US20100291001A1

US20100291001A1 - Metalloproteinase-binding proteins

Info

Publication number: US20100291001A1
Application number: US12/339,487
Authority: US
Inventors: Daniel T. Dransfield; Kristin Rookey; Robert C. Ladner
Original assignee: Dyax Corp
Current assignee: Dyax Corp
Priority date: 2003-11-19
Filing date: 2008-12-19
Publication date: 2010-11-18
Also published as: WO2005051299A2; WO2005051299A3; US7514534B2; US20060036076A1

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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

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

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.
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.
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.
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.
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 K_Dof less than 1×10⁻⁷M, 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 includes at least one human CDR or at least one human framework.
In one embodiment, the protein inhibits MMP-26 proteolytic activity.
In one embodiment, the heavy chain variable domain sequence includes
(a) a CDR1 that includes

- 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),
- (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);

(b) a CDR2 that includes

- 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

(c) a CDR3 that includes F-D-I.
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.
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.
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).
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.
In one embodiment, the light chain variable domain sequence is a κ light chain and includes
(a) a CDR1 that includes

(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,;

(b) a CDR2 that includes

(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

(c) a CDR3 that includes

	(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,.

In one embodiment, the light chain variable domain sequence is a X light chain and includes
(a) a CDR1 that includes

(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,;

(b) a CDR2 that includes
(SEQ ID NO: 240)

(RDNE)-(VDN)-(GSTDN)-(KDNEQ)-R-P-S,,

or

(SEQ ID NO: 241)

(RE)-(VDN)-(TDN)-(KQ)-R-P-S,;

andor
(c) a CDR3 that includes

(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,.

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.
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.
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).
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.
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).
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.
In one embodiment, CDR3 of the heavy chain variable domain sequence further includes a dityrosine sequence.
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).
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).
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).
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).
The protein can also bind to MMP-26, e.g., and not substantially inhibit its enzymatic activity.
In one embodiment, the heavy chain variable domain sequence includes
(a) a CDR1 that includes
P-Y-F-M-F,, (SEQ ID NO: 202)

or

(ALVEP)-Y-(SMWFD)-M-(YFKDNP),; (SEQ ID NO: 255)

and/or
(b) a CDR2 that includes

(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,.

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.
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).
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.
In one embodiment, the light chain variable domain sequence is a κ light chain and includes
(a) a CDR1 that includes

(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,

(b) a CDR2 that includes

(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),.

(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),;

(b) a CDR2 that includes

(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,;

- and/or

(c) a CDR3 that includes

(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,.

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.
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).
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.
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.
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).
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).
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.
In one embodiment, the protein binds MMP-26 with a K_Dthat is at least 2, 4, 5, 10, 20, 50, or 100 better than its K_Dfor 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 K_Dthat is at least 2, 4, 5, 10, 20, 50, or 100 better than its K_Dfor MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, or MMP-14.
In one embodiment, the protein inhibits MMP-26 with a K_iof less than 1×10⁻⁷M, 3×10⁻⁸, 1×10⁻⁸, 3×10⁻⁹, 1×10⁻⁹, 3×10¹⁰, 1×10¹⁰, 3×10¹¹, or 1×10⁻¹²M. In an embodiment, the protein inhibits MMP-26 with a K_ithat is at least 2, 4, 5, 10, 20, 50, or 100 better than its K_ifor another metalloproteinase, e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, or MMP-14.
In one embodiment, the protein reduces cell metastasis in vivo.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The protein can include additional features described herein.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 K_dof less than 1×10⁻⁷M, 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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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 K_dof less than 1×10⁻⁷M, 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. The antibody can be an antibody described herein.
The method can further include, prior to, during, or after the administering, evaluating cells of the subject for MMP-26 protein, mRNA, or activity.
The method can further include monitoring the subject for a metastatis.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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′)₂fragment, 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.
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.
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.
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, 10⁻⁷or 10⁻⁸M 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.
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]).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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

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):

(SEQ ID NO: 213)

MQLVILRVTIFLPWCFAVPVPPAADHKGWDFVEGYFHQFFLTKKESPLLT

QETQTQLLQQFHRNGTDLLDMQMHALLHQPHCGVPDGSDTSISPGRCKWN

KHTLTYRIINYPHDMKPSAVKDSIYNAVSIWSNVTPLIFQQVQNGDADIK

VSFWQWAHEDGWPFDGPGGILGHAFLPNSGNPGVVHFDKNEHWSASDTGY

NLFLVATHEIGHSLGLQHSGNQSSIMYPTYWYHDPRTFQLSADDIQRIQH

LYGEKCSSDIP

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:

(SEQ ID NO: 214)

TSISPGRCKWNKHTLTYRIINYPHDMKPSAVKDSIYNAVSIWSNVTPLIF

QQVQNGDADIKVSFWQWAHEDGWPFDGPGGILGHAFLPNSGNPGVVHFDK

NEHWSASDTGYNLFLVATHEIGHSLGLQHSGNQSSIMYPTYWYHDPRTFQ

LSADDIQRIQHLYGEKCSSDIP

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.
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).
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

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.
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.
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

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”).
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.
A variety of formats can be used for display libraries. Examples include the following.
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.
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).
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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 10¹⁰members. “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.
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.
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:

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.
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.
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.
For the cell ELISAs, cells are washed once in PBS and resuspended at a concentration of 1×10⁶to 2×10⁶cells/mL of PBS. A final concentration of 1-2×10⁵cells 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

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.
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.
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.
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.
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

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.
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.
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.
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.
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.
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.
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 naïve.
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.
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.
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 naïve.
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.
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).
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.
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.
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

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.
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 K_ican 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.
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.
In one embodiment, a protein (e.g., an antibody described herein) has a K_iof less than 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, or 10⁻¹¹M in an assay described herein.

Exemplary Biological Assays

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).
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.
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.
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.
In one embodiment, a protein (e.g., an antibody described herein) has a statistically significant effect in an assay described herein.

Secondary Screening Methods

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.
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.
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.
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.
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.
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.
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.
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).
Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (K_d), and kinetic parameters, including K_onand K_off, 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 K_off. 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 K_off. 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.
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).
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

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, 3^rdEdition, 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.
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.
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, 3^rdEdition, 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.
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.
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.
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.
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.
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.
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).
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).
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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

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.
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.
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.
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.
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.
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.
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.
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/m²or 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.
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.
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.
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).
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.
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/m²or 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.
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.
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.
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

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.
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.
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.
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); C_1-4alkyl-terminated polymers; and bis-activated polyethylene oxides (glycols) can be used for crosslinking. See, e.g., U.S. Pat. No. 5,951,974
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.
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.
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.
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).
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.
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

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.
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).
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
The term “sarcoma” is recognized by those skilled in the art and refers to malignant tumors of mesenchymal derivation.
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.
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.
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.
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.
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′-(trifluoromethyl)propionanilide.
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).
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.
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.
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.
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.
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.
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, Lu¹¹⁷may also be used as both an imaging and cytotoxic agent.
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).
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.
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.
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.
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.
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.
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.
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

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).
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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
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.
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

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.
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.
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.
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.
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.
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.
The following invention is further illustrated by the following examples, which should not be construed as limiting.

EXAMPLES

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

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:

Mat2.cat.top.ncoI

(SEQ ID NO: 218)

5′-GCCATCCATGGCGACCTCCATCTCGCCAGG

Mat2.bot.HindIII:

((SEQ ID NO: 219)

5′-GCAGCAAGCTTCATCATCATCATCATCATAGGTATGTCAGATGAACA

TTTTTCTCC

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

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

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 3×10¹¹pfu. 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.
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

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
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:

(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′,.

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

Reformatted MMP-26 antibodies were expressed transiently in HEK293T (GenHunter) cells using Lipofectamine 2000 (Invitrogen). Briefly, 6×10⁶cells 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

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 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

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 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

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 4

JEG-3 cell invasion through MATRIGEL ™
basement membrane matrix in vitro

% 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

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.
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.
The following Table provides inhibition data with the A1-orig antibody at various concentrations using a fluorescently labeled substrate:

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

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 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

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 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)

At least some members of Group I HC can include the following sequence in the region of CDR1:
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, X₁could be any of the amino-acid types given; “(AWFNQ)” means that X₃could 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.
At least some members of Group I HC can include one of the following sequences in the region of CDR2:

(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.

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 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)

At least some members of Group I LC κ sequences can include one of the following sequences in the region of CDR1:

(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,

At least some members of Group I LC κ sequences can include one of the following sequences in the region of CDR2:

(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).

At least some members of Group I LC κ sequences can include one of the following sequences in the region of CDR3:

(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.

At least some members of Group I LC λ sequences can include one of the following sequences in the region of CDR1:

(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

At least some members of Group I LC λ sequences can include one of the following sequences in the region of CDR2:

(SEQ ID NO: 240)

	(RDNE)-(VDN)-(GSTDN)-(KDNEQ)-R-P-S,
	or

(SEQ ID NO: 241)

(RE)-(VDN)-(TDN)-(KQ)-R-P-S.

At least some members of Group I LC λ sequences can include one of the following sequences in the region of CDR3:

(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 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

At least some members of Group II HC sequences can include one of the following sequences in the region of CDR1:

	P-Y-F-M-F,	(SEQ ID NO: 202)
	or

	(ALVEP)-Y-(SMWFD)-M-(YFKDNP).	(SEQ ID NO: 243)

At least some members of Group II HC sequences can include one of the following sequences in the region of CDR2:

(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 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

At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR1:

(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,.

At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR2:

(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).

At least some members of Group II LC κ sequences can include one of the following sequences in the region of CDR3:

(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.

(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).

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)

Group II LC (e.g., κ or λ) can include one or more of the following sequences in the region of CDR2:

(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)

At least some members of Group II LC λ sequences can include one of the following sequences in the region of CDR3:
(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.
Fab's from Group I can be classified as described below:
Set 1: a04(lambda) and b04(kappa).
Set 2 includes a11, c05, A1-orig, d02, and d04.
Set 3 includes b10, c01, c08, d04, and d06.
Set 4 includes c11 and d07
Some features that may be present in inhibitors include:
(SEQ ID NO: 294)

Cons: I-G-P-S-G-G-I-T-X-Y-A-D-S-V-K-G
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.
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.
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.
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

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.
The germline-encoded amino acid sequences referred to below are provided as:


	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

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:

(SEQ ID NO: 295)

	GVHSQSELTQ PPSVSLAPGQ TARITCGGNN IGTKSVHWYQ

	QKPGQAPVLV VYDDSDRPSG IPERFSGSNS GNTATLTISR

	VEAGDEADYY CQVWDSGSDH QVFGGGTKLT VLGQPKAAPS

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).


	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.


	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)

Mutation Location

E1D, V3Q, L4M, A9S, T10S FR1

CDR1

FR2

CDR2

FR3

CDR3

L96R FR4

Name: A06 is related to O2 (VKI)
Mutation Location

FR1

CDR1

FR2

CDR2

FR3

CDR3

Y96N FR4

Name: A07 is related to V1-2 (VL1).
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).


	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).


	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).

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).
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)
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.


	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.

Mutation Location

E1D FR1

V3Q

L4M

L33F CDR1

FR2

G50D CDR2

FR3

CDR3

FR4

Name: B10 is related to O2.


	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.

Mutation Location

1SQ FR1

Y2S

V3E

V13L

S31aT CDR1

FR2

CDR2

FR3

CDR3

S94G

P95cQ

V96- FR4

Name: B12 is related to V2-14.


	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.


	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.


	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.

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.
Mutation Location

E1D FR1

V3Q

L4M

CDR1

FR2

CDR2

FR3

.95aE CDR3

FR4

Name: B06 is related to L8.
Mutation Location

L4M FR1

CDR1

FR2

CDR2

E70D FR3

Q79E

Q90E CDR3

L91S

N92Y

Y94T

.95aF

K107R FR4

Name: B07 is related to L6.
Mutation Location

E1D FR1

V3Q

L4M

CDR1

FR2

CDR2

FR3

CDR3

L96. FR4

Name: B08 is related to V1-22.
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:

(SEQ ID NO: 299)

Mutation Location

S1Q FR1

Y2S

V3E

V13L

S31aT CDR1

FR2

CDR2

FR3

S94G CDR3

P95cQ

V96. FR4

Name: C01 is related to L12.
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.
Mutation Location

A3E FR1

CDR1

FR2

CDR2

FR3

F95b. CDR3

FR4

Name: C11 is related to V1-2.
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.
Mutation Location

S1Q FR1

Y2S

G16A

CDR1

FR2

Q50E CDR2

S52T

A80V FR3

A90V CDR3

V96. FR4

Name: C03 is related to V1-16.
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.


	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.

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.
Mutation Location

FR1

CDR1

FR2

A50P CDR2

V83F FR3

K90Q CDR3

Y91A

N92D

A94F

.95aP

L96. FR4

K107R

Name: C07 is related to O2.
Mutation Location

FR1

S31N CDR1

K39R FR2

Q55E CDR2

S56R

P80S FR3

T94P CDR3

.95aP

FR4

Name: C08 is related to O2.
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.
Mutation Location

E1D FR1

V3Q

L4M

S31aT CDR1

Y49S FR2

CDR2

FR3

CDR3

FR4

Name: D01 is related to V1-17.
Mutation Location

V3E FR1

V19I

CDR1

L39F FR2

G41E

Y49S

S50R CDR2

N52D

FR3

A90S CDR3

P95a.

FR4

Name: D02 is related to L8.
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.


	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.


	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.


	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.

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.


	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.


	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.

Mutation Location

FR1

CDR1

FR2

CDR2

FR3

CDR3

FR4

Name: D08 is related to O2.
Mutation Location

FR1

S31D CDR1

K45D FR2

CDR2

Y87F FR3

T94I CDR3

K107T FR4

Name: D09 is related to V2-1.
Mutation Location

S1Q FR1

Y2S

CDR1

FR2

CDR2

FR3

T95A CDR3

A95a.

FR4

Name: H6-O is related to V1-2.


	Mutation	Location

	A3E	FR1
	G29A	CDR1
	M47I	FR2
	S51aN	CDR2
	G64A	FR3
		CDR3
	N97S, F98L	FR4

Example

This example includes an analysis of HC germline differences In each example the isolate name is written under the GLG.
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:


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)

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.
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

The A02 heavy chain variable domain can include:
(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

JH is 4

Isolate: a04

S31E, A33N, S35A, A50R, S51aG, G52S, S56K, Y58K

JH is 3

Isolate: a05

S31V, A33S, S35N, A50Y, S51aV, G52P, S56N, Y58P

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

JH is 3

Isolate: a08

S31V, A33D, S35P, A50V, S51aY, G52P, S56F, Y58R

JH is 6

Isolate: a09

S31W, A33D, S35Y, A50S, S51aY, G52S, S56Y, Y58A

JH is 5

Isolate: a10

S31A, A33R, S35F, A50S, S51aW, G52P, S56T, Y58S

JH is 4

Isolate: a11

S31W, A33T, S35M, A50R, G52P, S56H, Y58L, K75z

JH is 4

Isolate: a12

S31A, A33W, S35D, A50V, S51aY, G52P, Y58N

JH is 3

Isolate: b01

S31T, A33D, S35L, A50S, G52P, Y58S, M82L

JH is 3

Isolate: b02

S31D, A33F, S35K, A50S, S51aY, G52P, S56P, Y58K

JH is 6

Isolate: b03

S31P, A33E, S35Q, A500, S51aG, G52S, S56-, T57-, Y58-,

Y59-, A60-N.B. 5 AA deletion in CDR2.
JH is 4

Isolate: b04

S31F, A33W, S35M, A50G, G52S, S56F, Y58K

JH is 3

Isolate: b05

S31E, A33W, S35P, A50R, S51aY, G52P, S56V, Y58T

JH is 6

Isolate: b06

S31Q, A33F, S35K, A50S, G52P, S56L, Y58Q

JH is 4

Isolate: b07

S31Q, A33Q, S35I, A50V, S51aV, G52P, S56I, Y58N

JH is 4

Isolate: b08

S31R, A33M, S35N, A50V, S51aW, G52S, S56K, Y58L, N82aK

JH is 6

Isolate: b09

S31P, A33F, S35F, A50S, S51aG, G52S, S56D, Y58S

JH is 4
The b09 heavy chain variable domain can include:
(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

JH is 4

Isolate: b11

S31P, A33F, S35F, A50S, S51aG, G52S, S56D, Y58S, K75z

JH is 4

Isolate: b12

S31L, A33V, S35Y, A50Y, G52S, S56I, Y58H

JH is 5

Isolate: c01

S31N, A33G, A50V, S51aG, G52P, S56I, Y58M

JH is 4

Isolate: c02

S31H, A33S, S35R, A50Y, S51aV, G52P, S56F, Y58Q

JH is 4

Isolate: c03

S31L, A33M, S35K, A50V, G52S, S56Y, Y58Q, S82bN

JH is 3

Isolate: c04

S31M, A33F, S35V, A50W, S51aG, G52S, S56E, Y58P

JH is 4

Isolate: c05

A33Q, S35D, A50R, S51aV, G52P, S56D, Y58T

JH is 4

Isolate: c06

S31F, A33F, S35F, A50Y, S51aG, G52P, S56P, Y58N

JH is 4

Isolate: c07

S31Y, A33V, S35M, A50V, S51aR, G52P, S56I, Y58T, M82T

JH is 4

Isolate: c08

S31Y, A33E, S35M, A50S, G52P, S56P, Y58M, A88G

JH is 6

Isolate: c09

S31Q, A33F, S35N, A50Y, G52-, S53-, S56R, Y58P N.B. 2 AA deletion in CDR2.
JH is 4

Isolate: c10

S31V, A33V, S35M, A500, S51aV, G52P, S56K, Y58H

JH is 4

Isolate: c11

S31E, A33P, S35W, A50W, S51aY, G52P, S56N, Y58D

JH is 4

Isolate: c12

S31A, A33N, S35M, A50R, S51aY, G52P, S56Y, Y58L

JH is 4

Isolate: d01

S31M, A33L, S35F, A50V, G52S, S56E, Y58S

JH is 4

Isolate: d02

S31W, A33D, S35A, A50R, S51aV, G52P, S56H, Y58S, S74F

JH is 4

Isolate: d03

S31W, A33R, S35D, A50V, S51aG, G52S, S56M

JH is 3

Isolate: d04

S31D, A33Q, S35M, A50R, G52P, S56M, Y58R, Q81P

JH is 4

Isolate: d05

S31H, A33N, S35A, A50R, S51aR, G52S, S56L, Y58V

JH is 4

Isolate: d06

S31G, A33I, S35E, A50V, S51aV, G52S, S56F, Y58M

JH is 4

Isolate: d07

S31E, A33N, S35F, A50Y, S51aY, G52S, Y58D

JH is 4

Isolate: d08

S31F, S35W, A50R, S51aY, G52S, S56K, Y58W

JH is 3

Isolate: d09

S31H, A33N, S35H, A50G, S51aV, G52S, S56N, Y58G, Y90D

JH is 5
A1-orig

S31Y, A33R, A50S, S51aG, G52P, S56D, Y58L

JH is 4
D6-orig

S31M, A33S, S35R, A50S, S51aY, G52P, Y58E

JH is 4
H6-orig

S31Q, A33W, S35N, A500, S51aG, G52P, S56I

JH is 4
Other embodiments of the invention are within the following claims.

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 K_Dof less than 5×10⁻⁷M 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

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 comprises

R-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

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

(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

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

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

(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

(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 K_ithat is at least 20 better than its K_ifor 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; 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.

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; and

contacting 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.