 45Ala Pro Lys
Leu Ile Ile Tyr Glu Val Asn Lys Arg Pro Ser Gly Val 50
55 60Pro Asp Arg Phe Ser Ala Ser Lys Ser Gly Asn Thr
Ala Ser Leu Thr65 70 75
80Val Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser
85 90 95Tyr Ala Gly Ser Asn Ser
Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr 100
105 110Val Leu Gly Gln Pro Lys Ala Ala Pro 115
12011396DNAHomo sapiens 11gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
cagtactgga tgaattgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt
atcggtcctt ctggtggcat tacttattat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac
actgcagtct actattgtgc gagaggtgag 300gaagatggct acaattctga ctactggggc
cagggaaccc tggtcaccgt ctcaagcgcc 360tccaccaagg gcccatcggt cttcccgcta
gcgccc 39612132PRTHomo sapiens 12Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Gln Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Gly Pro Ser Gly
Gly Ile Thr 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 Gly Glu Glu Asp Gly Tyr Asn Ser
Asp Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro
13013363DNAHomo sapiens 13ggcgtgcact ctgacatcca gatgacccag tctccaggca
ccctgtcttt gtctccaggg 60gaaagagcca ccctctcctg cagggccagt cagattgttc
gcagcaccta cttagcctgg 120tatcagcaga aacctggcca ggctcccagg ctcctcatct
atggtacatc cagcagggcc 180actggcgtcc cagacaggtt cagtggcagt gggtctggga
cagacttcac tctcaccatc 240agcggactgg agcctgaaga ttttgcacta tactactgtc
agcggtatgg tgactcacct 300ccgatcacct tcggccaagg gacacgactg gagattacac
gaactgtggc tgcaccatct 360gtc
36314121PRTHomo sapiens 14Gly Val His Ser Asp Ile
Gln Met Thr Gln Ser Pro Gly Thr Leu Ser1 5
10 15Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ile 20 25 30Val
Arg Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35
40 45Pro Arg Leu Leu Ile Tyr Gly Thr Ser
Ser Arg Ala Thr Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65
70 75 80Ser Gly Leu Glu Pro
Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Arg Tyr 85
90 95Gly Asp Ser Pro Pro Ile Thr Phe Gly Gln Gly
Thr Arg Leu Glu Ile 100 105
110Thr Arg Thr Val Ala Ala Pro Ser Val 115
12015390DNAHomo sapiens 15gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct gcttacaata
tgttttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt atcggttctt
ctggtggcat tgctccttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagagccgcg 300tacgaggtgg agaactggtt cgacccctgg ggccagggaa
ccctggtcac cgtctcaagc 360gcctccacca agggcccatc ggtcttcccg
39016130PRTHomo sapiens 16Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30Asn
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Gly Ser Ser Gly Gly Ile
Ala Pro 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 Ala Ala Tyr Glu Val Glu Asn Trp Phe
Asp Pro Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro 13017363DNAHomo
sapiens 17ggcgtgcact cacagagcgc tttgactcag ccaccctcag cgtctgggac
ccccgggcag 60agggtcacca tctcttgttc tggaggcagc tccaacatcg gaagtaattt
tgtttactgg 120taccggcagc tcccaggaac ggcccccaaa ctcctcatct ataggaatta
tcagcggccc 180tcaggggtcc ctgaccgatt ctcgggttcc aagtctggca cctcagcctc
cctggccatc 240agtgggctcc tgtccgaaga tgaggctgat tattactgcg cagcatggga
tgacaacgtg 300ggtggggtct tcggatctgg gaccaaggtc accgtcctgg gtcagcccaa
ggccaacccc 360act
36318121PRTHomo sapiens 18Gly Val His Ser Gln Ser Ala Leu Thr
Gln Pro Pro Ser Ala Ser Gly1 5 10
15Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser
Asn 20 25 30Ile Gly Ser Asn
Phe Val Tyr Trp Tyr Arg Gln Leu Pro Gly Thr Ala 35
40 45Pro Lys Leu Leu Ile Tyr Arg Asn Tyr Gln Arg Pro
Ser Gly Val Pro 50 55 60Asp Arg Phe
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65 70
75 80Ser Gly Leu Leu Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp 85 90
95Asp Asp Asn Val Gly Gly Val Phe Gly Ser Gly Thr Lys Val
Thr Val 100 105 110Leu Gly Gln
Pro Lys Ala Asn Pro Thr 115 12019390DNAHomo
sapiens 19gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct atttactcta tggattgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttcttct atctattctt ctggtggcgc
tactcgttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc
gaggtgtagc 300tggctacaat tagtaccgat gcacccttgg ggccagggaa ccctggtcac
cgtctcaagc 360gcctccacca agggcccatc ggtcttcccg
39020130PRTHomo sapiens 20Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile
Tyr 20 25 30Ser Met Asp Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ser Ile Tyr Ser Ser Gly Gly Ala Thr Arg 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 Cys Ser Trp Leu Gln Leu Val Pro Met His Pro Trp
Gly Gln 100 105 110Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125Phe Pro 13021366DNAHomo sapiens
21ggcgtgcact cacagagcgc tttgactcag ccaccctcag cgtctgggac ccccgggcag
60agggtcacca tctcttgttc tggaggctac tccaacatgg gaagcaatta tgcacactgg
120taccagcagg tcccaggaac ggcccccaaa ctcctcatct ataacaataa tcagaggccc
180tcaggggtcc ctgaccgatt ctctggctcc aagtctggca cctcagcctc cctagccatc
240agtgggctcc ggtccgagga tgaggctgat tattactgtg cagcatggga tgaaaacctg
300agtggtccgg tcttcggaac tgggaccaag gtcaccgtcc taggtcagcc caaggccaac
360cccact
36622122PRTHomo sapiens 22Gly Val His Ser Gln Ser Ala Leu Thr Gln Pro Pro
Ser Ala Ser Gly1 5 10
15Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Tyr Ser Asn
20 25 30Met Gly Ser Asn Tyr Ala His
Trp Tyr Gln Gln Val Pro Gly Thr Ala 35 40
45Pro Lys Leu Leu Ile Tyr Asn Asn Asn Gln Arg Pro Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65 70
75 80Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp 85 90
95Asp Glu Asn Leu Ser Gly Pro Val Phe Gly Thr Gly Thr Lys Val Thr
100 105 110Val Leu Gly Gln Pro Lys
Ala Asn Pro Thr 115 12023402DNAHomo sapiens
23gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct gagtacaata tggcttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctcgt atcggttctt ctggtggcaa gactaagtat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagagatgaa
300gcccccgact acggtgacga cgcggaagct tttgatatct ggggccaagg gacaatggtc
360accgtctcaa gcgcctccac caagggccca tcggtcttcc cg
40224134PRTHomo sapiens 24Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr
20 25 30Asn Met Ala Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Arg Ile Gly Ser Ser Gly Gly Lys Thr Lys 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 Asp Glu Ala Pro Asp Tyr Gly Asp Asp Ala Glu Ala Phe Asp
100 105 110Ile Trp Gly Gln Gly Thr
Met Val Thr Val Ser Ser Ala Ser Thr Lys 115 120
125Gly Pro Ser Val Phe Pro 13025357DNAHomo sapiens
25ggcgtgcact ctgacatcca gatgacccag tctccatcct ccctgtcttt gtctccaggg
60gaaagagcca ccctctcctg cagggccagt cagagtgtta gcagctactt agcctggtac
120caacagaaac ctggccaggc tcccaggctc ctcatctatg atgcatccaa cagggccact
180ggcatcccag ccaggttcag tggcagtggg tctgggacag acttcactct caccatcagc
240agcctagagc ctgaagattt tgcagtttat tactgtcagc agcgtagcaa ctggcctcgg
300actttcggcg gagggaccaa ggtggagatc aaacgaactg tggctgcacc atctgtc
35726119PRTHomo sapiens 26Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser1 5 10
15Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
20 25 30Val Ser Ser Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40
45Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70
75 80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Arg Ser 85 90
95Asn Trp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110Thr Val Ala Ala Pro Ser
Val 11527387DNAHomo sapiens 27gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
gtttactcta tgaattgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttat
atcgttcctt ctggtggcaa tactccttat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac
actgcagtct actattgtgc aagagatggg 300gcggctacgg tggacttaga ctactggggc
cagggaaccc tggtcaccgt ctcaagcgcc 360tccaccaagg gcccatcggt cttcccg
38728129PRTHomo sapiens 28Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Val Tyr 20 25
30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Tyr Ile Val Pro Ser Gly
Gly Asn Thr Pro 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 Asp Gly Ala Ala Thr Val Asp Leu
Asp Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro29363DNAHomo sapiens
29ggcgtgcact ctgacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga
60gacagagtca ccatcacttg ccgggcaagt cagagcatta gcagctattt aaattggtat
120cagcagaaac cagggaaagc ccctaagctc ctgatctatg ctgcatccag tttgcaaagt
180ggggtcccat caaggttcag tggcagtgga tctgggacag atttcactct caccatcagc
240agtctgcaac ctgaagattt tgcaacttac tactgtcaac agagttacag tacccctccg
300gagaacactt ttggccaggg gaccaagctg gagatcaaac gaactgtggc tgcaccatct
360gtc
36330121PRTHomo sapiens 30Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser1 5 10
15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
20 25 30Ile Ser Ser Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40
45Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70
75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr 85 90
95Ser Thr Pro Pro Glu Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
100 105 110Lys Arg Thr Val Ala Ala
Pro Ser Val 115 12031387DNAHomo sapiens
31gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct ccttaccata tgggttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctggt atctatcctt ctggtggctg gactaattat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagagatggg
300tatagcagtg gctggttccg gtactggggc cagggaaccc tggtcaccgt ctcaagcgcc
360tccaccaagg gcccatcggt cttcccg
38732129PRTHomo sapiens 32Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr
20 25 30His Met Gly Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Tyr Pro Ser Gly Gly Trp Thr Asn 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 Asp Gly Tyr Ser Ser Gly Trp Phe Arg Tyr Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro33369DNAHomo sapiens 33ggcgtgcact cacagagcga
attgactcag cctccctccg cgtccgggtc tcctggacag 60tcagtcacca tctcctgcac
tggaaccagc agtgacgttg gtggttataa ctatgtctcc 120tggtatcaac aacacccaga
caaagccccc aaactcctga tttatgaggt cactcagcgg 180ccctcagggg tccctgatcg
cttctctggc tccaggtctg gcaacacggc ctccctgacc 240gtctctgggc tccaggctga
ggatgaggct gattattact gcagctcata tgcaggcagg 300aacaatcttt atgtcttcgg
acctgggacc aaggtcaccg tcctaggtca gcccaaggcc 360aaccccact
36934123PRTHomo sapiens
34Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly1
5 10 15Ser Pro Gly Gln Ser Val
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp 20 25
30Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His
Pro Asp Lys 35 40 45Ala Pro Lys
Leu Leu Ile Tyr Glu Val Thr Gln Arg Pro Ser Gly Val 50
55 60Pro Asp Arg Phe Ser Gly Ser Arg Ser Gly Asn Thr
Ala Ser Leu Thr65 70 75
80Val Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser
85 90 95Tyr Ala Gly Arg Asn Asn
Leu Tyr Val Phe Gly Pro Gly Thr Lys Val 100
105 110Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr
115 12035405DNAHomo sapiens 35gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct gagtacaata tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg
ggtttctgtt atctctcctt ctggtggcgg tactctttat 180gctgactccg ttaaaggtcg
cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagagatcta 300aataacagct cgcccccgga
ttccaatgat gcttttgata tctggggccg agggacaatg 360gtcaccgtct caagcgcctc
caccaagggc ccatcggtct tcccg 40536135PRTHomo sapiens
36Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr 20 25
30Asn Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Val Ile
Ser Pro Ser Gly Gly Gly Thr Leu 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 Asp Leu Asn Asn
Ser Ser Pro Pro Asp Ser Asn Asp Ala Phe 100
105 110Asp Ile Trp Gly Arg Gly Thr Met Val Thr Val Ser
Ser Ala Ser Thr 115 120 125Lys Gly
Pro Ser Val Phe Pro 130 13537366DNAHomo sapiens
37ggcgtgcact cacagagcgt cttgactcag ccaccctcag tgtctgggac ccccggacag
60agggtcacca tctcttgttc tggaggctac cccaacatgg gaagcaatta tgcacactgg
120taccagcaac tcccaggaac ggcccccaaa ctcctcatct ataacgataa tcagcggccc
180tcaggggtcc ctgaccgatt ctctggctcc aagtctggca cctcagcctc cctggccatc
240agtgggctcc ggtccgagga tgaggctgat tattactgtg cagcatggga tgacagcctg
300agtggtccgg tgttcggcgg agggaccaag ctgaccgtcc taggtcagcc caaggctgcc
360ccctcg
36638122PRTHomo sapiens 38Gly Val His Ser Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Gly1 5 10
15Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Tyr Pro Asn
20 25 30Met Gly Ser Asn Tyr Ala His
Trp Tyr Gln Gln Leu Pro Gly Thr Ala 35 40
45Pro Lys Leu Leu Ile Tyr Asn Asp Asn Gln Arg Pro Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65 70
75 80Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp 85 90
95Asp Asp Ser Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr
100 105 110Val Leu Gly Gln Pro Lys
Ala Ala Pro Ser 115 12039402DNAHomo sapiens
39gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct gtttacgata tgccttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctgtt atctatcctt ctggtggctt tactcgttat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc ggcagatccg
300acgatacagc tatgggccta ctactacggt atggacgtct ggggccaagg gaccacggtc
360accgtctcaa gcgcctccac caagggccca tcggtcttcc cg
40240134PRTHomo sapiens 40Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr
20 25 30Asp Met Pro Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Val Ile Tyr Pro Ser Gly Gly Phe Thr Arg 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 Ala Asp Pro Thr Ile Gln Leu Trp Ala Tyr Tyr Tyr Gly Met Asp
100 105 110Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120
125Gly Pro Ser Val Phe Pro 13041363DNAHomo sapiens
41ggcgtgcact cacagagcga attgactcag ccaccctcag cgtctgggac ccccgggcag
60agggtcacca tctcttgttc tggaggcagc tccaacatcg gaagtaattt tgtttactgg
120taccggcagc tcccaggaac ggcccccaaa ctcctcatct ataggaatta tcagcggccc
180tcaggggtcc ctgaccgatt ctcgggttcc aagtctggca cctcagcctc cctggccatc
240agtgggctcc tgtccgaaga tgaggctgat tattactgcg cagcatggga tgacaacgtg
300ggtggggtct tcggatctgg gaccaaggtc accgtcctgg gtcagcccaa ggccaacccc
360act
36342121PRTHomo sapiens 42Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro Pro
Ser Ala Ser Gly1 5 10
15Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asn
20 25 30Ile Gly Ser Asn Phe Val Tyr
Trp Tyr Arg Gln Leu Pro Gly Thr Ala 35 40
45Pro Lys Leu Leu Ile Tyr Arg Asn Tyr Gln Arg Pro Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65 70
75 80Ser Gly Leu Leu Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp 85 90
95Asp Asp Asn Val Gly Gly Val Phe Gly Ser Gly Thr Lys Val Thr Val
100 105 110Leu Gly Gln Pro Lys Ala
Asn Pro Thr 115 12043405DNAHomo sapiens
43gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct tggtacgata tgtattgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttcttct atctattctt ctggtggcta tactgcttat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gaaagatcgt
300gatccttgta gtagaaccac ctgctataac tggttcgacc cctggggcca gggaaccctg
360gtcaccgtct caagcgcctc caccaagggc ccatcggtct tcccg
40544135PRTHomo sapiens 44Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr
20 25 30Asp Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Tyr Ser Ser Gly Gly Tyr Thr Ala 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 Lys Asp Arg Asp Pro Cys Ser Arg Thr Thr Cys Tyr Asn Trp Phe
100 105 110Asp Pro Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120
125Lys Gly Pro Ser Val Phe Pro 130
13545366DNAHomo sapiens 45ggcgtgcact cacagagcga attgactcag ccaccctcag
cgtctgggac ccccgggcag 60agggtcacca tctcttgttc tggaggcagc tccaacatcg
gaagtaatta tgtctcctgg 120taccagcagc tcccaggaac ggcccccaaa ctcctcatct
ataataataa tcagcggccc 180tcaggggtcc ctgaccgatt ctctggctcc aagtctggca
cctcagcctc cctggccatc 240agtgggctcc ggtccgagga tgaggctgat tattactgtg
cagcatggga tgacagcctg 300agttctgctg tgttcggagg aggcacccag ctgaccgtcc
tcggtcagcc caaggctgcc 360ccctcg
36646122PRTHomo sapiens 46Gly Val His Ser Gln Ser
Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly1 5
10 15Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly
Gly Ser Ser Asn 20 25 30Ile
Gly Ser Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala 35
40 45Pro Lys Leu Leu Ile Tyr Asn Asn Asn
Gln Arg Pro Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65
70 75 80Ser Gly Leu Arg Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp 85
90 95Asp Asp Ser Leu Ser Ser Ala Val Phe Gly Gly
Gly Thr Gln Leu Thr 100 105
110Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 115
12047393DNAHomo sapiens 47gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct gcttaccgta
tgttttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttct atctggcctt
ctggtggcac tacttcttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagagatcgg 300ggctatgata gtagtggtta ttttgactac tggggccagg
gaaccctggt caccgtctca 360agcgcctcca ccaagggccc atcggtcttc ccg
39348131PRTHomo sapiens 48Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30Arg
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ser Ile Trp Pro Ser Gly Gly Thr
Thr Ser 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 Asp Arg Gly Tyr Asp Ser Ser Gly Tyr
Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro 13049360DNAHomo
sapiens 49ggcgtgcact cacagagcgc tttgactcag ccaccctcgg tgtcactggc
cccaggacag 60acggccagga ttacctgtgg gggaaacaac attggaacta aaagtgttca
ctggtaccag 120cagaagccag gccaggcccc tgtgctggtc gtctatgatg acagcgaccg
gccctcaggg 180atccctgagc gattctctgg ctccaattct gggaacacgg ccaccctgac
catcagcagg 240gtcgaagccg gggatgaggc cgactattat tgtcaggtgt gggatagtgg
tagtgatcat 300caggtcttcg gcggagggac caagctgacc gtcctaggtc agcccaaggc
tgccccctcg 36050120PRTHomo sapiens 50Gly Val His Ser Gln Ser Ala Leu
Thr Gln Pro Pro Ser Val Ser Leu1 5 10
15Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn
Ile Gly 20 25 30Thr Lys Ser
Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val 35
40 45Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser
Gly Ile Pro Glu Arg 50 55 60Phe Ser
Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg65
70 75 80Val Glu Ala Gly Asp Glu Ala
Asp Tyr Tyr Cys Gln Val Trp Asp Ser 85 90
95Gly Ser Asp His Gln Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 110Gly Gln
Pro Lys Ala Ala Pro Ser 115 12051422DNAHomo
sapiensmisc_feature(226)..(226)n is a, c, g, or t 51gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct tggtacacta tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg
ggtttctcgt atctctcctt ctggtggcca tactctttat 180gctgactccg ttaaaggtcg
cttcactatc tctagagaca actctnagaa tactctctac 240ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagagacact 300tgggacgatt actatgatag
tagtggttat tacaacgatt ttgactactg gggccaggga 360accctggtca ccgtctcaag
cgcctccacc aagggcccat cggtcttccc gctagcgccc 420tg
42252140PRTHomo sapiens
52Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25
30Thr Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Arg Ile
Ser Pro Ser Gly Gly His Thr Leu Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glx
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 Asp Thr Trp Asp
Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Asn 100
105 110Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala 115 120 125Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 130 135
14053360DNAHomo sapiens 53ggcgtgcact ctgacatcca gatgacccag
tctccaggca ccctgtcttt gtctccaggg 60gaaagagcca ccctctcctg cagggccagt
cagagtgtta gtcgtagcta cttaggctgg 120taccagcaga aacctggcca ggctcccagg
ctcctcatct atggtgcatc caacagggcc 180actggcatcc cagacaggtt cagtggcagt
gggtctggga cagacttcac tctcaccatc 240agcagactgg agcctgaaga ttttgcagtg
tattactgtc agcagtacgg tatctcaccc 300ctcaccttcg gccctgggac caaagtggat
atcaaacgaa ctgtggctgc accatctgtc 36054120PRTHomo sapiens 54Gly Val His
Ser Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser1 5
10 15Leu Ser Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser 20 25
30Val Ser Arg Ser Tyr Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala
35 40 45Pro Arg Leu Leu Ile Tyr Gly
Ala Ser Asn Arg Ala Thr Gly Ile Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65
70 75 80Ser Arg Leu Glu
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 85
90 95Gly Ile Ser Pro Leu Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Lys 100 105
110Arg Thr Val Ala Ala Pro Ser Val 115
12055390DNAHomo sapiens 55gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct gcttactgga
tggattgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctgtt atctatcctt
ctggtggctc tactaattat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagagagggg 300atagccgcag cagcaccaat ggacgtctgg ggcaaaggga
ccacggtcac cgtctcaagc 360gcctccacca agggcccatc ggtcttcccg
39056130PRTHomo sapiens 56Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30Trp
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Val Ile Tyr Pro Ser Gly Gly Ser
Thr Asn 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 Glu Gly Ile Ala Ala Ala Ala Pro Met
Asp Val Trp Gly Lys 100 105
110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro 13057366DNAHomo
sapiens 57ggcgtgcact ctgacatcca gatgacccag tctccaggca ccctgtcttt
gtctccaggg 60gaaagagcca ccctctcctg cagggccagt cagagtgtta gcagcagcta
ctttgcctgg 120taccagcaga aacctggcca ggctcccagg ctcctcatct atgatgcatc
cagcagggcc 180actggcatcc cagacaggtt cagtggcagt gggtctggga cagacttcac
tctcaccatc 240agcagactgg agcctgaaga ttttgcagtg tattactgtc agcagtatgg
tagctcacct 300ccgatgtaca cttttggcca ggggaccaag ctggagatca aacgaactgt
ggctgcacca 360tctgtc
36658122PRTHomo sapiens 58Gly Val His Ser Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser1 5 10
15Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser 20 25 30Val Ser Ser Ser
Tyr Phe Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35
40 45Pro Arg Leu Leu Ile Tyr Asp Ala Ser Ser Arg Ala
Thr Gly Ile Pro 50 55 60Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65 70
75 80Ser Arg Leu Glu Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Tyr 85 90
95Gly Ser Ser Pro Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu 100 105 110Ile Lys Arg
Thr Val Ala Ala Pro Ser Val 115 12059405DNAHomo
sapiens 59gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct acttacgata tgctttgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttcttct atctctcctt ctggtggctc
tacttcttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagctga acagcttaag ggctgaggac actgcagtct actattgtgc
gagagagaaa 300gcgtcggatc tttcggggac ttactctgag gcccttgacc actggggcca
gggaaccctg 360gtcaccgtct caagcgcctc caccaagggc ccatcggtct tcccg
40560135PRTHomo sapiens 60Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr
Tyr 20 25 30Asp Met Leu Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ser Ile Ser Pro Ser Gly Gly Ser Thr Ser 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 Leu Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Glu Lys Ala Ser Asp Leu Ser Gly Thr Tyr Ser Glu
Ala Leu 100 105 110Asp His Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115
120 125Lys Gly Pro Ser Val Phe Pro 130
13561360DNAHomo sapiens 61ggcgtgcact ctgacatcca gatgacccag
tctccatcct ccctgtctgc atctgtggga 60gacagagtcg ccatcacttg ccgtgcaagt
cagagcatcg acacctattt aaattggtat 120cagcagaaac cagggaaagc ccctaaactc
ctgatctatg ctgcatccaa gttggaagac 180ggggtcccat caagattcag tggcagtgga
actgggacag atttcactct caccatcaga 240agtctgcaac ctgaagattt tgcaagttat
ttctgtcaac agagctactc tagtccaggg 300atcactttcg gccctgggac caaggtggag
atcaaacgaa ctgtggctgc accatctgtc 36062120PRTHomo sapiens 62Gly Val His
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser1 5
10 15Ala Ser Val Gly Asp Arg Val Ala Ile
Thr Cys Arg Ala Ser Gln Ser 20 25
30Ile Asp Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45Lys Leu Leu Ile Tyr Ala Ala
Ser Lys Leu Glu Asp Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Thr Gly Thr Asp Phe Thr Leu Thr Ile Arg65
70 75 80Ser Leu Gln Pro
Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Ser Tyr 85
90 95Ser Ser Pro Gly Ile Thr Phe Gly Pro Gly
Thr Lys Val Glu Ile Lys 100 105
110Arg Thr Val Ala Ala Pro Ser Val 115
12063405DNAHomo sapiens 63gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct gattacttta
tgaagtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttct atctatcctt
ctggtggccc tactaagtat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagagagcgt 300agcagtggct ggtacggtta ctactactac ggtatggacg
tctggggcca agggaccacg 360gtcaccgtct caagcgcctc caccaagggc ccatcggtct
tcccg 40564135PRTHomo sapiens 64Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asp Tyr 20 25
30Phe Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Tyr Pro Ser Gly Gly
Pro Thr Lys 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 Glu Arg Ser Ser Gly Trp Tyr Gly Tyr
Tyr Tyr Tyr Gly Met 100 105
110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
115 120 125Lys Gly Pro Ser Val Phe Pro
130 13565354DNAHomo sapiens 65ggcgtgcact ctgacatcca
gatgacccag tctccatcct tcctgtctgc ttctgtaggg 60gacagagtca ccatcacttg
ccgggccagt cagggcatta gggatttttt aggctggtat 120caacaaaaac cagggaaagc
ccctaatcaa ctgatctatg ctgcatccat tttgcaaagt 180ggggtcccat caaggttcag
cggcagtgga tctgggacag atttcactct cacgatcacc 240agcctgcagc ctgaggattt
tgcaacttat ttctgtcaac agcttaatgg ctaccgcgcc 300ttcggccaag ggacacgact
ggaaataaag cgaactgtgg ctgcaccatc tgtc 35466118PRTHomo sapiens
66Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser1
5 10 15Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly 20 25
30Ile Arg Asp Phe Leu Gly Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45Asn Gln Leu
Ile Tyr Ala Ala Ser Ile Leu Gln Ser Gly Val Pro Ser 50
55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Thr65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Leu Asn
85 90 95Gly Tyr Arg Ala Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr 100
105 110Val Ala Ala Pro Ser Val 11567390DNAHomo
sapiens 67gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct ccttacgaga tgcagtgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt atcggttctt ctggtggtga
ctccgttaaa 180ggtcgcttca ctatctctag agacaactct aagaatactc tctacttgca
gatgaacagc 240ttaagggctg aggacactgc agtctactat tgtgcgagag agagggtaga
ttgtagtggt 300ggtggctgcg ggagctactt tgactactgg ggccagggaa ccctggtcac
cgtctcaagc 360gcctccacca agggcccatc ggtcttcccg
39068130PRTHomo sapiens 68Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro
Tyr 20 25 30Glu Met Gln Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Gly Ser Ser Gly Gly Asp Ser Val Lys
Gly Arg Phe Thr 50 55 60Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser65 70
75 80Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Glu Arg Val 85 90
95Asp Cys Ser Gly Gly Gly Cys Gly Ser Tyr Phe Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125Phe Pro 13069357DNAHomo sapiens
69ggcgtgcact ctgacatcca gatgacccag tctccatctt ccgtgtctgc atctgtagga
60gacagagtca cgatcacttg tcgggcgagt cagggtatta gcaagagctt agcctggtat
120cagcagaaac cagggaaagc ccctaaactc ctggtctatg gtgcattcag tttggaaagt
180ggggtcccat caagattcag cggcactgga gctgggacag atttcattct caccatcagc
240aggctgcagc ctgaagactt tgcaacttat tattgtcaac aggctaacag tttcccgctc
300actttcggcg gagggaccaa ggtggagatc aaacgaactg tggctgcacc atctgtc
35770119PRTHomo sapiens 70Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Val Ser1 5 10
15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
20 25 30Ile Ser Lys Ser Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40
45Lys Leu Leu Val Tyr Gly Ala Phe Ser Leu Glu Ser Gly Val Pro
Ser 50 55 60Arg Phe Ser Gly Thr Gly
Ala Gly Thr Asp Phe Ile Leu Thr Ile Ser65 70
75 80Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ala Asn 85 90
95Ser Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110Thr Val Ala Ala Pro Ser
Val 11571384DNAHomo sapiens 71gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
ttttactgga tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt
atctcttctt ctggtggctt tactaagtat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actccaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac
actgcagtct actattgtgc gagggagacc 300agccggaggg cttttgatat ctggggccaa
gggacaatgg tcaccgtctc aagcgcctcc 360accaagggcc catcggtctt cccg
38472128PRTHomo sapiens 72Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Phe Tyr 20 25
30Trp Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Ser Ser Ser Gly
Gly Phe Thr Lys 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 Glu Thr Ser Arg Arg Ala Phe Asp
Ile Trp Gly Gln Gly Thr 100 105
110Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 12573363DNAHomo sapiens
73ggcgtgcact ctgacatcca gatgacccag tctccaggca ccctgtcttt gtctccaggg
60gaaagagcca ccctctcctg cagggccagt cagagtgtta gcagcagcta cttagcctgg
120taccagcaga aacctggcca ggctcccagg ctcctcatct atggtgcatc cagcagggcc
180actggcatcc cagacaggtt cagtggcagt gggtctggga cagacttcac tctcaccatc
240agcagactgg agcctgaaga ttttgcagtg tattactgtc agcagtatgg tagctcacct
300gagatcacct tcggccaagg gacacgactg gagattaaac gaactgtggc tgcaccatct
360gtc
36374121PRTHomo sapiens 74Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro
Gly Thr Leu Ser1 5 10
15Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
20 25 30Val Ser Ser Ser Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40
45Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65 70
75 80Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr 85 90
95Gly Ser Ser Pro Glu Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
100 105 110Lys Arg Thr Val Ala Ala
Pro Ser Val 115 12075411DNAHomo sapiens
75gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct gagtactgga tgccttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctcgt atctatcctt ctggtggcgt tactacttat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagagggggg
300gattacgatt tttggagtgt acaatactac tactactaca tggacgtctg gggcaaaggg
360accacggtca ccgtctcaag cgcctccacc aagggcccat cggtcttccc g
41176137PRTHomo sapiens 76Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr
20 25 30Trp Met Pro Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Arg Ile Tyr Pro Ser Gly Gly Val Thr Thr 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 Gly Gly Asp Tyr Asp Phe Trp Ser Val Gln Tyr Tyr Tyr Tyr
100 105 110Tyr Met Asp Val Trp Gly
Lys Gly Thr Thr Val Thr Val Ser Ser Ala 115 120
125Ser Thr Lys Gly Pro Ser Val Phe Pro 130
13577360DNAHomo sapiens 77ggcgtgcact ctgacatcca gatgacccag tctccatcct
tcctgtctgc atctgtagga 60gacagagtca ccatcacttg ccgggccagt cagggcatta
gcagttattt agcctggtat 120cagcaaaaac cagggaaagc ccctaagctc ctgatctatg
ctgcatccac tttgcaaagt 180ggggtcccat caaggttcag cggcagtgga tctggaacag
atttcactct caccatcagc 240agtctggaac ctgaagattt tgcaacttac tactgtcaag
agagttacag tacccccttc 300tttactttcg gccctgggac caaagtggat atcagacgaa
ctgtggctgc accatctgtc 36078120PRTHomo sapiens 78Gly Val His Ser Asp
Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser1 5
10 15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly 20 25
30Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser
Thr Leu Gln Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Glu Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Ser Tyr 85
90 95Ser Thr Pro Phe Phe Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Arg 100 105
110Arg Thr Val Ala Ala Pro Ser Val 115
12079393DNAHomo sapiens 79gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct cagtacttta
tgaagtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttct atctctcctt
ctggtggcct tactcagtat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagaggtggt 300atagaagcac ctgggtcccc ctctgactac tggggccagg
gaaccctggt caccgtctca 360agcgcctcca ccaagggccc atcggtcttc ccg
39380131PRTHomo sapiens 80Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Gln Tyr 20 25 30Phe
Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ser Ile Ser Pro Ser Gly Gly Leu
Thr Gln 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 Gly Gly Ile Glu Ala Pro Gly Ser Pro
Ser Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro 13081357DNAHomo
sapiens 81ggcgtgcact ctgacatcca gatgacccag tctccagcca ccctgtcttt
gtctccaggg 60gaaagagcca ccctctcctg cagggccagt cagagtgtta gcagctactt
agcctggtac 120caacagaaac ctggccaggc tcccaggctc ctcatctatg atgcatccaa
cagggccact 180ggcatcccag ccaggttcag tggcagtggg tctgggacag acttcactct
caccatcagc 240agcctagagc ctgaagattt tgcagtttat tactgtcagc agcgtagcaa
ctggcctcgg 300actttcggcg gagggaccaa ggtggagatc aaacgaactg tggctgcacc
atctgtc 35782119PRTHomo sapiens 82Gly Val His Ser Asp Ile Gln Met
Thr Gln Ser Pro Ala Thr Leu Ser1 5 10
15Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser 20 25 30Val Ser Ser
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35
40 45Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Glu Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Arg Ser 85 90
95Asn Trp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg 100 105 110Thr Val
Ala Ala Pro Ser Val 11583396DNAHomo sapiens 83gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg
cttccggatt cactttctct cagtaccaga tgatttgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttctgtt atcgttcctt ctggtggcat tactaattat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga
acagcttaag ggctgaggac actgcagtct actattgtgc gagaggtggg 300gtagaggcag
tggatagttc gtcgcctgac tactggggcc agggaaccct ggtcaccgtc 360tcaagcgcct
ccaccaaggg cccatcggtc ttcccg 39684132PRTHomo
sapiens 84Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 20
25 30Gln Met Ile Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ser Val Ile Val Pro Ser Gly Gly Ile Thr Asn 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
Gly Gly Val Glu Ala Val Asp Ser Ser Ser Pro Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro 13085366DNAHomo sapiens 85ggcgtgcact cacagagcga
attgactcag ccccactctg tgtcggggtc tccggggaag 60acggtaacca tctcctgcac
ccgcagcagt ggcagcattg ccggcaacta tgtgcagtgg 120taccagcagc gcccgggcag
ttcccccacc actgtgatct atgaggataa caaaagaccc 180tctggggtcc ctgatcggtt
ctctggctcc atcgacagct cctccaactc tgcctccctc 240atcatctctg gactgaagac
tgaggacgag gctgactact actgtcattc ttatgatacc 300agcaatcagg tattcggcgg
agggaccaaa ctgaccgtcc taggtcagcc caaggctgcc 360ccctcg
36686122PRTHomo sapiens
86Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro His Ser Val Ser Gly1
5 10 15Ser Pro Gly Lys Thr Val
Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser 20 25
30Ile Ala Gly Asn Tyr Val Gln Trp Tyr Gln Gln Arg Pro
Gly Ser Ser 35 40 45Pro Thr Thr
Val Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly Val Pro 50
55 60Asp Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn
Ser Ala Ser Leu65 70 75
80Ile Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys His
85 90 95Ser Tyr Asp Thr Ser Asn
Gln Val Phe Gly Gly Gly Thr Lys Leu Thr 100
105 110Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 115
12087393DNAHomo sapiens 87gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct cgttacatga tgaattgggt tcgccaagct 120cctggtaaag gtttggagtg
ggtttctgtt atctggtctt ctggtggcaa gactctttat 180gctgactccg ttaaaggtcg
cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga agagcttaag
ggctgaggac actgcagtct actattgtgc gaggggtggt 300tacaacaact actactactc
tatggacgtc tggggccaag ggaccacggt caccgtctca 360agcgcctcca ccaagggccc
atcggtcttc ccg 39388131PRTHomo sapiens
88Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25
30Met Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Val Ile
Trp Ser Ser Gly Gly Lys Thr Leu 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 Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Gly Tyr Asn
Asn Tyr Tyr Tyr Ser Met Asp Val Trp Gly 100
105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125Val Phe
Pro 13089357DNAHomo sapiens 89ggcgtgcact ctgacatcca gatgacccag
tctccttcct ccctgtctgc ctctgtagga 60gacagagtca ccatcgcgtg ccggacaagt
cagaacgtta ataggtacct gaattggtat 120caacataaac tcggccaggc ccctaaactc
ctgatctacg gtgcaaccat tttgcagagt 180ggggtcccat caaggttccg tggcagtgga
tctgggacag atttcatcct caccatcacc 240aatctgcaac ctgaagattt tgcagtttac
tactgtcaac agacttacag tcccccactg 300acgttcggcc aagggaccaa ggcggaattt
aaaggaactg tggctgcacc atctgtc 35790119PRTHomo sapiens 90Gly Val His
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser1 5
10 15Ala Ser Val Gly Asp Arg Val Thr Ile
Ala Cys Arg Thr Ser Gln Asn 20 25
30Val Asn Arg Tyr Leu Asn Trp Tyr Gln His Lys Leu Gly Gln Ala Pro
35 40 45Lys Leu Leu Ile Tyr Gly Ala
Thr Ile Leu Gln Ser Gly Val Pro Ser 50 55
60Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr Ile Thr65
70 75 80Asn Leu Gln Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Tyr 85
90 95Ser Pro Pro Leu Thr Phe Gly Gln Gly Thr
Lys Ala Glu Phe Lys Gly 100 105
110Thr Val Ala Ala Pro Ser Val 11591369DNAHomo sapiens
91gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct tcttacgcta tgatgtgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttcttgg atcgttcctt ctggtggcac tactttttat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagaggcctg
300taccggtggg gccagggaac cctggtcacc gtctcaagcg cctccaccaa gggcccatcg
360gtcttcccg
36992123PRTHomo sapiens 92Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ala Met Met Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Trp Ile Val Pro Ser Gly Gly Thr Thr Phe 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 Gly Leu Tyr Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro 115 12093360DNAHomo sapiens
93ggcgtgcact cacagagcga attgactcag ccaccctcgg tgtcactggc cccaggacag
60acggccagga ttacctgtgg gggaaacaac attggaacta aaagtgttca ctggtaccag
120cagaagccag gccaggcccc tgtgctggtc gtctatgatg acagcgaccg gccctcaggg
180atccctgagc gattctctgg ctccaattct gggaacacgg ccaccctgac catcagcagg
240gtcgaagccg gggatgaggc cgactattat tgtcaggtgt gggatagtgg tagtgatcat
300caggtcttcg gcggagggac caagctgacc gtcctaggtc agcccaaggt tgccccctcg
36094120PRTHomo sapiens 94Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro Pro
Ser Val Ser Leu1 5 10
15Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly
20 25 30Thr Lys Ser Val His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val 35 40
45Leu Val Val Tyr Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu
Arg 50 55 60Phe Ser Gly Ser Asn Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg65 70
75 80Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys
Gln Val Trp Asp Ser 85 90
95Gly Ser Asp His Gln Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110Gly Gln Pro Lys Val Ala
Pro Ser 115 12095378DNAHomo
sapiensmisc_feature(226)..(226)n is a, c, g, or t 95gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct ccttacttta tgttttgggt tcgccaagct 120cctggtaaag gtttggagtg
ggtttcttct atcggttctt ctggtggcga tacttcttat 180gctgactccg ttaaaggtcg
cttcactatc tctagagaca actctnagaa tactctctac 240ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagaggcctg 300taccggtggg gccagggaac
cctggtcacc gtctcaagcg cctccaccaa gggcccatcg 360gtcttcccgc tagcgccc
37896126PRTHomo sapiens
96Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25
30Phe Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Ser Ile
Gly Ser Ser Gly Gly Asp Thr Ser Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glx
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 Gly Leu Tyr Arg
Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100
105 110Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro 115 120 12597360DNAHomo
sapiens 97ggcgtgcact cacagagcgc tttgactcag ccaccctcgg tgtcagtggc
cccaggacag 60acggccagga tttcctgtgg gggcgacaac attgacacta aaaatgtaca
gtggtaccag 120cagaggccag gccaggcccc tgtgctggtc gtctatgata atagcgaccg
gccctcagcg 180atccctgagc gattctctgg ctccaactct gggaccacgg ccaccctgac
catcagcagg 240gtcgaggccg gggatgaggc cgactattac tgtcaggtgt ttgatggtag
gagtgatcat 300ccggtgttcg gcggagggac caagctgacc gttcctgggt cagcccaagg
ctgccccctc 36098120PRTHomo sapiens 98Gly Val His Ser Gln Ser Ala Leu
Thr Gln Pro Pro Ser Val Ser Val1 5 10
15Ala Pro Gly Gln Thr Ala Arg Ile Ser Cys Gly Gly Asp Asn
Ile Asp 20 25 30Thr Lys Asn
Val Gln Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val 35
40 45Leu Val Val Tyr Asp Asn Ser Asp Arg Pro Ser
Ala Ile Pro Glu Arg 50 55 60Phe Ser
Gly Ser Asn Ser Gly Thr Thr Ala Thr Leu Thr Ile Ser Arg65
70 75 80Val Glu Ala Gly Asp Glu Ala
Asp Tyr Tyr Cys Gln Val Phe Asp Gly 85 90
95Arg Ser Asp His Pro Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Pro 100 105 110Gly Ser
Ala Gln Gly Cys Pro Leu 115 12099411DNAHomo
sapiens 99gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct ctttacgtta tgtattgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttcttat atctcttctt ctggtggcat
tactcattat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc
gagaggctct 300attgtagtag taccagctgc tatacggagc aacaactggt tcgacccctg
gggccaggga 360accctggtca ccgtctcaag cgcctccacc aagggcccat cggtcttccc g
411100137PRTHomo sapiens 100Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Leu Tyr 20 25 30Val Met Tyr
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Tyr Ile Ser Ser Ser Gly Gly Ile Thr His
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 Gly Ser Ile Val Val Val Pro Ala Ala Ile Arg
Ser Asn Asn 100 105 110Trp Phe
Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115
120 125Ser Thr Lys Gly Pro Ser Val Phe Pro
130 135101357DNAHomo sapiens 101ggcgtgcact ctgacatcca
gatgacccag tctccttcca ccctgtctgc atctgtagga 60gacagagtca ccatcacttg
ccgggccagt cagagtattg gaaactggtt ggcctggtat 120cagcagaaac caggggaagc
ccctcacctc ctgatctatc aggcgtctag tttagaaggt 180ggggtcccat caaggttcag
cggcagtggg tctgggacaa aattcactct caacatcagc 240agcctgcagc ctgatgactt
tgcaacttat tactgccaac agtataattc ttattcgtac 300acttttggcc aggggaccaa
gctggacatc aaacgaactg tggctgcacc atctgtc 357102119PRTHomo sapiens
102Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser1
5 10 15Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 20 25
30Ile Gly Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Glu Ala Pro 35 40 45His Leu Leu
Ile Tyr Gln Ala Ser Ser Leu Glu Gly Gly Val Pro Ser 50
55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Lys Phe Thr
Leu Asn Ile Ser65 70 75
80Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn
85 90 95Ser Tyr Ser Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Asp Ile Lys Arg 100
105 110Thr Val Ala Ala Pro Ser Val
115103393DNAHomo sapiens 103gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct aattacggta
tgtcttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctgtt atcggtcctt
ctggtggcat tactatgtat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gaccgggtct 300agcagtggct ggtaccctaa ctttgactac tggggccagg
gaaccctggt caccgtctca 360agcgcctcca ccaagggccc atcggtcttc ccg
393104131PRTHomo sapiens 104Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asn Tyr 20 25
30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Val Ile Gly Pro Ser Gly Gly
Ile Thr Met 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 Thr Gly Ser Ser Ser Gly Trp Tyr Pro Asn
Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro
130105366DNAHomo sapiens 105ttctattctc acagtgcaca agacatccag atgacccagt
ctccatcctc cctgtctgca 60tctgtaggag atagagtcac catcacttgc cgggcaagtc
agaccattag cacctattta 120gtttggtatc agcagaaacc cgagaaagcc cctacgctcc
tgatctccgg tgcatccact 180ttgcaaagtg gggtcccaaa caggttcaga ggcagtggat
ctgggacaga cttcactctc 240gccatctcca gtcttcaacc tgaagatttt gcaacttact
actgtcaaca gagttacact 300tcccctagaa cgttcggcca agggaccaag gtggaaatca
aacgaactgt ggctgcacca 360tctgtc
366106122PRTHomo sapiens 106Phe Tyr Ser His Ser
Ala Gln Asp Ile Gln Met Thr Gln Ser Pro Ser1 5
10 15Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala 20 25
30Ser Gln Thr Ile Ser Thr Tyr Leu Val Trp Tyr Gln Gln Lys Pro Glu
35 40 45Lys Ala Pro Thr Leu Leu Ile Ser
Gly Ala Ser Thr Leu Gln Ser Gly 50 55
60Val Pro Asn Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu65
70 75 80Ala Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 85
90 95Gln Ser Tyr Thr Ser Pro Arg Thr Phe Gly Gln
Gly Thr Lys Val Glu 100 105
110Ile Lys Arg Thr Val Ala Ala Pro Ser Val 115
120107384DNAHomo sapiens 107gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct cattactcta
tgcgttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttat atcgttcctt
ctggtggctt tactcagtat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagaggcacg 300cacctcccgg gggttgacta ctggggccag ggaaccctgg
tcaccgtctc aagcgcctcc 360accaagggcc catcggtctt cccg
384108128PRTHomo sapiens 108Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser His Tyr 20 25
30Ser Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Tyr Ile Val Pro Ser Gly Gly
Phe Thr Gln 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 Gly Thr His Leu Pro Gly Val Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125109366DNAHomo sapiens
109ggcgtgcact cacagagcgc tttgactcag ccaccctcag cgtctgggac ccccgggcag
60agggtcacca tctcttgttc tggaagcaac tccaacatcg gaggtaatat tgtaatctgg
120ctccagcagc tcccaggaac ggcccccaaa ctcatgattt atgatgtcag tgatcggccc
180tcaggggtcc ctgaccgatt ctctggctcc aagtctggca cctcagcctc cctggccatc
240agtgggctcc agtctgagga tgaggccgat tattattgtg cagcctggga tgacagcctg
300aatggttggg tgttcggcgg agggaccaag ctgaccgtcc taagtcagcc caaggctgcc
360ccctcg
366110122PRTHomo sapiens 110Gly Val His Ser Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly1 5 10
15Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn
20 25 30Ile Gly Gly Asn Ile Val Ile
Trp Leu Gln Gln Leu Pro Gly Thr Ala 35 40
45Pro Lys Leu Met Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65 70
75 80Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp 85 90
95Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
100 105 110Val Leu Ser Gln Pro Lys
Ala Ala Pro Ser 115 120111369DNAHomo sapiens
111gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct ctttacatga tgaagtgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctgtt atctcttctt ctggtggcta tactcagtat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acaacttaag ggctgaggac actgcagtct actattgtgc gagagggtgg
300gacgtctggg gcaaagggac cacggtcacc gtctcaagcg cctccaccaa gggcccatcg
360gtcttcccg
369112123PRTHomo sapiens 112Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr
20 25 30Met Met Lys Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Val Ile Ser Ser Ser Gly Gly Tyr Thr Gln 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 Asn Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Trp Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser
100 105 110Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro 115 120113366DNAHomo sapiens
113ggcgtgcact cacagagcgc tttgactcag ccaccctcag cgtctgggac ccccgggcag
60agggtcacca tctcctgttc tggaaccagc tccaacatcg gaagtcatta tgtattctgg
120tatcagcagc tcccaggaac ggcccccaaa ctcctcatcc ataggaatga tgagcggccc
180tcaggggtcc ctgaccgctt ctctggctcc aagtctggca cctccgcctc cctggccatc
240agtggcctcc agtctgagga tgaggctgat tattactgtg ctacgtggga tgacaaccta
300aatggtccgg tattcggcgg agggaccaag ctgaccggcc ctgggtcagc ccaaggctgc
360cccctc
366114122PRTHomo sapiens 114Gly Val His Ser Gln Ser Ala Leu Thr Gln Pro
Pro Ser Ala Ser Gly1 5 10
15Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn
20 25 30Ile Gly Ser His Tyr Val Phe
Trp Tyr Gln Gln Leu Pro Gly Thr Ala 35 40
45Pro Lys Leu Leu Ile His Arg Asn Asp Glu Arg Pro Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65 70
75 80Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Thr Trp 85 90
95Asp Asp Asn Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr
100 105 110Gly Pro Gly Ser Ala Gln
Gly Cys Pro Leu 115 120115393DNAHomo sapiens
115gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct atgtacttta tggtttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttcttgg atcggttctt ctggtggcga gactccttat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc aagagggtac
300agcagtggct ggtatgtaat gggagactac tggggccagg gaaccctggt caccgtctca
360agcgcctcca ccaagggccc atcggtcttc ccg
393116131PRTHomo sapiens 116Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr
20 25 30Phe Met Val Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Trp Ile Gly Ser Ser Gly Gly Glu Thr Pro 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 Gly Tyr Ser Ser Gly Trp Tyr Val Met Gly Asp Tyr Trp Gly
100 105 110Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro 130117366DNAHomo sapiens 117ggcgtgcact
cacagagcga attgactcag ccaccctcag tgtctgggac ccccgggcag 60agggtcacca
tctcttgttc tggaagcagt tccaacatcg gaagtgagta tgtgtactgg 120ttccagcagc
tcccaggaac ggcccccaga ctcctcatct ataggaatga tcagcggccc 180tcaggggtcc
ctgaccgatt ctctggctcc aagtctggca cctcagcctc cctggccatc 240agtgggctcc
ggtccgagga tgagactgat tattactgta caacatggga tgacagcctg 300agtggtccgg
tgttcggcgg agggaccaag ctgaccgtcc taggtcagcc caaggctgcc 360ccctcg
366118122PRTHomo
sapiens 118Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro Pro Ser Val Ser
Gly1 5 10 15Thr Pro Gly
Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn 20
25 30Ile Gly Ser Glu Tyr Val Tyr Trp Phe Gln
Gln Leu Pro Gly Thr Ala 35 40
45Pro Arg Leu Leu Ile Tyr Arg Asn Asp Gln Arg Pro Ser Gly Val Pro 50
55 60Asp Arg Phe Ser Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile65 70 75
80Ser Gly Leu Arg Ser Glu Asp Glu Thr Asp Tyr Tyr Cys Thr
Thr Trp 85 90 95Asp Asp
Ser Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr 100
105 110Val Leu Gly Gln Pro Lys Ala Ala Pro
Ser 115 120119405DNAHomo sapiens 119gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg
cttccggatt cactttctct tcttaccaga tggattgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttctcgt atcgttcctt ctggtggcga tactacttat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga
acagcttaag ggctgaggac actgcagtct actattgtgc gagacatgtc 300tactatgata
gtagtgatta tttccccaac ccgtttgact actggggcca gggaaccctg 360gtcaccgtct
caagcgcctc caccaagggc ccatcggtct tcccg
405120135PRTHomo sapiens 120Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gln Met Asp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Arg Ile Val Pro Ser Gly Gly Asp Thr Thr 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 His Val Tyr Tyr Asp Ser Ser Asp Tyr Phe Pro Asn Pro Phe
100 105 110Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120
125Lys Gly Pro Ser Val Phe Pro 130
135121357DNAHomo sapiens 121ggcgtgcact ctgacatcca gatgacccag tctccatcct
ccctgtctgc gtctgtagga 60gacagagtca ccatcacttg ccgggcgagt cagggcatta
gcaattattt agcctggtat 120cagcagaaac cagggaaagt tcctaagctc ctgatctatc
ctgcatccac tttgcaaagt 180ggggtcccat caaggttcag cggcagtgga tctgggacag
atttcactct caccatcagc 240agcctgcagc ctgaagattt tgcaacttat tattgtcaac
aggctgacag tttcccgccc 300accttcggcg gagggaccac ggtggagatc agacgaactg
tggctgcacc atctgtc 357122119PRTHomo sapiens 122Gly Val His Ser Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser1 5
10 15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly 20 25
30Ile Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro
35 40 45Lys Leu Leu Ile Tyr Pro Ala Ser
Thr Leu Gln Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp 85
90 95Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Thr
Val Glu Ile Arg Arg 100 105
110Thr Val Ala Ala Pro Ser Val 115123399DNAHomo sapiens
123gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct ttttacttta tgttttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttcttat atcggtcctt ctggtggccc tactaattat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagacattac
300cccagggagt accagctgcc cgggtcgttc gacccctggg gccagggaac cctggtcacc
360gtctcaagcg cctccaccaa gggcccatcg gtcttcccg
399124133PRTHomo sapiens 124Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr
20 25 30Phe Met Phe Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Tyr Ile Gly Pro Ser Gly Gly Pro Thr Asn 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 His Tyr Pro Arg Glu Tyr Gln Leu Pro Gly Ser Phe Asp Pro
100 105 110Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120
125Pro Ser Val Phe Pro 130125360DNAHomo sapiens
125ggcgtgcact ctgacatcca gatgacccag tctccatctt ccctgtctgc atctgtagga
60gacagagtca ccatcacttg ccgggcaagt cagagtatta gtaactattt aaattggtat
120cagcagagac cagggaaggc ccctaagctc ctgatctatg ctgcatccag tttggaaaga
180ggggtcccat caaggttcag tggcagtgga tctgggacag atttcactct caccatcagc
240agtctgcaat ctgaagattt tgcaacttac tactgtcaac agagttacag tccccctcct
300ctcactttcg gcggagggac caaactagag atcaaacgaa ctgtggctgc accatctgtc
360126120PRTHomo sapiens 126Gly Val His Ser Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser1 5 10
15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
20 25 30Ile Ser Asn Tyr Leu Asn Trp
Tyr Gln Gln Arg Pro Gly Lys Ala Pro 35 40
45Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Arg Gly Val Pro
Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70
75 80Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr 85 90
95Ser Pro Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110Arg Thr Val Ala Ala Pro
Ser Val 115 120127393DNAHomo sapiens 127gaagttcaat
tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg
cttccggatt cactttctct tattacgtta tgatgtgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttctgtt atccgtcctt ctggtggcat tactacttat 180gctgactccg
ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagacga
acagcttaag ggctgaggac actgcagtct actattgtgc gaaaatcgac 300tacggtggta
actcgttcta ctttgactac tggggccagg gaaccctggt caccgtctca 360agcgcctcca
ccaagggccc atcggtcttc ccg
393128131PRTHomo sapiens 128Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr
20 25 30Val Met Met Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Val Ile Arg Pro Ser Gly Gly Ile Thr Thr 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 Thr Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Lys Ile Asp Tyr Gly Gly Asn Ser Phe Tyr Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro 130129357DNAHomo sapiens 129ggcgtgcact
ctgacatcca gatgacccag tctccaccct ccctgtctgc attagtaggg 60gacagagtca
ccatcacttg ccgggcaagt cagagcataa gcagatatgt gaattggtat 120cagcagaaac
cagggaaagc ccctaaggtc ctgatctatg ctgcatccat agtagaaaat 180ggggtcccat
ctaggttcag tggcagtgga tctgggacag atttcagtct caccatcagc 240agtctgcaac
ctgaagattt tgcaacttac tactgtcaac aaacttacag tactccgctc 300actttcggcg
gagggaccaa gctggcgatc aaacgaactg tggctgcacc atctgtc
357130119PRTHomo sapiens 130Gly Val His Ser Asp Ile Gln Met Thr Gln Ser
Pro Pro Ser Leu Ser1 5 10
15Ala Leu Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
20 25 30Ile Ser Arg Tyr Val Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40
45Lys Val Leu Ile Tyr Ala Ala Ser Ile Val Glu Asn Gly Val Pro
Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser65 70
75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Thr Tyr 85 90
95Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Ala Ile Lys Arg
100 105 110Thr Val Ala Ala Pro Ser
Val 115131426DNAHomo sapiens 131gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
tattacgaga tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttct
atctctcctt ctggtggccc tactatgtat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac
actggagtct actattgtgc gagaaagatg 300gggcgtgtag gatattgtag tagtaccagc
tgctatcggg atgactacta cggtatggac 360gtctggggcc aagggaccac ggtcaccgtc
tcaagcgcct ccaccaaggg cccatcggtc 420ttcccg
426132142PRTHomo sapiens 132Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Tyr Tyr 20 25
30Glu Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Ser Pro Ser Gly
Gly Pro Thr Met 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 Gly Val Tyr Tyr Cys 85
90 95Ala Arg Lys Met Gly Arg Val Gly Tyr Cys
Ser Ser Thr Ser Cys Tyr 100 105
110Arg Asp Asp Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
115 120 125Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 130 135
140133360DNAHomo sapiens 133ggcgtgcact ctgacatcca gatgacccag tctccaggca
ccctgtcttt gtctccaggg 60gaaagagcaa ccctctcctg cagggccagt cagagtgtta
gcagcaccta tttagcctgg 120taccagcaga aacctggcca ggctcccagg ctcctcatct
ctggtgcatc cagcagggcc 180actggcatcc cagacaggtt cagtggcagt gggtctggga
cagacttcac tctcaccatc 240agcagactgg agcctgaaga ttttgcagtg tattactgtc
agcagtatgg tagctcaccg 300tacacttttg gccaggggac caagctggag atcaaacgaa
ctgtggctgc accatctgtc 360134120PRTHomo sapiens 134Gly Val His Ser Asp
Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser1 5
10 15Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser 20 25
30Val Ser Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
35 40 45Pro Arg Leu Leu Ile Ser Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65
70 75 80Ser Arg Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 85
90 95Gly Ser Ser Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105
110Arg Thr Val Ala Ala Pro Ser Val 115
120135402DNAHomo sapiens 135gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct cagtacttta
tgaattgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttat atttctggtg
gccgtactcc ttatgctgac 180tccgttaaag gtcgcttcac tatctctaga gacaactcta
agaatactct ctacttgcag 240atgaacagct taagggctga ggacactgca gtctactatt
gtgcgatcct tctgggaccg 300agcagctcca atcacccttt cctggggccc tggggccagg
gaaccctggt caccgtctca 360agcgcctcca ccaagggccc atcggtcttc ccgctagcgc
cc 402136134PRTHomo sapiens 136Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Gln Tyr 20 25
30Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Tyr Ile Ser Gly Gly Arg Thr
Pro Tyr Ala Asp Ser Val Lys Gly 50 55
60Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln65
70 75 80Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ile 85
90 95Leu Leu Gly Pro Ser Ser Ser Asn His Pro Phe
Leu Gly Pro Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro
130137366DNAHomo sapiens 137ggcgtgcact cacagagcga attgactcag cctgcctccg
tgtctgggtc tcctggacag 60tcgatcacca tctcctgcac tggaaccagc agtgatgttg
ggagttataa ccttgtctcc 120tggtaccaac agcacccagg caaagccccc aaactcatga
tttatgaggg cagtaagcgg 180ccctcagggg tttctaatcg cttctctggc tccaagtctg
gcaacacggc ctccctgaca 240atctctgggc tccaggctga ggacgaggct gattattact
gctgctcata tgcaggtagt 300agcacttatg tcttcggaac tgggaccaag gtcaccgtcc
taggtcagcc caaggccaac 360cccact
366138122PRTHomo sapiens 138Gly Val His Ser Gln
Ser Glu Leu Thr Gln Pro Ala Ser Val Ser Gly1 5
10 15Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr
Gly Thr Ser Ser Asp 20 25
30Val Gly Ser Tyr Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys
35 40 45Ala Pro Lys Leu Met Ile Tyr Glu
Gly Ser Lys Arg Pro Ser Gly Val 50 55
60Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr65
70 75 80Ile Ser Gly Leu Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser 85
90 95Tyr Ala Gly Ser Ser Thr Tyr Val Phe Gly Thr
Gly Thr Lys Val Thr 100 105
110Val Leu Gly Gln Pro Lys Ala Asn Pro Thr 115
120139393DNAHomo sapiens 139gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct gtttacgtta
tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt atcgttcctt
ctggtggcaa gactcattat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagaccggac 300tacggtggta attcgcgccc ccttgagtac tggggccagg
gaaccctggt caccgtctca 360agcgcctcca ccaagggccc atcggtcttc ccg
393140131PRTHomo sapiens 140Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Val Tyr 20 25
30Val Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Val Pro Ser Gly Gly
Lys Thr His 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 Pro Asp Tyr Gly Gly Asn Ser Arg Pro
Leu Glu Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro
130141369DNAHomo sapiens 141ggcgtgcact cacagagcga attgactcag cctccctccg
cgtccgggtc tcctggacag 60tcagtcacca tctcctgcac tggaaccagc agtgacgttg
gtggttataa ctatgtctcc 120tggtatcaac aacacccaga caaagccccc aaactcctga
tttatgaggt cactcagcgg 180ccctcagggg tccctgatcg cttctctggc tccaggtctg
gcaacacggc ctccctgacc 240gtctctgggc tccaggctga ggatgaggct gattattact
gcagctcata tgcaggcagg 300aacaatcttt atgtcttcgg acctgggacc aaggtcaccg
tcctaggtca gcccaaggcc 360aaccccact
369142123PRTHomo sapiens 142Gly Val His Ser Gln
Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly1 5
10 15Ser Pro Gly Gln Ser Val Thr Ile Ser Cys Thr
Gly Thr Ser Ser Asp 20 25
30Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Asp Lys
35 40 45Ala Pro Lys Leu Leu Ile Tyr Glu
Val Thr Gln Arg Pro Ser Gly Val 50 55
60Pro Asp Arg Phe Ser Gly Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr65
70 75 80Val Ser Gly Leu Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser 85
90 95Tyr Ala Gly Arg Asn Asn Leu Tyr Val Phe Gly
Pro Gly Thr Lys Val 100 105
110Thr Val Leu Gly Gln Pro Lys Ala Asn Pro Thr 115
120143390DNAHomo sapiens 143gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct gagtacccta
tgtggtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttcttgg atctatcctt
ctggtggcaa tactgattat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gattccctat 300tgtagtagtt ccagctgccc cctacactgg ggccagggaa
ccctggtcac cgtctcaagc 360gcctccacca agggcccatc ggtcttcccg
390144130PRTHomo sapiens 144Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Glu Tyr 20 25
30Pro Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Trp Ile Tyr Pro Ser Gly Gly
Asn Thr Asp 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 Ile Pro Tyr Cys Ser Ser Ser Ser Cys Pro
Leu His Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro 130145354DNAHomo
sapiens 145ggcgtgcact cacagagcga attgactcag ccaccctcag tgtccgtgtc
cccagcacag 60acagccagca tcacctgctc tggagataaa ttgggggata aatatgcttg
ctggtatcag 120cagaagccag gccagtcccc tgtactggtc atctatgaag ataccaagcg
gccctcaggg 180atccctgagc gattctctgg ctccaattct gggaacacag ccactctgac
catcagcggg 240acccaggtta tggatgaggc tgactattac tgtcaggtgt gggacagcag
cactgcggta 300ttcggcggag ggaccaagct gaccgtcctg ggtcagccca aggctgcccc
ctcg 354146118PRTHomo sapiens 146Gly Val His Ser Gln Ser Glu Leu
Thr Gln Pro Pro Ser Val Ser Val1 5 10
15Ser Pro Ala Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys
Leu Gly 20 25 30Asp Lys Tyr
Ala Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val 35
40 45Leu Val Ile Tyr Glu Asp Thr Lys Arg Pro Ser
Gly Ile Pro Glu Arg 50 55 60Phe Ser
Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly65
70 75 80Thr Gln Val Met Asp Glu Ala
Asp Tyr Tyr Cys Gln Val Trp Asp Ser 85 90
95Ser Thr Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gln 100 105 110Pro Lys
Ala Ala Pro Ser 115147396DNAHomo sapiens 147gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct gcttacaata tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg
ggtttctcgt atctatcctt ctggtggcta tactctttat 180gctgactcgg ttaaaggtcg
cttcactatc tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag
ggctgaggac actgcagtct actattgtgc gagacaaaaa 300cttatgattc gggcagttcg
cccgtttgac tactggggcc agggaaccct ggtcaccgtc 360tcaagcgcct ccaccaaggg
cccatcggtc ttcccg 396148132PRTHomo sapiens
148Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25
30Asn Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Arg Ile
Tyr Pro Ser Gly Gly Tyr Thr Leu 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 Gln Lys Leu Met
Ile Arg Ala Val Arg Pro Phe Asp Tyr Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro 115 120 125Ser Val
Phe Pro 130149366DNAHomo sapiens 149ggcgtgcact cacagagcga attgactcag
ccaccgtcag cgtccgggac ccccgggcag 60aggatcacca tctcttgttc tggaagcagc
tccaacatcg gaagtaatta tgtatactgg 120taccaacagt tcccagagac ggcccccaaa
ctcctcatct ctagaaatga tcagcggccc 180tcaggggtcc ctgaccgatt ctctggctcc
aagtctggca cctcagcctc cctggccatc 240agtgggctcc ggtccgaaga tgaggctgat
tattactgtg catcatggga tgacagcctg 300agtggtgtgg ttttcggcgg agggaccaag
ctgaccgtcc taggtcagcc caaggctgcc 360ccctcg
366150122PRTHomo sapiens 150Gly Val His
Ser Gln Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly1 5
10 15Thr Pro Gly Gln Arg Ile Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn 20 25
30Ile Gly Ser Asn Tyr Val Tyr Trp Tyr Gln Gln Phe Pro Glu Thr Ala
35 40 45Pro Lys Leu Leu Ile Ser Arg
Asn Asp Gln Arg Pro Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile65
70 75 80Ser Gly Leu Arg
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp 85
90 95Asp Asp Ser Leu Ser Gly Val Val Phe Gly
Gly Gly Thr Lys Leu Thr 100 105
110Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 115
120151396DNAHomo sapiens 151gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct atgtacctta
tgttttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctgtt atctcttctt
ctggtggcga gacttcttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagacaggtc 300agtgactgga cgcgcctcta ctcctttgac tactggggcc
agggaaccct ggtcaccgtc 360tcaagcgcct ccaccaaggg cccatcggtc ttcccg
396152132PRTHomo sapiens 152Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Met Tyr 20 25
30Leu Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Val Ile Ser Ser Ser Gly Gly
Glu Thr Ser 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 Gln Val Ser Asp Trp Thr Arg Leu Tyr
Ser Phe Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro
130153363DNAHomo sapiens 153ggcgtgcact ctgacatcca gatgacccag tctccatcct
tcctgtctgc atctgtagga 60gacagagtca ccatcacttg ccgggccagt cagggcatta
gcacttattt agcctggtat 120cagcaaaaac cagggaaagc ccctaaggtc ctcatctata
ctgcatccac tttgcaaagt 180ggggtcccat caaggttcag cggcagtgga tctgggacag
aattcactct cacaatcagc 240agcctgcagc ctgaagattt tgcaacttac tactgtcaac
agagttacat tacccctccg 300gaggtcactt tcggccctgg gaccaaagtg gatatcaaac
gaactgtggc tgcaccatct 360gtc
363154121PRTHomo sapiens 154Gly Val His Ser Asp
Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser1 5
10 15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly 20 25
30Ile Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45Lys Val Leu Ile Tyr Thr Ala Ser
Thr Leu Gln Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr 85
90 95Ile Thr Pro Pro Glu Val Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile 100 105
110Lys Arg Thr Val Ala Ala Pro Ser Val 115
120155387DNAHomo sapiens 155gaagttcaat tgttagagtc tggtggcggt cttgttcagc
ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct tggtacgata
tggcttgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctcgt atcgttcctt
ctggtggcca tacttcttat 180gctgactccg ttaaaggtcg cttcactatc tctagagaca
actttaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac actgcagtct
actattgtgc gagaagggca 300agtcgtcctg agttttttga ctactggggc cagggagccc
tggtcaccgt ctcaagcgcc 360tccaccaagg gcccatcggt cttcccg
387156129PRTHomo sapiens 156Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Trp Tyr 20 25
30Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Val Pro Ser Gly Gly
His Thr Ser Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe 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 Arg Ala Ser Arg Pro Glu Phe Phe Asp
Tyr Trp Gly Gln Gly 100 105
110Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro157363DNAHomo sapiens
157ggcgtgcact ctgacatcca gatgacccag tctccatctg ccatgtctgc atctgtcgga
60gacagagtca ccatcacttg tcgggcgagt caggtcatga tcaattatat agcctggttt
120cggcagaaac cagggaaagt ccctgagcgc ctgatctatg cagcatccac tctgcaaaat
180ggggtcccat caaggttcag cggcagtggg tctgggacag acttcactct caccatcagc
240agactagaac ctgaggattt tgcagtttat tactgtcagc accgtatcac ctggcctccg
300gcgctcactt tcggcggagg gaccacggtg gagatcaaac gaactgtggc tgcaccatct
360gtc
363158121PRTHomo sapiens 158Gly Val His Ser Asp Ile Gln Met Thr Gln Ser
Pro Ser Ala Met Ser1 5 10
15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val
20 25 30Met Ile Asn Tyr Ile Ala Trp
Phe Arg Gln Lys Pro Gly Lys Val Pro 35 40
45Glu Arg Leu Ile Tyr Ala Ala Ser Thr Leu Gln Asn Gly Val Pro
Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70
75 80Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln His Arg Ile 85 90
95Thr Trp Pro Pro Ala Leu Thr Phe Gly Gly Gly Thr Thr Val Glu Ile
100 105 110Lys Arg Thr Val Ala Ala
Pro Ser Val 115 120159387DNAHomo sapiens
159gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct tggtaccgta tggattgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctgtt atcggttctt ctggtggcat gacttattat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagacgggta
300gtcgggggcg ccggtatgga cgtctggggc caagggacca cggtcaccgt ctcaagcgcc
360tccaccaagg gcccatcggt cttcccg
387160129PRTHomo sapiens 160Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr
20 25 30Arg Met Asp Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Val Ile Gly Ser Ser Gly Gly Met Thr 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 Arg Val Val Gly Gly Ala Gly Met Asp Val Trp Gly Gln Gly
100 105 110Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro161360DNAHomo sapiens 161ggcgtgcact ctgacatcca
gatgacccag tctccatcct ccctgtctgc atctgtggga 60gacagagtcg ccatcacttg
ccgcgcaagt cagagcatcg acacctattt aaattggtat 120cagcagaaac cagggaaagc
ccctaaactc ctgatctatg ctgcatccaa gttggaagac 180ggggtcccat caagattcag
tggcagtgga actgggacag atttcactct caccatcaga 240agtctgcaac ctgaagattt
tgcaagttat ttctgtcaac agagctactc tagtccaggg 300atcactttcg gccctgggac
caaggtggag atcaaacgaa ctgtggctgc accatctgtc 360162120PRTHomo sapiens
162Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser1
5 10 15Ala Ser Val Gly Asp Arg
Val Ala Ile Thr Cys Arg Ala Ser Gln Ser 20 25
30Ile Asp Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45Lys Leu Leu
Ile Tyr Ala Ala Ser Lys Leu Glu Asp Gly Val Pro Ser 50
55 60Arg Phe Ser Gly Ser Gly Thr Gly Thr Asp Phe Thr
Leu Thr Ile Arg65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Ser Tyr
85 90 95Ser Ser Pro Gly Ile Thr
Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100
105 110Arg Thr Val Ala Ala Pro Ser Val 115
120163381DNAHomo sapiens 163gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
gattaccaga tgatgtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctcgt
atctctcctt ctggtggcat gactcgttat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgccgatga acagcttaag ggctgaggac
actgcagtct actattgtgc gagatcgggg 300ccgtactact ttgactactg gggccaggga
accctggtca ccgtctcaag cgcctccacc 360aagggcccat cggtcttccc g
381164127PRTHomo sapiens 164Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25
30Gln Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Ser Pro Ser Gly
Gly Met Thr Arg 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 Pro Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Ser Gly Pro Tyr Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125165363DNAHomo sapiens
165ggcgtgcact cacagagcgt cttgactcag cctgactccg tgtctgggtc tcctgggcag
60tcgatcacca tctcctgcac tggcagcagt catgacattg gttcctatga ctatgtctcc
120tggtatcagc accacccagg gaaagccccc aaattcatac tttatgatgt ctataatcgg
180ccctcaggtg tttctgatcg cttctctggc tccaagtctg gcaacacggc ctccctgact
240atctctgggc tccagcctga cgacgaggct gactattttt gtatgtccta tacaatcaca
300acgcttctct tcggaactgg gaccagggtc accgtcctga gtcagcccaa ggccaacccc
360act
363166121PRTHomo sapiens 166Gly Val His Ser Gln Ser Val Leu Thr Gln Pro
Asp Ser Val Ser Gly1 5 10
15Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Ser Ser His Asp
20 25 30Ile Gly Ser Tyr Asp Tyr Val
Ser Trp Tyr Gln His His Pro Gly Lys 35 40
45Ala Pro Lys Phe Ile Leu Tyr Asp Val Tyr Asn Arg Pro Ser Gly
Val 50 55 60Ser Asp Arg Phe Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr65 70
75 80Ile Ser Gly Leu Gln Pro Asp Asp Glu Ala Asp
Tyr Phe Cys Met Ser 85 90
95Tyr Thr Ile Thr Thr Leu Leu Phe Gly Thr Gly Thr Arg Val Thr Val
100 105 110Leu Ser Gln Pro Lys Ala
Asn Pro Thr 115 120167375DNAHomo sapiens
167gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct cattacaata tggcttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctcgt atccgttctt ctggtggcct tactgtttat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagagtggct
300ggccctgggt actggggcca gggaaccctg gtcaccgtct caagcgcctc caccaagggc
360ccatcggtct tcccg
375168125PRTHomo sapiens 168Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr
20 25 30Asn Met Ala Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Arg Ile Arg Ser Ser Gly Gly Leu Thr Val 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 Val Ala Gly Pro Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125169357DNAHomo sapiens 169ggcgtgcact ctgacatcca gatgacccag tctccatcct
ccctgtctgc atctgtagga 60gacagagtca ctatcacttg ccggacaagt caaatcatta
acacctattt aaattggtat 120caacaaaaac cgggaaaagc ccctaaactc ctgatctatg
ctgcctccac tttacagggt 180ggggtcccgt caagattcag tggcagtgga tccgggacag
acttcactct caccatcaag 240agtctgcaac ctgacgactt tgcaacttac tattgtcaac
agagttatac ttccccgcga 300acattcggcc aagggaccaa ggtggaaatc aaacgaactg
tggctgcacc atctgtc 357170119PRTHomo sapiens 170Gly Val His Ser Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser1 5
10 15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Thr Ser Gln Ile 20 25
30Ile Asn Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45Lys Leu Leu Ile Tyr Ala Ala Ser
Thr Leu Gln Gly Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Lys65
70 75 80Ser Leu Gln Pro Asp
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr 85
90 95Thr Ser Pro Arg Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val 115171402DNAHomo sapiens
171gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct ggttacatta tggagtgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttctgtt atcgtttctt ctggtggctt tactatgtat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagagttggg
300gattccaagg gcgggtacta ccttgactac tggggccagg gaaccctggt caccgtctca
360agcgcctcca ccaagggccc atcggtcttc ccgctagcgc cc
402172134PRTHomo sapiens 172Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr
20 25 30Ile Met Glu Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Val Ile Val Ser Ser Gly Gly Phe Thr Met 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 Val Gly Asp Ser Lys Gly Gly Tyr Tyr Leu Asp Tyr Trp Gly
100 105 110Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro 130173366DNAHomo sapiens
173ggcgtgcact cacagagcga attgactcag cctgcctccg tgtctgggtc tcctggacag
60tcgatcacca tctcctgcac tggaaccaac agtgacattg gtggttataa ttatgtctcc
120tggtaccaac aacacccggg caaagtcccc aaactcttga tttttgaggt caataatcgg
180ccctcagggg tttctagtcg cttctctggc tccaagtctg gcgacacggc ctccctgacc
240atctctgggc tccaacctga ggacgaggct gtttattact gcggctcatt tacagtcagc
300gtcacctatg tcttcggaac tgggaccaag gtcaccgtcc tgggtcagcc caaggccaac
360cccact
366174122PRTHomo sapiens 174Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro
Ala Ser Val Ser Gly1 5 10
15Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Asn Ser Asp
20 25 30Ile Gly Gly Tyr Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys 35 40
45Val Pro Lys Leu Leu Ile Phe Glu Val Asn Asn Arg Pro Ser Gly
Val 50 55 60Ser Ser Arg Phe Ser Gly
Ser Lys Ser Gly Asp Thr Ala Ser Leu Thr65 70
75 80Ile Ser Gly Leu Gln Pro Glu Asp Glu Ala Val
Tyr Tyr Cys Gly Ser 85 90
95Phe Thr Val Ser Val Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr
100 105 110Val Leu Gly Gln Pro Lys
Ala Asn Pro Thr 115 120175398DNAHomo sapiens
175gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt
60tcttgcgctg cttccggatt cactttctct gagtacaata tgttttgggt tcgccaagct
120cctggtaaag gtttggagtg ggtttcttat atctattctt ctggtggctc tactgattat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac
240ttgcagatga acagcttaag ggctgaggac actgcagtct actattgtgc gagagtaggt
300atagcagctc gtccgttcga cccctggggc cagggaaccc tggtcaccgt ctcaagcgcc
360tccaccaagg gcccatcggt cttcccgcta gcgccctg
398176132PRTHomo sapiens 176Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr
20 25 30Asn Met Phe Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Tyr Ile Tyr Ser Ser Gly Gly Ser Thr Asp 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 Val Gly Ile Ala Ala Arg Pro Phe Asp Pro Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro 130177357DNAHomo sapiens 177ggcgtgcact
ctgacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga 60gacagagtca
ccatcacttg ccgggcaagt cagagcatta gcgactattt aaattggtat 120cagcagaaac
cagggaaagc ccctgacctc ctgatctatg ctgcatccag tttgcaaagt 180ggggtcccat
caaggttcag tggcagtgga tctgggacag atttcactct caccgtcagc 240agtctgcaac
ctgaagattt tgcaacttac ttctgtcaac agagttactc tattcctctc 300actttcggcg
gcgggaccaa ggttgagatc actcgaactg tggctgcacc atctgtc
357178119PRTHomo sapiens 178Gly Val His Ser Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser1 5 10
15Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
20 25 30Ile Ser Asp Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40
45Asp Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Val Ser65 70
75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe
Cys Gln Gln Ser Tyr 85 90
95Ser Ile Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Thr Arg
100 105 110Thr Val Ala Ala Pro Ser
Val 115179387DNAHomo sapiens 179gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
ttttacgcta tgtggtgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctcgt
atctattctt ctggtggcaa gacttggtat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac
actgcagtct actattgtgc gagagtgggg 300atgtccacct atgcttttga tatctggggc
caagggacaa tggtcaccgt ctcaagcgcc 360tccaccaagg gcccatcggt cttcccg
387180129PRTHomo sapiens 180Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Phe Tyr 20 25
30Ala Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Tyr Ser Ser Gly
Gly Lys Thr Trp 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 Val Gly Met Ser Thr Tyr Ala Phe
Asp Ile Trp Gly Gln Gly 100 105
110Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro181354DNAHomo sapiens
181ggcgtgcact cacagagcga attgactcag ccaccctcag tgtccgtgtc cccaggacag
60acagccagca tcacctgctc tggagataaa ttgggggata aatatgcttg ctggtatcag
120cagaagccag gccagtcccc tgtgctggtc atctatcaag atagcaagcg gccctcaggg
180atccctgagc gattctctgg ctccaactct gggaacacag ccactctgac catcagcggg
240acccaggcta tggatgaggc tgactattac tgtcaggcgt gggacagcag cgctgtggta
300ttcggcggag ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcg
354182118PRTHomo sapiens 182Gly Val His Ser Gln Ser Glu Leu Thr Gln Pro
Pro Ser Val Ser Val1 5 10
15Ser Pro Gly Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly
20 25 30Asp Lys Tyr Ala Cys Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Val 35 40
45Leu Val Ile Tyr Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu
Arg 50 55 60Phe Ser Gly Ser Asn Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly65 70
75 80Thr Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys
Gln Ala Trp Asp Ser 85 90
95Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110Pro Lys Ala Ala Pro Ser
115183399DNAHomo sapiens 183gaagttcaat tgttagagtc tggtggcggt
cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt cactttctct
cattacaata tgcattgggt tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt
atcgtttctt ctggtggcaa tactggttat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag ggctgaggac
actgcagtcg actattgtgc gagagtggta 300cggtatagca gtggctggta ctactggttc
gacccctggg gccagggaac cctggtcacc 360gtctcaagcg cctccaccaa gggcccatcg
gtcttcccg 399184133PRTHomo sapiens 184Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser His Tyr 20 25
30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Gly Ile Val Ser
Ser Gly Gly Asn Thr Gly 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 Asp Tyr Cys
85 90 95Ala Arg Val Val Arg Tyr Ser
Ser Gly Trp Tyr Tyr Trp Phe Asp Pro 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 115 120 125Pro Ser Val Phe
Pro 1301854PRTHomo sapiens 185Ala Ser Asp Phe11864PRTHomo sapiens
186Ala Ser Asp Phe11874PRTHomo sapiens 187Ala Ser Asp Phe11884PRTHomo
sapiens 188Ala Ser Asp Phe11894PRTHomo sapiens 189Ala Ser Asp
Phe11904PRTHomo sapiens 190Ala Ser Asp Phe11914PRTHomo sapiens 191Ala Ser
Asp Phe11924PRTHomo sapiens 192Ala Ser Asp Phe11934PRTHomo sapiens 193Ala
Ser Asp Phe11944PRTHomo sapiens 194Ala Ser Asp Phe11954PRTHomo sapiens
195Ala Ser Asp Phe11964PRTHomo sapiens 196Ala Ser Asp Phe11974PRTHomo
sapiens 197Ala Ser Asp Phe11984PRTHomo sapiens 198Ala Ser Asp
Phe11994PRTHomo sapiens 199Ala Ser Asp Phe12004PRTHomo sapiens 200Ala Ser
Asp Phe12014PRTHomo sapiens 201Ala Ser Asp Phe12024PRTHomo sapiens 202Ala
Ser Asp Phe12034PRTHomo sapiens 203Ala Ser Asp Phe12044PRTHomo sapiens
204Ala Ser Asp Phe12054PRTHomo sapiens 205Ala Ser Asp Phe12064PRTHomo
sapiens 206Ala Ser Asp Phe12074PRTHomo sapiens 207Ala Ser Asp
Phe12084PRTHomo sapiens 208Ala Ser Asp Phe12094PRTHomo sapiens 209Ala Ser
Asp Phe12104PRTHomo sapiens 210Ala Ser Asp Phe12114PRTHomo sapiens 211Ala
Ser Asp Phe12124PRTHomo sapiens 212Ala Ser Asp Phe12134PRTHomo sapiens
213Ala Ser Asp Phe12144PRTHomo sapiens 214Ala Ser Asp Phe12154PRTHomo
sapiens 215Ala Ser Asp Phe12164PRTHomo sapiens 216Ala Ser Asp
Phe12174PRTHomo sapiens 217Ala Ser Asp Phe12184PRTHomo sapiens 218Ala Ser
Asp Phe12194PRTHomo sapiens 219Ala Ser Asp Phe12204PRTHomo sapiens 220Ala
Ser Asp Phe12214PRTHomo sapiens 221Ala Ser Asp Phe12224PRTHomo sapiens
222Ala Ser Asp Phe12234PRTHomo sapiens 223Ala Ser Asp Phe12244PRTHomo
sapiens 224Ala Ser Asp Phe12254PRTHomo sapiens 225Ala Ser Asp
Phe12264PRTHomo sapiens 226Ala Ser Asp Phe12274PRTHomo sapiens 227Ala Ser
Asp Phe12284PRTHomo sapiens 228Ala Ser Asp Phe12294PRTHomo sapiens 229Ala
Ser Asp Phe12304PRTHomo sapiens 230Ala Ser Asp Phe12314PRTHomo sapiens
231Ala Ser Asp Phe12324PRTHomo sapiens 232Ala Ser Asp Phe12334PRTHomo
sapiens 233Ala Ser Asp Phe12344PRTHomo sapiens 234Ala Ser Asp
Phe12354PRTHomo sapiens 235Ala Ser Asp Phe12364PRTHomo sapiens 236Ala Ser
Asp Phe12374PRTHomo sapiens 237Ala Ser Asp Phe12384PRTHomo sapiens 238Ala
Ser Asp Phe12394PRTHomo sapiens 239Ala Ser Asp Phe12404PRTHomo sapiens
240Ala Ser Asp Phe12414PRTHomo sapiens 241Ala Ser Asp Phe12424PRTHomo
sapiens 242Ala Ser Asp Phe12434PRTHomo sapiens 243Ala Ser Asp
Phe12444PRTHomo sapiens 244Ala Ser Asp Phe12454PRTHomo sapiens 245Ala Ser
Asp Phe12464PRTHomo sapiens 246Ala Ser Asp Phe12474PRTHomo sapiens 247Ala
Ser Asp Phe12484PRTHomo sapiens 248Ala Ser Asp Phe12494PRTHomo sapiens
249Ala Ser Asp Phe12504PRTHomo sapiens 250Ala Ser Asp Phe12514PRTHomo
sapiens 251Ala Ser Asp Phe12524PRTHomo sapiens 252Ala Ser Asp
Phe12534PRTHomo sapiens 253Ala Ser Asp Phe12544PRTHomo sapiens 254Ala Ser
Asp Phe12554PRTHomo sapiens 255Ala Ser Asp Phe12564PRTHomo sapiens 256Ala
Ser Asp Phe12574PRTHomo sapiens 257Ala Ser Asp Phe12584PRTHomo sapiens
258Ala Ser Asp Phe12594PRTHomo sapiens 259Ala Ser Asp Phe12604PRTHomo
sapiens 260Ala Ser Asp Phe12614PRTHomo sapiens 261Ala Ser Asp
Phe12624PRTHomo sapiens 262Ala Ser Asp Phe12634PRTHomo sapiens 263Ala Ser
Asp Phe12644PRTHomo sapiens 264Ala Ser Asp Phe12654PRTHomo sapiens 265Ala
Ser Asp Phe12664PRTHomo sapiens 266Ala Ser Asp Phe12674PRTHomo sapiens
267Ala Ser Asp Phe12684PRTHomo sapiens 268Ala Ser Asp Phe12694PRTHomo
sapiens 269Ala Ser Asp Phe127096PRTHomo sapiens 270Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65
70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85
90 9527196PRTHomo sapiens 271Ser Tyr Val Leu Thr
Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5
10 15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile
Gly Ser Lys Ser Val 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr
35 40 45Asp Asp Ser Asp Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65
70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85
90 9527296PRTHomo sapiens 272Gln 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 Asn 20 25
30Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45Ile Tyr Ser 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 Ser Gly Leu Arg65
70 75 80Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 9527395PRTHomo sapiens 273Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5
10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65
70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro 85
90 9527495PRTHomo sapiens 274Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Tyr 20 25 30Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro 85
90 9527596PRTHomo sapiens 275Gln Ser Ala Leu Thr Gln
Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1 5
10 15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30Asn
Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45Met Ile Tyr Glu Val Ser Lys Arg Pro
Ser Gly Val Pro Asp Arg Phe 50 55
60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu65
70 75 80Gln Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser 85
90 9527695PRTHomo sapiens 276Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro 85
90 9527796PRTHomo sapiens 277Asn Phe Met Leu Thr Gln
Pro His Ser Val Ser Glu Ser Pro Gly Lys1 5
10 15Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser
Ile Ala Ser Asn 20 25 30Tyr
Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val 35
40 45Ile Tyr Glu Asp Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe Ser 50 55
60Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly65
70 75 80Leu Lys Thr Glu Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85
90 9527895PRTHomo sapiens 278Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Ser Trp 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Asp Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser 85
90 9527996PRTHomo sapiens 279Gln Ser Ala Leu Thr Gln
Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln1 5
10 15Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30Asn
Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45Met Ile Tyr Glu Val Ser Lys Arg Pro
Ser Gly Val Pro Asp Arg Phe 50 55
60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu65
70 75 80Gln Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser 85
90 9528096PRTHomo sapiens 280Gln 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 Asn 20 25
30Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45Ile Tyr Ser 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 Ser Gly Leu Gln65
70 75 80Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 9528195PRTHomo sapiens 281Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Ser Asn Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr
Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro 85
90 9528296PRTHomo sapiens 282Gln 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 Asn 20 25 30Tyr
Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45Ile Tyr Ser 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 Ser Gly Leu Arg65
70 75 80Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 9528395PRTHomo sapiens 283Asp Ile Gln Leu Thr
Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro 85
90 9528495PRTHomo sapiens 284Asn Ile Gln Met Thr Gln
Ser Pro Ser Ala Met Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Arg Gln Gly
Ile Ser Asn Tyr 20 25 30Leu
Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys His Leu Ile 35
40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro 85
90 9528596PRTHomo sapiens 285Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5
10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30Asn
Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45Met Ile Tyr Glu Val Ser Asn Arg Pro
Ser Gly Val Ser Asn Arg Phe 50 55
60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80Gln Ala Glu Asp Glu
Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85
90 9528695PRTHomo sapiens 286Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5
10 15Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu
Gly Asp Lys Tyr Ala 20 25
30Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr
35 40 45Gln Asp Ser Lys Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65
70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala 85
90 9528796PRTHomo sapiens 287Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser
Thr 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 9528817PRTHomo sapiens 288Ala Glu Tyr Phe Gln
His Trp Gly Gln Gly Thr Leu Val Thr Val Ser1 5
10 15Ser28917PRTHomo sapiens 289Tyr Trp Tyr Phe Asp
Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser1 5
10 15Ser29015PRTHomo sapiens 290Ala Phe Asp Ile Trp
Gly Gln Gly Thr Met Val Thr Val Ser Ser1 5
10 1529115PRTHomo sapiens 291Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser1 5 10
1529216PRTHomo sapiens 292Asn Trp Phe Asp Pro Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser1 5 10
1529320PRTHomo sapiens 293Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val1 5 10
15Thr Val Ser Ser 202944PRTHomo sapiens
294Ala Ser Asp Phe1295120PRTHomo sapiens 295Gly Val His Ser Gln Ser Glu
Leu Thr Gln Pro Pro Ser Val Ser Leu1 5 10
15Ala Pro Gly Gln Thr Ala Arg Ile Thr Cys Gly Gly Asn
Asn Ile Gly 20 25 30Thr Lys
Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val 35
40 45Leu Val Val Tyr Asp Asp Ser Asp Arg Pro
Ser Gly Ile Pro Glu Arg 50 55 60Phe
Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg65
70 75 80Val Glu Ala Gly Asp Glu
Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser 85
90 95Gly Ser Asp His Gln Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110Gly
Gln Pro Lys Ala Ala Pro Ser 115 12029696PRTHomo
sapiens 296Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20
25 30Phe Met Phe Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Gly Ser Ser Gly Gly Asp Thr Ser 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
952974PRTHomo sapiens 297Gly Leu Tyr Arg129896PRTHomo sapiens 298Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Pro Tyr 20 25
30Phe Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ser Ile Gly Ser
Ser Gly Gly Asp Thr Ser 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 95299120PRTHomo sapiens 299Gly
Val His Ser Gln Ser Glu Leu Thr Gln Pro Pro Ser Val Ser Leu1
5 10 15Ala Pro Gly Gln Thr Ala Arg
Ile Thr Cys Gly Gly Asn Asn Ile Gly 20 25
30Thr Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Val 35 40 45Leu Val Val Tyr
Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg 50 55
60Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Arg65 70 75
80Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser
85 90 95Gly Ser Asp His Gln Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110Gly Gln Pro Lys Ala Ala Pro Ser 115
120
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