# Patent

## Title
Nucleic acid encoding hepatocyte growth factor (HGF) binding proteins

## Abstract

The present invention provides a family of binding proteins that bind and neutralize the activity of hepatocyte growth factor (HGF), in particular human HGF. The binding proteins can be used as diagnostic and/or therapeutic agents. With regard to their therapeutic activity, the binding proteins can be used to treat certain HGF responsive disorders, for example, certain HGF responsive tumors.

## Background
RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 13/589,664, filed Aug. 20, 2012, which is a divisional of U.S. patent application Ser. No. 13/051,48, filed Mar. 18, 2011, now U.S. Pat. No. 8,273,35, which is a divisional of U.S. patent application Ser. No. 12/632,765, filed Dec. 7, 2009, now U.S. Pat. No. 7,943,344, which is a divisional of U.S. patent application Ser. No. 11/757,059, filed Jun. 1, 2007, now U.S. Pat. No. 7,649,083, which claims the benefit of and priority to U.S. Provisional Application Nos. 60/810,714, filed Jun. 2, 2006, and 60/860,509, filed Nov. 21, 2006, the entire disclosures of which are incorporated by reference herein.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created Apr. 12, 2013, is named AVO-001BDV4-Sequence-Listing.txt and is 285,673 bytes in size.
FIELD OF THE INVENTION
The field of the invention is molecular biology, immunology and oncology. More particularly, the field is antibody-based binding proteins that bind human hepatocyte growth factor (HGF).
BACKGROUND
Hepatocyte Growth Factor (HGF), also known as Scatter Factor (SF), is a multi-functional heterodimeric protein produced predominantly by mesenchymal cells, and is an effector of cells expressing the Met tyrosine kinase receptor (Bottaro et al. (1991) SCIENCE 251: 802-804, Rubin et al. (1993) BIOCHIM. BIOPHYS. ACTA 1155: 357-371). The human Met receptor is also known as “c-Met.” Mature HGF contains two polypeptide chains, the α-chain and the β-chain. Published studies suggest it is the α-chain that contains HGF's c-Met receptor binding domain.
When it binds to its cognate receptor, HGF mediates a number of cellular activities. The HGF-Met signaling pathway plays a role in liver regeneration, wound healing, neural regeneration, angiogenesis and malignancies. See, e.g., Cao et al. (2001) PROC. NATL. ACAD. SCI. USA 98: 7443-7448, Burgess et al. (2006) CANCER RES. 66: 1721-1729, and U.S. Pat. Nos. 5,997,868 and 5,707,624. Investigators have been developing a number of HGF modulators, including antibodies, to treat various disorders that involve HGF activity, for example, certain HGF responsive cancers. See, e.g., International Application Publication No. WO 2005/017107.
The basic structure common to all antibodies is shown schematically in FIG. 1. Antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called heavy or H chains and two of the polypeptide chains are called light or L chains. The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by a number of interchain disulfide bonds. A light chain is composed of one variable region (VL in FIG. 1) and one constant region (CL in FIG. 1), while the heavy chain is composed of one variable region (VH in FIG. 1) and at least three constant regions (CH1, CH2 and CH3 in FIG. 1). The variable regions determine the specificity of the antibody and the constant regions have other functions.
Amino acid and structural information indicate that each variable region comprises three hypervariable regions (also known as complementarity determining regions or CDRs) flanked by four relatively conserved framework regions or FRs. The three CDRs, referred to as CDR1, CDR2, and CDR3, are responsible for the binding specificity of individual antibodies. When antibodies are to be used as diagnostic and therapeutic agents, typically it is desirable to create antibodies that have the highest binding specificity and affinity to the target molecule. It is believed that differences in the variable regions can have profound effects on the specificity and affinity of the antibody.
U.S. Pat. No. 5,707,624 describes the use of anti-HGF antibodies in the treatment of Kaposi's sarcoma. Similarly, U.S. Pat. No. 5,997,868 describes treating a tumor by administering an anti-HGF antibody to the patient to be treated so as to block the ability of endogeneous HGF to promote angiogenesis in the tumor. More recently, investigators propose that antibodies that bind the β-chain of HGF may have potential as therapeutic agents in patients with HGF-dependent tumors (Burgess (2006) supra).
Notwithstanding, there is still a need for additional HGF modulators that can be used as therapeutic and diagnostic agents.
SUMMARY OF THE INVENTION
The invention is based, in part, upon the discovery of a family of binding proteins that specifically bind HGF, in particular, human HGF. The binding proteins are antibody-based in so far as they contain antigen (i.e., HGF) binding sites based on the CDRs of a family of antibodies that specifically bind HGF. The CDRs confer the binding specificity of the binding proteins to HGF. The binding proteins can be used as diagnostic and therapeutic agents. When used as a therapeutic agent, the binding proteins are engineered (e.g., humanized) so as to reduce or eliminate the risk of inducing an immune response against the binding protein when administered to the recipient (e.g., a human).
The binding proteins neutralize the activity of HGF and, therefore, can be used as a therapeutic agent. In certain embodiments, the binding proteins prevent HGF from binding to its cognate receptor, c-Met, thereby neutralizing HGF activity. In other embodiments, the binding proteins bind to HGF and neutralize its biological activity but without preventing HGF from binding to the c-Met receptor. Because HGF has been implicated in the growth and proliferation of cancer cells, the binding proteins can be used to inhibit the proliferation of cancer cells. Furthermore, when administered to a mammal, the binding proteins can inhibit or reduce tumor growth in the mammal.
These and other aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims.



DESCRIPTION OF THE DRAWINGS
The invention can be more completely understood with reference to the following drawings.
FIG. 1 is a schematic representation of a typical antibody.
FIG. 2 is a schematic diagram showing the amino acid sequence defining the complete immunoglobulin heavy chain variable region of the antibodies denoted as 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6 and 3D11. The amino acid sequences for each antibody are aligned against one another and the regions defining the signal peptide, CDR1, CDR2, and CDR3 are identified in boxes. The unboxed sequences represent FR sequences.
FIG. 3 is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences for each of the immunoglobulin heavy chain variable region sequences presented in FIG. 2.
FIG. 4 is a schematic diagram showing the amino acid sequence defining the complete immunoglobulin light chain variable region of the antibodies 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6, and 3D11. The amino acid sequences for each antibody are aligned against one another and the regions defining the signal peptide, CDR1, CDR2, and CDR3 are identified in boxes. The unboxed sequences represent FR sequences.
FIG. 5 is a schematic diagram showing the CDR1, CDR2, and CDR3 sequences for each of the immunoglobulin light chain variable region sequences presented in FIG. 4.
FIG. 6 is a graph summarizing results from an experiment to measure tumor inhibitory activity of anti-HGF antibodies 1D3, 1F3, 1A3 and 2B8 in a U87MG xenograft model. Diamonds correspond to PBS; triangles correspond to anti-HGF antibody 1A3; X corresponds to anti-HGF antibody 1D3; squares correspond to anti-HGF antibody 1F3, and circles correspond to anti-HGF antibody 2B8.
FIG. 7 is a graph summarizing results from an experiment to measure tumor inhibitory activity of anti-HGF antibodies 1D3, 1F3, 1A3 and 2B8 in a U 18 xenograft model. Diamonds correspond to IgG; squares correspond to anti-HGF antibody 1F3, triangles to anti-HGF antibody 1D3; X corresponds to anti-HGF antibody 1A3; and circles correspond to anti-HGF antibody 2B8.
FIG. 8 is a table summarizing surface plasmon resonance data on antigen-binding affinity and kinetics of interaction between human HGF and chimeric, chimeric/humanized, or humanized 2B8 antibodies. The table lists the pairs of Kappa light chain and IgG1 heavy chain tested. Those antibodies with standard deviations (STDEV) listed were analyzed in three independent experiments.
FIG. 9 is a bar chart summarizing experimental data indicating that Hu2B8 binds an epitope mutually exclusive to murine monoclonal antibody 2B8. Humanized or chimeric 2B8 was captured on an anti-human Fc chip. HGF then was bound to the humanized or chimeric 2B8. The ability of mouse 2B8 or the control antibody (polyclonal goat anti-HGF antibody) to bind the captured HGF was measured. Both humanized 2B8 antibodies and chimeric 2B8 prevent murine 2B8 from binding HGF. White bars correspond to the chimeric 2B8 antibody; gray bars correspond to the humanized Hu2B8 antibody (kappa variable region Kv1-39.1 and heavy chain variable region Hv5-51.1); black bars correspond to the humanized Hu2B8 antibody (kappa variable region Kv3-15.1 and heavy chain variable region Hv5-51.1).



DETAILED DESCRIPTION OF THE INVENTION
The invention is based, in part, upon the discovery of a family of binding proteins that specifically bind, and neutralize the activity of, HGF, in particular, human HGF. The binding proteins can be used in a variety of diagnostic and therapeutic applications. The binding proteins are based upon the antigen binding sites of certain monoclonal antibodies that have been selected for their ability to bind, and neutralize the activity of, HGF. In particular, the binding proteins contain immunoglobulin variable region CDR sequences that together define a binding site for HGF.
In view of the neutralizing activity of these antibodies, they are particularly useful in modulating the growth and/or proliferation of HGF responsive cells, for example, cancer cells. When used as a therapeutic agent, the binding proteins can be engineered so as to minimize or eliminate the risk of inducing an immune response against the binding proteins when administered to the recipient. Furthermore, depending upon the particular application, it is contemplated that the binding proteins can be conjugated to other moieties, for example, detectable labels, for example, radiolabels, and effector molecules, for example, other protein and small molecule-based therapeutics. Each of these features and aspects of the invention are discussed in more detail below.
I—Binding Proteins that Bind HGF
In one aspect, the invention provides an isolated binding protein that binds human HGF. The binding protein comprises (i) an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, and (ii) an immunoglobulin heavy chain variable region comprising three complementarity determining regions (CDRs), wherein the immunoglobulin light chain variable region and the immunoglobulin heavy chain variable region together define a single binding site for binding human HGF. CDRL1 comprises the amino acid sequence X1 X2 Ser X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15, wherein amino acid X1 is Arg, Lys, or Ser, X2 is Ala or Thr, X4 is Glu, Gln, or Ser, Xs is Asn, Asp, or Ser, X6 is Ile or Val, X7 is Asp, Lys, Ser, Val, or Tyr, X8 is a peptide bond or Tyr, X9 is a peptide bond or Asp, X10 is a peptide bond or Gly, X11 is a peptide bond or Asn, X12 is a peptide bond, Ile, or Ser, X13 is Asn or Tyr, X14 is Ile, Leu, Met, or Val, X15 is Ala, Asn, His, or Ser. CDRL2 comprises the amino acid sequence X16 X17 X18 X19 X20 X21 X22, wherein amino acid X16 is Ala, Asp, Arg, Gly, or Val, X17 is Ala, Thr, or Val, X18 is Asn, Ser, or Thr, X19 is Arg, Asn, Lys, or His, X20 is Leu or Arg, X21 is Ala, Asn, Glu, Val, or Pro, X22 is Asp, Ser, or*Thr. CDRL3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 Pro X30 Thr, wherein amino acid X23 is Leu, Gly, or Gln, X24 is His or Gln, X25 is Phe, Ser, Trp, or Tyr, X26 is Asp, Ile, Ser, Trp, or Tyr, X27 is Gly, Glu, Asn, or Ser, X28 is Asp, Asn, Phe, Thr, or Tyr, X30 is Leu, Phe, Pro, or Tyr.
In another aspect, the invention provides an isolated binding protein that binds human HGF comprising (i) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3 and (ii) an immunoglobulin light chain variable region comprising three complementarity determining regions (CDRs), wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for binding human HGF. CDRH1 comprises the amino acid sequence X1 Tyr X3 X4 X5, wherein amino acid X1 is Asp, Asn, Ser, or Thr, X3 is Phe, Ser, Trp, or Tyr, X4 is Ile, Leu, or Met, X5 is Asn, His, or Ser. CDRH2 comprises the amino acid sequence X6 Ile X8 X9 X10 X11 Gly X13 X14 X15 Tyr X17 X18 X19 X20 X21 X22, wherein amino acid X6 is Lys, Gln, Glu, Val, or Tyr, X8 is Asn, Gly, Ser, Trp, or Tyr, X9 is Ala, Pro or Ser, X10 is Gly or Thr, X11 is a peptide bond, Asp, Asn, Gly, or Ser, X13 is Asp, Asn, His, or Ser, X14 is Ser or Thr, X15 is Asn or Tyr, X17 is Asn or Pro, X18 is Ala, Asp, Gly, Gln, Glu, Pro, or Ser, X19 is Asn, Lys, Met, or Ser, X20 is Leu, Phe or Val, X21 is Lys, Met, or Gln, X22 is Asp, Gly or Ser. CDRH3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 X29 X30 X31 X32 X33 X34 Tyr, wherein amino acid X23 is Arg, Asn, Gln, or Glu, X24 is Gly, Leu, Arg, or Tyr, X25 is a peptide bond, Asp, or Gly, X26 is a peptide bond or Gly, X27 is a peptide bond or Tyr, X28 is a peptide bond, Leu, or Tyr, X29 is a peptide bond, Gly, Leu, Arg, or Val, X30 is a peptide bond, Asp, Gly, or Glu, X31 is a peptide bond, Asn, Arg, Ser, or Tyr, X32 is peptide bond, Ala, Gly, Ile, or Tyr, X33 is Met or Phe, X34 is Ala or Asp.
It is understood that the binding protein can comprise both the immunoglobulin light chain and the immunoglobulin heavy chain sequences or the fragments thereof, noted above. Furthermore, it is understood that the binding protein can be an intact antibody or an antigen binding fragment thereof, or a biosynthetic antibody site.
In certain embodiments, the CDR sequences of the immunoglobulin light chain and the immunoglobulin heavy chain are interposed with framework regions (FR).
In certain other embodiments, the CDR sequences of the immunoglobulin light chain and the immunoglobulin heavy chain are interposed between human or humanized framework regions.
In another aspect, the invention provides an isolated binding protein that specifically binds human HGF. The binding protein comprises: (a) an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3 and (b) immunoglobulin heavy chain variable region, wherein the immunoglobulin light chain variable region and the immunoglobulin heavy chain variable region together define a single binding site for binding human HGF. The CDRL1 comprises a sequence selected from the group consisting of SEQ ID NO. 8 (1A3), SEQ ID NO. 18 (2B8), SEQ ID NO. 28 (2F8), SEQ ID NO. 38 (3B6), SEQ ID NO. 48 (3D11), SEQ ID NO. 58 (1D3), SEQ ID NO. 68 (1F3), and SEQ ID NO. 78 (3A12). The CDRL2 comprises a sequence selected from the group consisting of SEQ ID NO. 9 (1A3), SEQ ID NO. 19 (2B8), SEQ ID NO. 29 (2F8), SEQ ID NO. 39 (3B6), SEQ ID NO. 49 (3D11), SEQ ID NO. 59 (1D3), SEQ ID NO. 69 (1F3), SEQ ID NO. 79 (3A12) and SEQ ID NO. 206 (LRMR2B8LC). The CDRL3 comprises a sequence selected from the group consisting of SEQ ID NO. 10 (1A3), SEQ ID NO. 20 (2B8), SEQ ID NO. 30 (2F8), SEQ ID NO. 40 (3B6), SEQ ID NO. 50 (3D11), SEQ ID NO. 60 (1D3), SEQ ID NO. 70 (1F3), and SEQ ID NO. 80 (3A12). Throughout the specification and claims, the sequences denoted by a particular SEQ ID NO. are followed in parentheses by the antibody that was the origin of the particular sequence. By way of example, SEQ ID NO. 8 (1A3) indicates that the sequence of SEQ ID NO. 8 is based upon the sequence present in antibody 1A3.
In one embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 8 (1A3), a CDRL2 comprising the sequence of SEQ ID NO. 9 (1A3), and a CDRL3 comprising the sequence of SEQ ID NO. 10 (1A3).
In another embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 18 (2B8), a CDRL2 comprising the sequence of SEQ ID NO. 19 (2B8) or SEQ ID NO. 206 (LRMR2B8LC), and a CDRL3 comprising the sequence of SEQ ID NO. 20 (2B8).
In another embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 28 (2F8), a CDRL2 comprising the sequence of SEQ ID NO. 29 (2F8), and a CDRL3 comprising the sequence of SEQ ID NO. 30 (2F8).
In another embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 38 (3B6), a CDRL2 comprising the sequence of SEQ ID NO. 39 (3B6), and a CDRL3 comprising the sequence of SEQ ID NO. 40 (3B6).
In another embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 48 (3D11), a CDRL2 comprising the sequence of SEQ ID NO. 49 (3D11), and a CDRL3 comprising the sequence of SEQ ID NO. 50 (3D11).
In another embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 58 (1D3), a CDRL2 comprising the sequence of SEQ ID NO. 59 (1D3), and a CDRL3 comprising the sequence of SEQ ID NO. 60 (1D3).
In another embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 68 (1F3), a CDRL2 comprising the sequence of SEQ ID NO. 69 (1F3), and a CDRL3 comprising the sequence of SEQ ID NO. 70 (1F3).
In another embodiment, the binding protein comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 78 (3A12), a CDRL2 comprising the sequence of SEQ ID NO. 79 (3A12), and a CDRL3 comprising the sequence of SEQ ID NO. 80 (3A12).
In each of the foregoing embodiments, the CDRL1, CDRL2, and CDRL3 sequences preferably are interposed between human or humanized immunoglobulin FRs. It is understood that the binding protein can be an intact antibody, an antigen binding fragment thereof, or a biosynthetic antibody site.
In another aspect, the invention provides an isolated binding protein that binds human HGF. The binding protein comprises (a) an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, and (b) an immunoglobulin light chain variable region, wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together define a single binding site for binding human HGF. The CDRH1 comprises a sequence selected from the group consisting of SEQ ID NO. 5 (1A3), SEQ ID NO. 15 (2B8), SEQ ID NO. 25 (2F8), SEQ ID NO. 35 (3B6), SEQ ID NO. 45 (3D11), SEQ ID NO. 55 (1D3), SEQ ID NO. 65 (1F3), and SEQ ID NO. 75 (3A12); the CDRH2 comprises a sequence selected from the group consisting of SEQ ID NO. 6 (1A3), SEQ ID NO. 16 (2B8), SEQ ID NO. 26 (2F8), SEQ ID NO. 36 (3B6), SEQ ID NO. 46 (3D11), SEQ ID NO. 56 (1D3), SEQ ID NO. 66 (1F3), SEQ ID NO. 76 (3A12), SEQ ID NO. 202 (Hu2B8 Hv1f.1), SEQ ID NO. 203 (Hu2B8 Hv5a.1 or Hu2B8 Hv5-51.1), SEQ ID NO. 204 (LR2B8HC) and SEQ ID NO. 205 (LRMR2B8HC); and the CDRH3 comprises a sequence selected from the group consisting of SEQ ID NO. 7 (1A3), SEQ ID NO. 17 (2B8), SEQ ID NO. 27 (2F8), SEQ ID NO. 37 (3B6), SEQ ID NO. 47 (3D11), SEQ ID NO. 57 (1D3), SEQ ID NO. 67 (1F3), and SEQ ID NO. 77 (3A12).
In one embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO. 5 (1A3); a CDRH2 comprising the sequence of SEQ ID NO. 6 (1A3); and a CDRH3 comprising the sequence of SEQ ID NO. 7 (1A3).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO. 15 (2B8); a CDRH2 comprising the sequence of SEQ ID NO. 16 (2B8), SEQ ID NO. 202 (Hu2B8 Hv1f.1), SEQ ID NO. 203 (Hu2B8 Hv5a.1 or Hu2B8 Hv5-51.1), SEQ ID NO. 204 (LR2B8HC) or SEQ ID NO. 205 (LRMR2B8HC); and a CDRH3 comprising the sequence of SEQ ID NO. 17 (2B8).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO. 25 (2F8); a CDRH2 comprising the sequence of SEQ ID NO. 26 (2F8); and a CDR3 comprising the sequence of SEQ ID NO. 27 (2F8).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ ID NO. 35 (3B6); a CDRH2 comprising the sequence of SEQ ID NO. 36 (3B6); and a CDRH3 comprising the sequence of SEQ ID NO. 37 (3B6).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO. 45 (3D11); a CDR comprising the sequence of SEQ ID NO. 46 (3D11); and a CDRH3 comprising the sequence of SEQ ID NO. 47 (3D11).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO. 55 (1 D3); a CDRH2 comprising the sequence of SEQ ID NO. 56 (1D3); and a CDRH3 comprising the sequence of SEQ ID NO. 57 (1D3).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO. 65 (1F3); a CDRH2 comprising the sequence of SEQ ID NO. 66 (1F3); and a CDRH3 comprising the sequence of SEQ ID NO. 67 (1F3).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising: a CDRH1 comprising the sequence of SEQ ID NO. 75 (3A12); a CDRH2 comprising the sequence of SEQ ID NO. 76 (3A12); and a CDRH3 comprising the sequence of SEQ ID NO. 77 (3A12).
In each of the foregoing embodiments, the CDRH1, CDRH2, and CDRH3 sequences preferably are interposed between human or humanized immunoglobulin FRs. It is understood that the binding protein can be an intact antibody, an antigen binding fragment thereof, or a biosynthetic antibody site.
In another aspect, the invention provides a binding protein that binds human HGF. The binding protein comprises an immunoglobulin heavy chain variable region selected from the group consisting of residues 20-141 of SEQ ID NO. 2 (1A3), residues 20-137 of SEQ ID NO. 12 (2B8), residues 20-137 of SEQ ID NO. 22 (2F8), residues 20-139 of SEQ ID. NO. 32 (3B6), residues 20-132 of SEQ ID NO. 42 (3D11), residues 20-141 of SEQ ID NO. 52 (1D3), residues 20-141 of SEQ ID NO. 62 (1F3), and residues 20-141 of SEQ ID NO. 72 (3A12) and an immunoglobulin light chain variable region selected from the group consisting of residues 21-127 of SEQ ID NO. 4 (1A3), residues 21-127 of SEQ ID NO. 14 (2B8), residues 20-131 of SEQ ID NO. 24 (2F8), residues 23-129 of SEQ ID NO. 34 (3B6), residues 23-128 of SEQ ID NO. 44 (3D11), residues 21-127 of SEQ ID NO. 54 (1D3), residues 21-127 of SEQ ID NO. 64 (1F3), and residues 21-127 of SEQ ID NO. 74 (3A12).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-141 of SEQ ID NO. 2 (1A3), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 21-127 of SEQ ID NO. 4 (1A3).
In one embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-137 of SEQ ID NO. 12 (2B8), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 21-127 of SEQ ID NO. 14 (2B8).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-137 of SEQ ID NO. 22 (2F8), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 20-131 of SEQ ID NO. 24 (2F8).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-139 of SEQ ID NO. 32 (3B6), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 23-129 of SEQ ID NO. 34 (3B6).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-132 of SEQ ID NO. 42 (3D11), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 23-128 of SEQ ID NO. 44 (3D11).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-141 of SEQ ID NO. 52 (1D3), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 21-127 of SEQ ID NO. 54 (1D3).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-141 of SEQ ID NO. 62 (1F3), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 21-127 of SEQ ID NO. 64 (1F3).
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of residues 20-141 of SEQ ID NO. 72 (3A12), and an immunoglobulin light chain variable region comprising the amino acid sequence of residues 21-127 of SEQ ID NO. 74 (3A12).
In each of the foregoing embodiments, the binding protein can be an intact antibody, an antigen binding fragment thereof, or a biosynthetic antibody site.
In another aspect, the invention provides an isolated binding protein that binds human HGF. The binding protein comprises (i) an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO. 173 (Hu2B8 Kv1-39.1 light chain variable region), SEQ ID NO. 179 (Hu2B8 Kv3-15.1 light chain variable region), SEQ ID NO. 193 (LR2B8LC light chain variable region), and SEQ ID NO. 199 (LRMR2B8LC light chain variable region); and (ii) an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO. 159 (Hu2B8 Hv1f.1 heavy chain variable region), SEQ ID NO. 165 (Hu2B8 Hv5a.1 heavy chain variable region), SEQ ID NO. 169 (Hu2B8 Hv5-51.1 heavy chain variable region), SEQ ID NO. 183 (LR2B8HC heavy chain variable region), and SEQ ID NO. 189 (LRMR2B8LC light chain variable region). The binding protein can be an intact antibody, an antigen binding fragment thereof, or a biosynthetic antibody site.
In another aspect, the invention provides an isolated binding protein that binds human HGF. The binding protein comprises (i) an immunoglobulin light chain selected from the group consisting of SEQ ID NO. 177 (Hu2B8 Kv1-39.1+kappa constant (Km(3) allotype (allele 2)), SEQ ID NO. 181 (Hu2B8 Kv3-15.1+Kappa constant (Km(3) allotype (allele 2)), SEQ ID NO. 197 (LR2BSLC+Kappa constant (Km(3) allotype (allele 1)), and SEQ ID NO. 201 (LRMR2BSLC+Kappa constant (Km(3) allotype (allele 1)); and (ii) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO. 163 (Hu2B8 Hv1f.1+IgG1 Constant (G1m(17,1) allotype)), SEQ ID NO. 167 (Hu2B8 Hv5a.1+IgG1 Constant (G1m(17,1) allotype)), SEQ ID NO. 171 (Hu2B8 Hv5-51.1+IgG1 Constant (G1m(17,1) allotype)), SEQ ID NO. 187 (LR2BSHC+IgG1 Constant (G1m(3) allotype) (allele 1)), and SEQ ID NO. 191 (LRMR2B8HC+IgG1 Constant (G1m(3) allotype) (allele 1)). The binding protein can be an intact antibody, an antigen binding fragment thereof, or a biosynthetic antibody site.
In another aspect, the invention provides an isolated binding protein that binds reduced human HGF. The binding protein comprises (i) an immunoglobulin light chain variable region comprising three CDRs, and (ii) an immunoglobulin heavy chain variable region comprising three CDRs. The CDRs typically are interposed between FRs. The CDRs of the immunoglobulin light chain and the immunoglobulin heavy chain together define a binding site that binds reduced human HGF, for example, the α-chain of reduced HGF. Reduced HGF refers to HGF treated with an amount of reducing agent, for example, dithiothreitol (DTT), 2-mercaptoethanol, or glutathione sufficient to reduce the disulfide linkage between the α-chain and the β-chain. Exemplary concentrations include, for example, 100 mM DTT and 5% 2-mercaptoethanol.
In certain embodiments, the binding protein comprises an immunoglobulin light chain variable region comprising at least one CDR selected from the group consisting of CDRL1, CDRL2 and CDRL3. Optionally, the binding protein comprises two CDRs, for example, CDRL1 and CDRL2, or CDRL1 and CDRL3, or CDRL2 and CDRL3. Optionally, the binding protein comprises all three CDRs, i.e., CDRL1, CDRL2 and CDRL3. CDRL1 comprises the amino acid sequence X1 X2 Ser X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15, wherein amino acid X1 is Arg or Lys, X2 is Ala or Thr, X4 is Glu or Gln, X5 is Asn, Ser, or Asp, X6 is Ile or Val, X7 is Tyr, Asp, or Lys, X8 is a peptide bond or Tyr, X9 is a peptide bond or Asp, X10 is a peptide bond or Gly, X11 is a peptide bond or Asn, X12 is a peptide bond or Ser, X13 is Asn or Tyr, X14 is Ile or Leu, X is Ala, Asn, or Ser. CDRL2 comprises the amino acid sequence X16 X17 X18 X19 Leu X21 X22, wherein amino acid X16 is Ala, Asp, Val, or Arg, X17 is Ala or Val, X18 is Asn, Ser, or Thr, X19 is Arg, Asn, or His, X21 is Ala, Glu, Val, or Pro, X22 is Asp or Ser. CDRL3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 Pro X30 Thr, wherein amino acid X23 is Leu or Gln, X24 is His or Gln, X25 is Phe, Ser, or Tyr, X26 is Asp, Ile, or Trp, X27 is Gly or Glu, X28 is Asp, Phe, or Thr, X30 is Phe, Pro, or Tyr.
In another embodiment, the binding protein comprises an immunoglobulin heavy chain variable region comprising at least one CDR selected from the group consisting of CDRH1, CDRH2, and CDRH3. Optionally, the binding protein comprises two CDRs, for example, CDRH1 and CDRH2, or CDRH1 and CDRH3, or CDRH1 and CDRH3. Optionally, the binding protein comprises all three CDRs, i.e., CDRH1, CDRH2 and CDRH3. CDRH1 comprises the amino acid sequence X1 Tyr X3 X4 X5, wherein amino acid X1 is Asp, Asn, Ser, or Thr, X3 is Phe, Trp, or Tyr, X4 is Ile or Met, X5 is Asn, His, or Ser. CDRH2 comprises the amino acid sequence X6 Ile X8 X9 Gly X11 Gly X13 X14 X15 Tyr X17 X18 X19 X20 Lys X22, wherein amino acid X6 is Lys, Gln, or Tyr, X8 is Gly, Ser, or Tyr, X9 is Pro or Ser, X11 is Asp, Gly, or Ser, X13 is Asp or Ser, X14 is Ser or Thr, X15 is Asn or Tyr, X17 is Asn or Pro, X18 is Ala, Asp, Gly, or Glu, X19 is Asn, Met, or Ser, X20 is Phe or Val, X22 is Asp or Gly. CDRH3 comprises the amino acid sequence X23 X24 X25 X26 X27 X28 X29 X30 X31 X32 X33 Asp Tyr, wherein amino acid X23 is Arg or Gln, X24 is Gly or Leu, X25 is Asp, Gly, or a peptide bond, X26 is Gly or a peptide bond, X27 is a peptide bond or Tyr, X28 is Leu, a peptide bond or Tyr, X29 is a Gly, Arg or Leu, X30 is Asp, Gly or Glu, X31 is a Tyr, Arg or Asn, X32 is Ala, Gly or Tyr, X33 is Met or Phe.
It is understood that the binding protein can comprise both the immunoglobulin heavy chain and the immunoglobulin light chain sequences or the fragments thereof, noted above. Furthermore, it is understood that the binding protein can be an intact antibody or an antigen binding fragment thereof, or a biosynthetic antibody site.
In certain embodiments, the binding protein comprises an immunoglobulin light chain variable region comprising (i) a CDRL1 having a sequence selected from the group consisting of SEQ ID NO. 8 (1A3), SEQ ID NO. 28 (2F8), SEQ ID NO. 38 (3B6), SEQ ID NO. 58 (1D3), and SEQ ID NO. 68 (1F3), (ii) a CDRL2 having a sequence selected from the group consisting of SEQ ID NO. 9 (1A3), SEQ ID NO. 29 (2F8), SEQ ID NO. 39 (3B6), SEQ ID NO. 59 (1D3), and SEQ ID NO. 69 (1F3), and (iii) a CDRL3 having a sequence selected from the group consisting of SEQ ID NO. 10 (1A3), SEQ ID NO. 30 (2F8), SEQ ID NO. 40 (3B6), SEQ ID NO. 60 (1D3), and SEQ ID NO. 70 (1F3). The CDR sequences can be interposed between human or humanized FRs. In other embodiments, the binding protein comprises an immunoglobulin light chain variable region comprising an amino acid sequence selected from the group consisting of residues 21-127 of SEQ ID NO. 4 (1A3), residues 20-131 of SEQ ID NO. 24 (2F8), residues 23-129 of SEQ ID NO. 34 (3B6), residues 21-127 of SEQ ID NO. 54 (1D3), and residues 21-127 of SEQ ID NO. 64 (1F3).
In certain other embodiments, the binding protein comprises an immunoglobulin heavy chain variable region comprising (i) a CDRH1 having a sequence selected from the group consisting of SEQ ID NO. 5 (1A3), SEQ ID NO. 25 (2F8), SEQ ID NO. 35 (3B6), SEQ ID NO. 55 (1D3), and SEQ ID NO. 65 (1F3), (ii) a CDRH2 having a sequence selected from the group consisting of SEQ ID NO. 6 (1A3), SEQ ID NO. 26 (2F8), SEQ ID NO. 36 (3B6), SEQ ID NO. 56 (1D3), and SEQ ID NO. 66 (1F3), and (iii) a CDRH3 having a sequence selected from the group consisting of SEQ ID NO. 7 (1A3), SEQ ID NO. 27 (2F8), SEQ ID NO. 37 (3B6), SEQ ID NO. 57 (1D3), and SEQ ID NO. 67 (1F3). The CDR sequences can be interposed between human or humanized FRs. In another embodiment, the immunoglobulin heavy chain variable region comprises an amino acid sequence selected from the group consisting of residues 20-141 of SEQ ID NO. 2 (1A3), residues 20-137 of SEQ ID NO. 22 (2F8), residues 20-139 of SEQ ID NO. 32 (3B6), residues 20-141 of SEQ ID NO. 52 (1D3), and residues 20-141 of SEQ ID NO. 62 (1F3).
In another aspect, the invention provides an isolated binding protein that binds human HGF and comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region. The isolated binding protein competes for binding to HGF with at least one reference antibody selected from the group consisting of (i) an antibody having an immunoglobulin light chain variable region of residues 20-131 of SEQ ID NO. 24 (2F8), and an immunoglobulin heavy chain variable region of residues 20-137 of SEQ ID NO. 22 (2F8), (ii) an antibody having an immunoglobulin light chain variable region of residues 23-129 of SEQ ID NO. 34 (3B6), and an immunoglobulin heavy chain variable region of residues 20-139 of SEQ ID NO. 32 (3B6), and (iii) an antibody having an immunoglobulin light chain variable region of residues 23-128 of SEQ ID NO. 44 (3D11), and an immunoglobulin heavy chain variable region of residues 20-132 of SEQ ID NO. 42 (3D11). Under certain circumstances, the binding protein binds the same epitope of HGF as one of the reference antibodies.
It is understood that each of the binding proteins discussed above can be an intact antibody, for example, a monoclonal antibody. Alternatively, the binding protein can be an antigen binding fragment of an antibody, or can be a biosynthetic antibody binding site. Antibody fragments include Fab, Fab′, (Fab′)2 or Fv fragments. Techniques for making such antibody fragments are known to those skilled in the art. A number of biosynthetic antibody binding sites are known in the art and include, for example, single Fv or sFv molecules, described, for example, in U.S. Pat. No. 5,476,786. Other biosynthetic antibody binding sites include bispecific or bifunctional binding proteins, for example, bispecific or bifunctional antibodies, which are antibodies or antibody fragments that bind at least two different antigens. For example, bispecific binding proteins can bind HGF, for example, human HGF, and another antigen of interest. Methods for making bispecific antibodies are known in art and, include, for example, by fusing hybridomas or by linking Fab′ fragments. See, e.g., Songsivilai et al. (1990) CLIN. EXP. IMMUNOL. 79: 315-325; Kostelny et al. (1992) J. IMMUNOL. 148: 1547-1553.
The binding proteins of the invention can bind hHGF containing a cysteine to arginine substitution at position 561 or a glycine to glutamate substitution at position 555.
In another aspect, the invention provides an isolated binding protein that binds human HGF with a kd of 4.0×10−5 s−1 or lower, 3.0×10−5 s−1 or lower, or 2.0×10−5 s−1 or lower. The isolated binding proteins can bind human HGF with a kd from 5.0×10−5 s−1 to 0.5×10−5 s−1, or from 4.0×105 s−1 to 1.0×10−5 s−1, or from 3.0×10−5 s−1 to 1.5×10−5 s−1. In another aspect, the invention provides an isolated binding protein that binds human HGF with a KD of 100 pM or lower, or 20 pM or lower, or 10 pM or lower, or 5 pM or lower. The isolated binding proteins can bind human HGF with a KD from 100 pM to 5 pM, or from 20 pM to 5 pM, or from 15 pM to 10 pM, or from 20 pM to 10 pM, or from 15 pM to 5 pM. Unless otherwise specified, KD values are determined by the methods, and under the conditions, described in Example 6.
In another aspect, the invention provides an isolated binding protein that binds human HGF, wherein the antibody binds to human HGF with lower KD at 37° C. than at 25° C. The binding protein binding optionally binds human HGF with a KD less than 5 μM at 37° C.
In other aspects and embodiments, the binding proteins can inhibit hHGF from binding to c-Met. For example, the binding proteins can have an IC50 (concentration at 50% of maximum inhibition) of at least about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 nM when assayed using the protocol described in Example 7(a). In certain other embodiments, the binding proteins can neutralize HGF BrdU incorporation in 4 MBr-5 cells (ATCC, Catalog No. CCL208) using the method described in Example 7(b).
The binding proteins have an IC50 of 50 nM or lower, preferably 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5 nM or lower, when assayed using the protocol described in Example 7(b). In certain other embodiments, the binding proteins can be used to inhibit HGF stimulated c-Met phosphorylation in PC-3 cells (ATCC, Manassus, Va. Catalog No. CRL-1435) using the assay described in Example 9. The binding proteins inhibit HGF-stimulated (1.25 nM) c-Met phosphorylation in PC-3 cells with an IC50 of 2 nM or less (Table 8), using the assay described in Example 9.
II—Production of Binding Proteins
Binding proteins of the invention can be produced in various ways using approaches know in the art. For example, DNA molecules encoding light chain variable regions and heavy chain variable regions can be chemically synthesized, using a commercial synthesizer and sequence information provided herein. Such synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired binding proteins. Production of defined gene constructs is within routine skill in the art. Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or PCR techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein or prior art sequence information regarding genes encoding the heavy and light chains of murine antibodies in hybridoma cells. Production and use of such probes is within ordinary skill in the art.
The nucleic acids encoding the desired binding proteins can be introduced (ligated) into expression vectors, which can be introduced into a host cell via standard transfection or transformation techniques known in the art. Exemplary host cells include, for example, E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce immunoglobulin protein. Transfected host cells can be grown under conditions that permit the host cells to express the genes of interest, for example, the genes that encode the immunoglobulin light or heavy chain variable regions. The resulting expression products can be harvested using techniques known in the art.
The particular expression and purification conditions will vary depending upon what expression system is employed. For example, if the gene is to be expressed in E. coli, it is first cloned into an expression vector. This is accomplished by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a signal sequence, e.g., a sequence encoding fragment B of protein A (FB). The resulting expressed fusion protein typically accumulates in refractile or inclusion bodies in the cytoplasm of the cells, and may be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the expressed proteins refolded and cleaved by the methods already established for many other recombinant proteins.
If the engineered gene is to be expressed in eukayotic host cells, for example, myeloma cells or CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, immunoglobulin enhancers, and various introns. This expression vector optionally can contain sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be transfected into myeloma cells or CHO cells using established transfection protocols. Such transfected cells can express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a protein domain having another function (e.g., cytotoxicity).
III—Modifications to the Binding Proteins
It is understood that the binding proteins can be modified to optimize performance depending upon the intended use of the binding proteins. For example, when the binding protein is being used as a therapeutic agent, the binding protein can be modified to reduce its immunogenicity in the intended recipient. Alternatively or in addition, the binding protein can be fused or coupled to another protein or peptide, for example, a growth factor, cytokine, or cytotoxin. Such modifications can be achieved by using routine gene manipulation techniques known in the art.
Various techniques for reducing the antigenicity of antibodies and antibody fragments are known in the art. These techniques can be used to reduce or eliminate the antigenicity of the binding proteins of the invention. For example, when the binding proteins are to be administered to a human, the binding proteins preferably are engineered to reduce their antigenicity in humans. This process often is referred to as humanization. Preferably, the humanized binding proteins have the same or substantially the same affinity for the antigen as the original non-humanized binding protein it was derived from.
In one well known humanization approach, chimeric proteins are created in which immunoglobulin constant regions of antibodies from one species, e.g., mouse, are replaced with immunoglobulin constant regions from a second, different species, e.g., a human. In this example, the resulting antibody is a mouse-human chimera, where the human constant region sequences, in principle, are less immunogenic than the counterpart murine sequences. This type of antibody engineering is described, for example, Morrison, et al. (1984) PROC. NAT. ACAD. SCI. 81: 6851-6855, Neuberger et al. (1984) NATURE 312: 604-608; U.S. Pat. No. 6,893,625 (Robinson); U.S. Pat. No. 5,500,362 (Robinson); and U.S. Pat. No. 4,816,567 (Cabilly).
In another approach, known as CDR grafting, the CDRs of the light and heavy chain variable regions of an antibody of interest are grafted into frameworks (FRs) from another species. For example, murine CDRs can be grafted into human FR sequences. In some embodiments, the CDRs of the light and heavy chain variable regions of an anti-HGF antibody are grafted into human FRs or consensus human FRs. In order to create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR grafting is described, for example, in U.S. Pat. No. 7,022,500 (Queen); U.S. Pat. No. 6,982,321 (Winter); U.S. Pat. No. 6,180,370 (Queen); U.S. Pat. No. 6,054,297 (Carter); U.S. Pat. No. 5,693,762 (Queen); U.S. Pat. No. 5,859,205 (Adair); U.S. Pat. No. 5,693,761 (Queen); U.S. Pat. No. 5,565,332 (Hoogenboom); U.S. Pat. No. 5,585,089 (Queen); U.S. Pat. No. 5,530,101 (Queen); Jones et al. (1986) NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332: 323-327; Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBS LETT 430: 92-94.
In an approach called “superhumanization,” antibodies in which human immunogenicity is reduced or eliminated are created by an alternative form of grafting. In superhumanization, human FR sequences are chosen from a set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. This approach is described, for example, in U.S. Pat. No. 6,881,557 (Foote) and in Tan et al. (2002) J. IMMUNOL 169:1119-1125.
Other approaches to reduce immunogenicity include, techniques are known as “reshaping,” “hyperchimerization,” or “veneering/resurfacing” to produce humanized antibodies. See, e.g., Vaswami et al. (1998) ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81: 105; Roguska et al. (1996) PROT. ENGINEER 9: 895-904; and U.S. Pat. No. 6,072,035 (Hardman). In the veneering/resurfacing approach, the surface accessible amino acid residues in the murine antibody are replaced by amino acid residues more frequently found at the same positions in a human antibody. This type of antibody resurfacing is described, for example, in U.S. Pat. No. 5,639,641 (Pedersen).
One exemplary approach for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMABTM technology (Vaccinex, Inc., Rochester, N.Y.), which involves a vaccinia virus-based vector to express antibodies in mammalian cells. High levels of combinatorial diversity of immunoglobulin heavy and light chains are said to be produced. See, e.g., U.S. Pat. No. 6,706,477 (Zauderer); U.S. Pat. No. 6,800,442 (Zauderer); and U.S. Pat. No. 6,872,518 (Zauderer).
Another exemplary approach for converting a mouse antibody into a form suitable for use in humans is technology practiced commercially by KaloBios Pharmaceuticals, Inc. (Palo Alto, Calif.). This technology involves the use of a proprietary human “acceptor” library to produce an “epitope focused” library for antibody selection.
Another exemplary approach for modifying a mouse antibody into a form suitable for medical use in humans is HUMAN ENGINEERINGTM (HETM) technology, which is practiced commercially by XOMA (US) LLC. See, e.g., International Application Publication No. WO 93/11794 and U.S. Pat. Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619.
Any suitable approach, including any of the above approaches, can be used to reduce or eliminate human immunogenicity of a binding protein of interest.
In addition, it is possible to create fully human antibodies in mice. In this approach, human antibodies are prepared using a transgenic mouse in which the mouse's antibody-producing genes have been replaced by a substantial portion of the human antibody producing genes. Such mice produce human immunoglobulin instead of murine immunoglobulin molecules. See, e.g., WO 98/24893 (Jacobovitz et al.) and Mendez et al. (1997) NATURE GENETICS 15: 146-156. Fully human anti-HGF monoclonal antibodies can be produced using the following approach. Transgenic mice containing human immunoglobulin genes are immunized with the antigen of interest, e.g., HGF. Lymphatic cells from the mice then are obtained from the mice, which are then fused with a myeloid-type cell line to prepare immortal hybridoma cell lines. The hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to HGF.
Binding proteins of the invention can be conjugated with other molecules, depending upon their intended use. For example, if the binding protein is going to be used as a therapeutic, then the binding protein can be conjugated with another agent, for example, an effector molecule that modulates or otherwise promotes the therapy. To the extent that the effector is non-protein based agent, for example, a small molecule drug, a radiolabel or toxin, then, the agent can be chemically coupled to the binding protein using standard in vitro coupling chemistries. If, on the other hand, the effector molecule is a protein or peptide, for example, an enzyme, receptor, toxin, growth factor, cytokine or other immunomodulator, then the binding protein can either be chemically coupled to the effector using in vitro coupling chemistries or can be coupled to the effector as a fusion protein. Fusion proteins can be constructed and expressed using the techniques similar to those discussed in section II.
IV—Use of Binding Proteins
The binding proteins described herein can be used as a diagnostic agent or a therapeutic agent.
(1) Therapeutic Applications
Because the binding proteins of the invention neutralize the activity of HGF, they can be used in various therapeutic applications. For example, certain binding proteins of the invention are useful in the prevention or treatment of hyperproliferative diseases or disorders, e.g., various forms of cancer.
The binding proteins can be used to inhibit or reduce the proliferation of tumor cells. In such an approach, the tumor cells are exposed to a therapeutically effective amount of the binding protein so as to inhibit or reduce proliferation of the tumor cell. In certain embodiments, the binding proteins inhibit tumor cell proliferation by at least 50%, 60%, 70%, 80%, 90%, 95% or 100%.
In certain embodiments, the binding protein is used to inhibit or reduce proliferation of a tumor cell wherein the binding protein reduces the ability of hHGF to bind to c-Met. In other embodiments, the binding protein is used to inhibit or reduce the proliferation of a tumor cell even when the binding protein binds hHGF but does not substantially inhibit hHGF binding to c-Met, as shown by antibody 3B6 in Tables 5 and 6.
In addition, the binding protein can be used to inhibit, or slow down tumor growth or development in a mammal. In such a method, an effective amount of the binding protein is administered to the mammal so as to inhibit or slow down tumor growth in the mammal. Accordingly, the binding proteins can be used to treat tumors, for example, in a mammal. The method comprises administering to the mammal a therapeutically effective amount of the binding protein. The binding protein can be administered alone or in combination with another pharmaceutically active molecule, so as to treat the tumor.
It is contemplated that the binding proteins of the invention can be used in the treatment of a variety of HGF responsive disorders, including, for example, HGF responsive tumor cells in lung cancer, breast cancer, colon cancer, prostate cancer, ovarian cancer, head and neck cancer, ovarian cancer, multiple myeloma, liver cancer, gastric cancer, esophageal cancer, kidney cancer, nasopharangeal cancer, pancreatic cancer, mesothelioma, melanoma and glioblastoma.
As used herein, “treat, “treating” and “treatment” refer to the treatment of a disease-state in a mammal, particularly in a human, and include: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, i.e., arresting its development; and/or (c) relieving the disease-state, i.e., causing regression of the disease state.
Generally, a therapeutically effective amount of active component will be in the range of from about 0.1 mg/kg to about 100 mg/kg, optionally from about 1 mg/kg to about 100 mg/kg, optionally from about 1 mg/kg to 10 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health status of the particular patient, the relative biological efficacy of the binding protein delivered, the formulation of the binding protein, the presence and types of excipients in the formulation, and the route of administration. The initial dosage administered may be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level, or the initial dosage may be smaller than the optimum and the daily dosage may be progressively increased during the course of treatment depending on the particular situation. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease condition being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. Formulation of monoclonal antibody-based drugs is within ordinary skill in the art. In some embodiments of the invention, the binding protein, e.g., monoclonal antibody, is lyophilized and reconstituted in buffered saline at the time of administration.
The binding proteins may be administered either alone or in combination with other pharmaceutically active ingredients. The other active ingredients, e.g., immunomodulators, can be administered together with the binding protein, or can be administered before or after the binding protein.
Formulations containing the binding proteins for therapeutic use, typically include the binding proteins combined with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” means buffers, carriers, and excipients, that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be “acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Pharmaceutically acceptable carriers, in this regard, are intended to include any and all buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
The formulations can be conveniently presented in a dosage unit form and can be prepared by any suitable method, including any of the methods well known in the pharmacy art. A pharmaceutical composition of the invention should be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration or non-parenteral administration, for example, intravenous, intradermal, inhalation, transdermal (topical), transmucosal, and rectal administration. Useful solutions for oral or parenteral administration can be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
Formulations suitable for oral administration can be in the form of: discrete units such as injectables, capsules, gelatin capsules, sachets, tablets, troches, or lozenges, each containing a predetermined amount of the binding protein; a powder or granular composition; a solution or a suspension in an aqueous liquid or non-aqueous liquid; or an oil-in-water emulsion or a water-in-oil emulsion.
Formulations suitable for parenteral administration include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
In general, compositions suitable for injectable use include aqueous solutions (where water soluble) or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted prior to or following lyophilization and reconstitution. Once the pharmaceutical composition has been formulated, it can be stored, for example, in vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
(2) Diagnostic Applications
Whenever the binding proteins are used for diagnostic purposes, either in vitro or in vivo, the binding proteins typically are labeled either directly or indirectly with a detectable moiety. The detectable moiety can be any moiety which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3Hydrogen (3H), 14Carbon (14C), 32Phosphorus (32P), 35Sulfur (35S), or 125Iodine (125I); a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; a spin probe, such as a spin label; or a colored particle, for example, a latex or gold particle. It is understood that the binding protein can be conjugated to the detectable moiety using a number of approaches known in the art, for example, as described in Hunter et al. (1962) NATURE 144: 945; David et al. (1974) BIOCHEMISTRY 13: 1014; Pain et al. (1981) J. IMMUNOL. METH. 40: 219; and Nygren (1982) J. HISTOCHEM. AND CYTOCHEM. 30: 407. The labels may be detected, e.g., visually or with the aid of a spectrophotometer or other detector.
The binding proteins can be employed in a wide range of immunoassay techniques available in the art. Exemplary immunoassays include, for example, sandwich immunoassays, competitive immunoassays, immunohistochemical procedures.
In a sandwich immunoassay, two antibodies that bind an analyte or antigen of interest are used, e.g., one immobilized onto a solid support, and one free in solution and labeled with a detectable moiety. When a sample containing the antigen is introduced into this system, the antigen binds to both the immobilized antibody and the labeled antibody, to form a “sandwich” immune complex on the surface of the support. The complexed protein is detected by washing away non-bound sample components and excess labeled antibody, and measuring the amount of labeled antibody complexed to protein on the support's surface. Alternatively, the antibody free in solution can be detected by a third antibody labeled with a detectable moiety which binds the free antibody. A detailed review of immunological assay design, theory and protocols can be found in numerous texts, including Butt, ed., (1984) PRACTICAL IMMUNOLOGY, Marcel Dekker, New York; Harlow et al. eds. (1988) ANTIBODIES, A LABORATORY APPROACH, Cold Spring Harbor Laboratory; and Diamandis et al., eds. (1996) IMMUNOASSAY, Academic Press, Boston.
It is contemplated that the labeled binding proteins are useful as in vivo imaging agents, whereby the binding proteins can target the imaging agents to particular tissues of interest in the recipient. A preferred remotely detectable moiety for in vivo imaging includes the radioactive atom Technetium-99m (99mTc), a gamma emitter with a half-life of about six hours. Non-radioactive moieties also useful in in vivo imaging include nitroxide spin labels as well as lanthanide and transition metal ions all of which induce proton relaxation in situ. In addition to immunoimaging, the complexed radioactive moieties may be used in standard radioimmunotherapy protocols to destroy the targeted cell. Preferred nucleotides for high dose radioimmunotherapy include the radioactive atoms 90Yttrium (90Yt), 131Iodine (131I) and 111Indium (111In). The binding protein can be labeled with 131I, 111In and 99mTC using coupling techniques known in the imaging arts. Similarly, procedures for preparing and administering the imaging agent as well as capturing and processing images are well known in the imaging art and so are not discussed in detail herein. Similarly, methods for performing antibody-based immunotherapies are well known in the art. See, for example, U.S. Pat. No. 5,534,254.
Throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Except where indicated otherwise, the order of steps or order for performing certain actions are immaterial so long as the invention remains operable. Moreover, unless otherwise noted, two or more steps or actions may be conducted simultaneously.
EXAMPLES
The following Examples discuss the production and characterization of a number of anti-hHGF monoclonal antibodies.
Example 1
Production of Anti-hHGF Monoclonal Antibodies
This Example describes the production of a number of anti-hHGF monoclonal antibodies.
Immunizations, fusions, and primary screens were conducted at MBS Inc. (Portland, Me.), following the Repetitive Immunization Multiple Sites (RIMMS) protocol. Five AJ mice and Five Balb/c mice were immunized with recombinant human HGF (R&D Systems, Minneapolis, Minn.; Catalog No. 294-HGN-025). Two mice with sera displaying highest anti-HGF activity by Enzyme Linked Immunosorbent Assay (ELISA) were chosen for subsequent fusion. Spleens and lymph nodes from the appropriate mice were harvested. B-cells then were harvested and fused with an myeloma line. Fusion products were serially diluted on one or more plates to near clonality. Supernatants from the resulting fusions were screened for their binding to hHGF by ELISA. Supernatants identified as containing antibodies to HGF were further characterized by in vitro functional testing as discussed in the following examples. A panel of hybridomas was selected and the hybridomas were subcloned and expanded. The monoclonal antibodies then were purified by affinity chromatography on Protein A/G resin under standard conditions.
Example 2
Sequence Analysis of Anti-hHGF Monoclonal Antibodies
This Example describes isotype and sequence analyses of the anti-hHGF monoclonal antibodies produced in Example 1.
a. Determination of HGF Murine Monoclonal Antibody Isotypes
The light-chain type and heavy chain isotype of each monoclonal antibody were determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit in accordance the manufacturer's instructions (Roche Applied Science).
All the antibodies were determined to contain a Kappa immunoglobulin light chain and an IgG1 immunoglobulin heavy chain.
b. Determination of Nucleotide Sequences Encoding Immunoglobulin Heavy and Light Chain Variable Regions
Total RNA was extracted from each monoclonal hybridoma cell line using the RNeasy Miniprep kit according to the manufacturer's instructions (Qiagen Venlo, The Netherlands). Full-length first strand cDNA was generated using the BD SMARTTM RACE cDNA Amplification Kit according to the manufacturer's instructions (Clontech) using the oligonucleotide primers BD SMART II A (5′ aagcagtggtatcaacgcagagtacgcggg 3′) (SEQ ID NO. 85) and 5′-RACE CDS Primer (5′ tttttttttttttttttttttttttvn 3′, where v=a, g, or c and n=a, g, c, or t) (SEQ ID NO. 86) for the purpose of 5′ RACE (Rapid Amplification of cDNA Ends).
The variable regions of the Kappa and Heavy (IgG1) immunoglobulin chains were amplified by PCR (Polymerase Chain Reaction) using the Expand High-Fidelity PCR System (Roche Applied Science) according to the manufacturer's instructions. Heavy chain variable regions were amplified with the 5′ oligonucleotide primer mix Universal Primer Mix A (mix of 5′ ctaatacgactcactatagggcaagcagtggtatcaacgcagagt 3′ (SEQ ID NO. 87) and 5′ ctaatacgactcactatagggc 3′ (SEQ ID NO. 88)) and a 3′ IgG1 Constant Region specific primer, either 5′ tatgcaaggcttacaaccaca 3′ (SEQ ID NO. 89) or 5′ gccagtggatagacagatgggggtgtcg 3′ (SEQ ID NO. 90). Kappa chain variable regions were amplified with the 5′ oligonucleotide primer mix Universal Primer Mix A and a 3′ Kappa Constant Region specific primer, either 5′ ctcattcctgttgaagctcttgacaat 3′ (SEQ ID NO. 91) or 5′ cgactgaggcacctccagatgtt 3′ (SEQ ID NO. 92).
Individual PCR products were fractionated by agarose gel electrophoresis and purified using the Qiaquick Gel Purification kit according to the manufacturer's instructions (Qiagen). The PCR products were subsequently cloned into the pCR2.1 TOPO plasmid using the topoisomerase based cloning kit TOPO TA Cloning® Kit (with pCR®2.1-TOPO® vector) according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.) and transformed into DH5 bacteria using standard transformation techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using T7 (5′ TAATACGACTCACTATAGGG 3′) (SEQ ID NO. 93), M13 Forward (5′ GTAAAACGACGGCCAGT 3′) (SEQ ID NO. 94), and M13 Reverse primers (5′ CAGGAAACAGCTATGACC 3′) (SEQ ID NO. 95) by Agencourt Bioscience using standard dideoxy DNA sequencing methods to identify the sequence of the variable region sequences. The sequences were analyzed using Vector NTI software (Invitrogen, Carlsbad, Calif.) and the IMGTN-Quest webserver (http://imgt.cines.fr/textes/vquest) to identify and confirm variable region sequences.
c. Determination of Nucleotide Sequences Encoding Immunoglobulin Heavy and Light Chain Constant Region Sequences for 1A3, 1D3, 1F3, and 2B8 Kappa and IgG1 Chains
Full Length cDNAs for the 1A3, 1D3, and 1F3 IgG1 chains were PCR amplified from the cDNA created above using the forward primer 5′ ggggacaagtttgtacaaaaaagcaggctgccaccatgaactttgggctcagattgattttcc 3′ (start codon underlined) (SEQ ID NO. 96) and the reverse primer 5′ ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagagg 3′ (stop codon underlined) (SEQ ID NO. 97). Full Length cDNA for the 2B8 IgG1 chain was amplified from the cDNA created above using the forward primer 5′ ggggacaagtttgtacaaaaaagcaggctgccaccatgggatggagctatatcatcctcttt 3′ (start codon underlined) (SEQ ID NO. 98) and reverse primer 5′ ggggaccactttgtacaagaaagctgggttcatttaccaggagagtgggagag 3′ (stop codon underlined) (SEQ ID NO. 99).
Full Length cDNA for the 2B8 Kappa Chain was amplified using the forward primer 5′ ggggacaagtttgtacaaaaaagcaggctgccaccatggaatcacagactctggtcttcata 3′ (start codon underlined) (SEQ ID NO. 100) and the reverse primer 5′ ggggaccactttgtacaagaaagctgggtctaacactcattcctgttgaagctc 3′ (stop codon underlined) (SEQ ID NO. 101). PCR fragments were subcloned into pDONR221 (Invitrogen, Carlsbad, Calif.) by Gateway BP recombination reaction (Invitrogen, Carlsbad, Calif.) and sequenced by Agencourt Bioscience using standard dideoxy DNA sequencing methods to identify the sequence of the constant region and further confirm variable region sequences.
d. Sequence Analysis
Variable Regions (normal text) were identified using IMGT/V-QUEST webserver software (http://imgt.cines.fr/textes/vquest/). Signal Peptide sequences were predicted based on identification of the in frame start codon (ATG) that was upstream of the identified Variable Region. Signal Peptide sequences were identified and are underlined below.
The last nucleotide of each variable region is the first base of the next codon generated by the variable/constant region junction. This nucleotide is included in the variable region because it is part of that exon. Amino acid sequences of the constant regions listed below include the translation of this junction codon.
In order to create the complete heavy or kappa chain antibody sequences, the variable region sequences noted below are combined with their respective constant region sequences (the signal sequences are underlined).








(1) 1A3 Heavy Chain Variable Region



(SEQ ID NO. 1)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc


 


121
tgtgcagcct ctgaattcac tttcagtaac tattacatgt cttgggttcg ccagactcca


 


181
gagaagaggc tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca


 


241
gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg


 


301
caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat


 


361
ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc


 


421
tcag


 








(2) 1A3 Kappa Light Chain Variable Region



(SEQ ID NO. 3)










1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt



 


61
gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga aactgtcacc


 


121
atcacatgtc gagcaagtga gaatatttat agtaatttag catggtatca gcagaaacag


 


181
ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct


 


301
gaagattttg ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg


 


361
gggaccaagc tggaaataaa ac


 








(3) 2B8 Heavy Chain Variable Region



(SEQ ID NO. 11)










1
atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag



 


61
gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc


 


121
tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct


 


181
ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat


 


241
gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg


 


301
caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt


 


361
ggtagcatct ttgactactg gggccaaggc accactctca cagtctcctc ag


 








(4) 2B8 Kappa Light Chain Variable Region



(SEQ ID NO. 13)










1
atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg



 


61
aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc


 


121
ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca


 


181
gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat


 


241
cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct


 


301
gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg


 


361
gggaccaggc tggaaataaa ac


 








(5) 2F8 Heavy Chain Variable Region



(SEQ ID NO. 21)










1
atggaatgga gctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag



 


61
gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt gaagatgtcc


 


121
tgcaaggctt ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct


 


181
ggacagggcc ttgagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat


 


241
gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac agcctacatg


 


301
cagctcagca gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg


 


361
ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc ag


 








(6) 2F8 Kappa Light Chain Variable Region



(SEQ ID NO. 23)










1
atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt



 


61
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc


 


121
atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat caactggtac


 


181
caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct


 


241
gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat


 


301
cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtattga ggatcctccc


 


361
acgttcggtg ctgggaccaa gctggagctg aaac


 








(7) 3B6 Heavy Chain Variable Region



(SEQ ID NO. 31)










1
atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag



 


61
gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc


 


121
tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct


 


181
ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat


 


241
ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg


 


301
cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg


 


361
ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcag


 








(8) 3B6 Kappa Light Chain Variable Region (2 possible ATG start codons



(uppercase))


(SEQ ID NO. 33)










1
ATGgacATGa ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc



 


61
aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga


 


121
gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag


 


181
aaaccaggga aatctcctaa gaccctgatc tatcgtgtaa acagattggt agatggggtc


 


241
ccatcaaggt tcagtggcag tggatctggg caagattctt ctctcaccat caccagcctg


 


301
gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc


 


361
ggagggggga ccaagctgga aataaagc


 








(9) 3D11 Heavy Chain Variable Region



(SEQ ID NO. 41)










1
atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag



 


61
gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact


 


121
tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca


 


181
ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg


 


241
tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa


 


301
atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct


 


361
tactggggcc aagggactct ggtcactgtc tctgcag


 








(10) 3D11 Kappa Light Chain Variable Region



(SEQ ID NO. 43)










1
atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc



 


61
agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag


 


121
gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag


 


181
tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct


 


241
gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag tagtatggag


 


301
gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt


 


361
gctgggacca agctggagct gaaac


 








(11) 1D3 Heavy Chain Variable Region



(SEQ ID NO. 51)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc


 


121
tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca


 


181
gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca


 


241
gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg


 


301
caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat


 


361
ggttattacg gggactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc


 


421
tcag


 








(12) 1D3 Kappa Light Chain Variable Region



(SEQ ID NO. 53)










1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt



 


61
gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc


 


121
atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag


 


181
ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct


 


301
gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg


 


361
gggaccaaac tggaaataaa ac


 








(13) 1F3 Heavy Chain Variable Region



(SEQ ID NO. 61)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc


 


121
tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca


 


181
gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca


 


241
gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg


 


301
caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat


 


361
ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc


 


421
tcag


 








(14) 1F3 Kappy Light Chain Variable Region



(SEQ ID NO. 63)










1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt



 


61
gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc


 


121
atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag


 


181
ggaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct


 


301
gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg


 


361
gggaccagac tggaattaa ac


 








(15) 3A12 Heavy Chain Variable Region



(SEQ ID NO. 71)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc


 


121
tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca


 


181
gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca


 


241
gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg


 


301
caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat


 


361
ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc


 


421
tcag


 








(16) 3A12 Kappa Light Chain Variable Region



(SEQ ID NO. 73)










1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt



 


61
gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc


 


121
atcacatgtc gagcaagtga gaatatttac attaatttag catggtatca gcagaaacag


 


181
ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct


 


301
gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg


 


361
gggaccaaac tagaaataaa ac


 








(17) Reference Mouse IgG1 Heavy Chain Constant Region (J00453)



(SEQ ID NO. 81)










1
ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact



 


61
ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct


 


121
ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg gagtctgacc


 


181
tctacactct gagcagctca gtgactgtcc cctccagccc tcggcccagc gagaccgtca


 


241
cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg


 


301
attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc


 


361
ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg


 


421
tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg


 


481
tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca


 


541
gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca


 


601
acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga


 


661
aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca


 


721
gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga


 


781
atgggcagcc agcggagaac tacaagaaca ctcagcccat catgaacacg aatggctctt


 


841
acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca


 


901
cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact


 


961
ctcctggtaa atga


 








(18) Mouse IgG1 Heavy Chain Constant Region Determined for 1A3, 1D3,



1F3, and 2B8 (derived from AJ strain mice)


(SEQ ID NO. 82)










1
ccaaaacgac acccccatct gtctatccac tggcccctgg atctgctgcc caaactaact



 


61
ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg acagtgacct


 


121
ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg cagtctgacc


 


181
tctacactct gagcagctca gtgactgtcc cctccagcac ctggcccagc gagaccgtca


 


241
cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt gtgcccaggg


 


301
attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc ttcatcttcc


 


361
ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg


 


421
tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat gatgtggagg


 


481
tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc cgctcagtca


 


541
gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa tgcagggtca


 


601
acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa ggcagaccga


 


661
aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag gataaagtca


 


721
gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag tggcagtgga


 


781
atgggcagcc agcggagaac tacaagaaca ctcagcccat catggacaca gatggctctt


 


841
acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga aatactttca


 


901
cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc ctctcccact


 


961
ctcctggtaa atga


 








(19) Reference Mouse Kappa Light Chain Constant Region (V00807) and Mouse



Kappa Light Chain Constant Region Determined for 1D3, 1F3, and 2B8 (derived


from AJ strain mice)


(SEQ ID NO. 83)










1
gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg



 


61
gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt


 


121
ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca


 


181
gcaaagacag cacctacagc atgagcagca ccctcacgtt gaccaaggac gagtatgaac


 


241
gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga


 


301
gcttcaacag gaatgagtgt tag


 








(20) Mouse Kappa Light Chain Constant Region Determined for 1A3 containing



one altered nucleotide compared to 1D3, 1F3, and 2B8 (underlined)


(SEQ ID NO. 84)










1
gggctgatgc tgcaccaact gtatccatct tcccaccatc cagtgagcag ttaacatctg



 


61
gaggtgcctc agtcgtgtgc ttcttgaaca acttctaccc caaagacatc aatgtcaagt


 


121
ggaagattga tggcagtgaa cgacaaaatg gcgtcctgaa cagttggact gatcaggaca


 


181
gcaaagacag cacctacagc atgagcagca ccctcatgtt gaccaaggac gagtatgaac


 


241
gacataacag ctatacctgt gaggccactc acaagacatc aacttcaccc attgtcaaga


 


301
gcttcaacag gaatgagtgt tag






Each of the amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 1 are set forth in FIG. 2. Each of the sequences are aligned with one another and the sequences defining the signal peptide, CDR1, CDR2 and CDR3 are identified by boxes. FIG. 3 shows an alignment of the separate CDR1, CDR2 and CDR3 sequences for each of the antibodies.
Each of the amino acid sequences defining the immunoglobulin light chain variable regions for each of the antibodies produced in Example I are set forth in FIG. 4. Each of the sequences are aligned with one another and the sequences defining the signal peptide, CDR1, CDR2 and CDR3 are identified by boxes. FIG. 5 shows an alignment of the separate CDR1, CDR2 and CDR3 sequences for each of the antibodies.
For convenience, Table 1 provides a concordance chart showing the correspondence between the antibody sequences discussed in this Example with those presented in the Sequence Listing.








TABLE 1





SEQ. ID NO.
Protein or Nucleic Acid
















1
Heavy Chain Variable Region 1A3-nucleic acid


2
Heavy Chain Variable Region 1A3-protein


3
Light (kappa) Chain Variable Region 1A3-nucleic acid


4
Light (kappa) Chain Variable Region 1A3-protein


5
Heavy Chain CDR1 1A3


6
Heavy Chain CDR2 1A3


7
Heavy Chain CDR3 1A3


8
Light (kappa) Chain CDR1 1A3


9
Light (kappa) Chain CDR2 1A3


10
Light (kappa) Chain CDR3 1A3


11
Heavy Chain Variable Region 2B8-nucleic acid


12
Heavy Chain Variable Region 2B8-protein


13
Light (kappa) Chain Variable Region 2B8-nucleic acid


14
Light (kappa) Chain Variable Region 2B8-protein


15
Heavy Chain CDR1 2B8


16
Heavy Chain CDR2 2B8


17
Heavy Chain CDR3 2B8


18
Light (kappa) Chain CDR1 2B8


19
Light (kappa) Chain CDR2 2B8


20
Light (kappa) Chain CDR3 2B8


21
Heavy Chain Variable Region 2F8-nucleic acid


22
Heavy Chain Variable Region 2F8-protein


23
Light (kappa) Chain Variable Region 2F8-nucleic acid


24
Light (kappa) Chain Variable Region 2F8-protein


25
Heavy Chain CDR1 2F8


26
Heavy Chain CDR2 2F8


27
Heavy Chain CDR3 2F8


28
Light (kappa) Chain CDR1 2F8


29
Light (kappa) Chain CDR2 2F8


30
Light (kappa) Chain CDR3 2F8


31
Heavy Chain Variable Region 3B6-nucleic acid


32
Heavy Chain Variable Region 3B6-protein


33
Light (kappa) Chain Variable Region 3B6-nucleic acid


34
Light (kappa) Chain Variable Region 3B6-protein


35
Heavy Chain CDR1 3B6


36
Heavy Chain CDR2 3B6


37
Heavy Chain CDR3 3B6


38
Light (kappa) Chain CDR1 3B6


39
Light (kappa) Chain CDR2 3B6


40
Light (kappa) Chain CDR3 3B6


41
Heavy Chain Variable Region 3D11-nucleic acid


42
Heavy Chain Variable Region 3D11-protein


43
Light (kappa) Chain Variable Region 3D11-nucleic acid


44
Light (kappa) Chain Variable Region 3D11-protein


45
Heavy Chain CDR1 3D11


46
Heavy Chain CDR2 3D11


47
Heavy Chain CDR3 3D11


48
Light (kappa) Chain CDR1 3D11


49
Light (kappa) Chain CDR2 3D11


50
Light (kappa) Chain CDR3 3D11


51
Heavy Chain Variable Region 1D3-nucleic acid


52
Heavy Chain Variable Region 1D3-protein


53
Light (kappa) Chain Variable Region 1D3-nucleic acid


54
Light (kappa) Chain Variable Region 1D3-protein


55
Heavy Chain CDR1 1D3


56
Heavy Chain CDR2 1D3


57
Heavy Chain CDR3 1D3


58
Light (kappa) Chain CDR1 1D3


59
Light (kappa) Chain CDR2 1D3


60
Light (kappa) Chain CDR3 1D3


61
Heavy Chain Variable Region 1F3-nucleic acid


62
Heavy Chain Variable Region 1F3-protein


63
Light (kappa) Chain Variable Region 1F3-nucleic acid


64
Light (kappa) Chain Variable Region 1F3-protein


65
Heavy Chain CDR1 1F3


66
Heavy Chain CDR2 1F3


67
Heavy Chain CDR3 1F3


68
Light (kappa) Chain CDR1 1F3


69
Light (kappa) Chain CDR2 1F3


70
Light (kappa) Chain CDR3 1F3


71
Heavy Chain Variable Region 3A12-nucleic acid


72
Heavy Chain Variable Region 3A12-protein


73
Light (kappa) Chain Variable Region 3A12-nucleic acid


74
Light (kappa) Chain Variable Region 3A12-protein


75
Heavy Chain CDR1 3A12


76
Heavy Chain CDR2 3A12


77
Heavy Chain CDR3 3A12


78
Light (kappa) Chain CDR1 3A12


79
Light (kappa) Chain CDR2 3A12


80
Light (kappa) Chain CDR3 3A12









Also, for convenience, the following sequences represent the actual or contemplated full length heavy and light chain sequences (i.e., containing both the variable and constant region sequences) for each of the antibodies described in this Example. It is noted that the constant regions of the murine antibodies 2F8, 3A12, 3B6, and 3D11 were not sequenced but are presumed to have the same constant region sequences as the 1D3, 1F3, and 2B8 antibodies, which were sequenced, as they were all derived from AJ strain mice. It is appreciated, however, that the variable region sequences described herein can be ligated to each of a number of other constant region sequences known to those skilled in the art to produce active full length immunoglobulin heavy and light chains.








(1) Nucleic Acid Sequence Encoding the Full Length 1A3 Heavy Chain



sequence (1A3 Heavy Chain Variable Region and IgG1 Constant Region)


(signal sequence underlined)


(SEQ ID NO. 122)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc


 


121
tgtgcagcct ctgaattcac tttcagtaac tattacatgt cttgggttcg ccagactcca


 


181
gagaagaggc tgcagtgggt cgcatacatt agtcctggtg gtggtagctc ctactatcca


 


241
gccagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg


 


301
caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgcaag acaaggggat


 


361
ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc


 


421
tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact


 


481
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg


 


541
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct


 


601
gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc


 


661
gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc


 


721
agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc


 


781
ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt


 


841
gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg


 


901
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca


 


961
gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg


 


1021
gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga


 


1081
ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa


 


1141
gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag


 


1201
tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc


 


1261
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact


 


1321
ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc


 


1381
cactctcctg gtaaatga


 








(2) Protein Sequence Defining the Full Length 1A3 Heavy Chain Sequence (1A3



Heavy Chain Variable Region and IgG1 Constant Region)(without signal sequence)


(SEQ ID NO. 123)










1
evqlvesggg lvqpggslkl scaaseftfs nyymswvrqt pekrlqwvay ispgggssyy



 


61
pasvkgrfti srdnakntly lqmsslksed tamyycarqg dgyygdyamd ywgqgtsvtv


 


121
ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq


 


181
sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf


 


241
ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr


 


301
syselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd


 


361
kvsltcmitd ffneditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn


 


421
tftcsvlheg lhnhhteksl shspgk


 








(3) Nucleic Acid Sequence Encoding the Full Length 1A3 Light Chain Sequence



(1A3 Kappa Variable Region and Constant Region)(signal sequence underlined)


(SEQ ID NO. 124)










1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt



 


61
gacatccaga tgactcagtc tccagcctcc ctatctgttt ctgtgggaga aactgtcacc


 


121
atcacatgtc gagcaagtga gaatatttat agtaatttag catggtatca gcagaaacag


 


181
gaaaaatctc ctcagctcct ggtctatgct gcaacaaact tagcagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct


 


301
gaagattttg ggacttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg


 


361
gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca


 


421
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac


 


481
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg


 


541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcatg


 


601
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca


 


661
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag


 








(4) Protein Sequence Defining the Full Length 1A3 Light Chain Sequence (1A3



Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 125)










1
diqmtqspas lsysvgetvt itcraseniy snlawyqqkq gkspqllvya atnladgvps



 


61
rfsgsgsgtq fslkinslqs edfgtyycqh fwgtpytfgg gtkleikrad aaptvsifpp


 


121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlm


 


181
ltkdeyerhn sytceathkt stspivksfn rnec


 








(5) Nucleic Acid Sequence Encoding the Full Length 2B8 Heavy Chain Sequence



(2B8 Heavy Chain Variable Region and IgG1 Constant Region)(signal sequence


underlined)


(SEQ ID NO. 126)










1
atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag



 


61
gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc


 


121
tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct


 


181
ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat


 


241
gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg


 


301
caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt


 


361
ggtaacatct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg


 


421
acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg


 


481
accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct


 


541
ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact


 


601
ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac


 


661
attgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt


 


721
tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag


 


781
cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc


 


841
agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca


 


901
gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt


 


961
cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca


 


1021
gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca


 


1081
caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc


 


1141
tacatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag


 


1201
ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc


 


1261
tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct


 


1321
gtgttacatg agggcctgca caaccaccat actgagaaaa gcctctccca ctctcctggt


 


1381
aaatga


 








(6) Protein Sequence Defining the Full Length 2B8 Heavy Chain Sequence (2B8



Heavy Chain Variable Region and IgG1 Constant Region)(without signal


sequence)


(SEQ ID NO. 127)










1
qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewige inptnghtny



 


61
nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq gttltvssak


 


121
ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn sgslssgvht fpavlqsdly


 


181
tlsssvtvps stwpsetvtc nvahpasstk vdkkivprdc gckpcictvp evssvfifpp


 


241
kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnstfrsvse


 


301
lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk eqmakdkvsl


 


361
tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf vysklnvqks nweagntftc


 


421
svlheglhnh htekslshsp gk


 








(7) Nucleic Acid Sequence Encoding the Full Length 2B8 Light Sequence



(2B8 Kappa Variable Region and Constant Region)(signal sequence


underlined)


(SEQ ID NO. 128)










1
atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg



 


61
aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc


 


121
ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca


 


181
gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat


 


241
cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct


 


301
gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg


 


361
gggaccaggc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca


 


421
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac


 


481
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg


 


541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg


 


601
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca


 


661
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag


 








(8) Protein Sequence Defining the Full Length 2B8 Light Chain Sequence (2B8



Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 129)










1
nivmtqspks msmsvgervt lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd



 


61
rftgsgsatd ftltissvra edladyhcgq synypytfgg gtrleikrad aaptvsifpp


 


121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt


 


181
ltkdeyerhn sytceathkt stspivksfn rnec


 








(9) Nucleic Acid Sequence Encoding the Full Length 2F8 Heavy Chain Sequence



(2F8 Heavy Chain Variable Region and IgG1 Constant Region)(signal sequence


underlined)


(SEQ ID NO. 130)










1
atggaatgga gctgggtctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactgccag



 


61
gtccagctga agcagtctgg agctgagctg gtgaggcctg ggacttcagt gaagatgtcc


 


121
tgcaaggctt ctggctacac cttcactacc tactatatac actgggtgaa tcagaggcct


 


181
ggacaaggcc ttaagtggat tggaaagatt ggtcctggaa gtggtagtac ttactacaat


 


241
gagatgttca aagacaaggc cacattgact gtagacacat cctccagcac agcctacatq


 


301
cagctcagca gcctgacatc tgacgactct gcggtctatt tctgtgcaag aaggggactg


 


361
ggacgtggct ttgactactg gggccaaggc accactctca cagtctcctc agccaaaacg


 


421
acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg


 


481
accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct


 


541
ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact


 


601
ctgagcagct caatgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac


 


661
gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt


 


721
tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag


 


781
cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc


 


841
agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca


 


901
gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt


 


961
cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca


 


1021
gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca


 


1081
caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc


 


1141
tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag


 


1201
ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc


 


1261
tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct


 


1321
gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt


 


1381
aaatga


 








(10) Protein Sequence Defining the Full Length 2F8 Heavy Chain Sequence (2F8



Heavy Chain Variable Region and IgG1 Constant Region)(without signal


sequence)


(SEQ ID NO. 131)










1
qvqlkqsgae lvrpgtsvkm sckasgytft tyyihwvnqr pgqglewigk igpgsgstyy



 


61
nemfkdkatl tvdtssstay mqlssltsdd savyfcarrg lgrgfdywgq gttltvssak


 


121
ttppsvypla pgsaaqtnsm vtlgclvkgy fpepvtvtwn sgslssgvht fpavlqsdly


 


181
tlsssvtvps stwpsetvtc nvahpasstk vdkkivprdc gckpcictvp evssvfifpp


 


241
kpkdvltitl tpkvtcvvvd iskddpevqf swfvddvevh taqtqpreeq fnstfrsvse


 


301
lpimhqdwln gkefkcrvns aafpapiekt isktkgrpka pqvytipppk eqmakdkvsl


 


361
tcmitdffpe ditvewqwng qpaenykntq pimdtdgsyf vysklnvqks nweagntftc


 


421
svlheglhnh htekslshsp gk


 








(11) Nucleic Acid Sequence Encoding the Full Length 2F8 Light Chain Sequence



(2F8 Kappa Variable Region and Constant Region)(signal sequence underlined)


(SEQ ID NO. 132)










1
atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt



 


61
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc


 


121
atctcctgca aggccagcca aagtgttgat tatgatggta atagttatat caactggtac


 


181
caacagaaac caggacagcc acccaaagtc ctcatctatg ttgcatccaa tctagaatct


 


241
gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat


 


301
cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtattga ggatcctccc


 


361
acgttcggtg ctgggaccaa gctggagctg aaacgggctg atgctgcacc aactgtatcc


 


421
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg


 


481
aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa


 


541
aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc


 


601
agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc


 


661
actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag


 








(12) Protein Sequence Defining the Full Length 2F8 Light Chain Sequence



(2F8 Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 133)










1
divltqspas lavslgqrat isckasqsvd ydgnsyinwy qqkpgqppkv liyvasnles



 


61
giparfsgsg sgtdftlnih pveeedaaty ycqgsiedpp tfgagtklel kradaaptvs


 


121
ifppsseqlt sggasvvcfl nnfypkdinv kwkidgserq ngvlnswtdq dskdstysms


 


181
stltltkdey erhnsytcea thktstspiv ksfnrnec


 








(13) Nucleic Acid Sequence Encoding the Full Lenght 3B6 Heavy Chain Sequence



(3B6 Heavy Chain Variable Region and IgG1 Constant Region)(signal sequence


underlined)


(SEQ ID NO. 134)










1
atggaatggc cttgtatctt tctcttcctc ctgtcagtaa ctgaaggtgt ccactcccag



 


61
gttcagctgc agcagtctgg ggctgaactg gtgaggcctg ggtcctcagt gaagatttcc


 


121
tgcaaggctt ctggctatgt attcagtagc tactggatga actgggtgaa gcagaggcct


 


181
ggacagggtc ttgagtggat tggacagatt tatcctggag atggtgatag taactacaat


 


241
ggaaacttca agggtaaagc cacactgact gcagacaaat cctccagtac agcctacatg


 


301
cagctcagca gcctaacatc tgaggactct gcggtctatt tctgtgcatc ccagctcggg


 


361
ctacgtgaga actactttga ctactggggc caaggcacca ctctcacagt ctcctcagcc


 


421
aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc


 


481
atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg


 


541
aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc


 


601
tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc


 


661
tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat


 


721
tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc


 


781
ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta


 


841
gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg


 


901
cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt


 


961
gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac


 


1021
agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag


 


1081
gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt


 


1141
ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat


 


1201
gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac


 


1261
ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc


 


1321
tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct


 


1381
cctggtaaat ga


 








(14) Protein Sequence Defining the Full Length 3B6 Heavy Chain Sequence (3B6



Heavy Chain Variable Region and IgG1 Constant Region)(without signal sequence)


(SEQ ID No. 135)










1
qvqlqqsgae lvrpgssvki sckasgyvfs sywmnwvkqr pgqglewigq iypgdgdsny



 


61
ngnfkgkatl tadkssstay mqlssltsed savyfcasql glrenyfdyw gqgttltvss


 


121
akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd


 


181
lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif


 


241
ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv


 


301
selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv


 


361
sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf


 


421
tcsvlheglh nhhtekslsh spgk


 








(15) Nucleic Acid Sequence Encoding the Full Length 3B6 Light Chain Sequence



(3B6 Kappa Variable Region and Constant Region)(signal sequence underlined)


(SEQ ID NO. 136)










1
ATGgacATGa ggacccctgc tcagtttctt ggaatcttgt tgctctggtt tccaggtatc



 


61
aaatgtgaca tcaagatgac ccagtctcca tcttccatgt atgcatctct aggagagaga


 


121
gtcacaatca cttgcaaggc gagtcaggac attaaaagct atttaagctg gttccagcag


 


181
aaaccaggga aatctcctaa gaccctgatc tatcatgtaa acagattggt agatggggtc


 


241
ccatcaaggt tcaatggcag tggatctggg caagattctt ctctcaccat caccagcctg


 


301
gagaatgaag atatgggaat ttattattgt ctacagtatg atgagtttcc gttcacgttc


 


361
ggagggggga ccaagctgga aataaagcgg gctgatgctg caccaactgt atccatcttc


 


421
ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac


 


481
ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc


 


541
gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc


 


601
ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac


 


661
aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgtta g


 








(16) Protein Sequence Defining the Full Length 3B6 Light Chain Sequence (3B6



Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 137)










1
dikmtqspss myaslgervt itckasqdik sylswfqqkp gkspktliyr vnrlvdgvps



 


61
rfsgsgsgqd ssltitslen edmgiyyclq ydefpftfgg gtkleikrad aaptvsifpp


 


121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt


 


181
ltkdeyerhn sytceathkt stspivksfn rnec


 








(17) Nucleic Acid Sequence Encoding the Full Length 3D11 Heavy Chain Sequence



(3D11 Heavy Chain Variable Region and IgG1 Constant Region)(signal sequence


underlined


(SEQ ID NO. 138)










1
atggctgtcc cggtgctgtt cctctgcctg gttgcatttc caagctgtgt cctgtcccag



 


61
gtacagctga aggagtcagg acctggcctg gtggcgccct cacagagcct gtccatcact


 


121
tgcactgtct ctgggttttc attaaccagc tatagtttac actgggttcg ccagcctcca


 


181
ggaaagggtc tggaatggct gggagtaata tgggctggtg gaaacacaaa ttataattcg


 


241
tctctcatgt ccagactgac catcaggaaa gacaactcca agagccaagt tttcttaaaa


 


301
atgaacagtc tgcaaactga tgacacagcc atgtactact gtgccagaga gaggtttgct


 


361
tactggggcc aagggactct ggtcactgtc tctgcagcca aaacgacacc cccatctgtc


 


421
tatccactgg cccctggatc tgctgcccaa actaactcca tggtgaccct gggatgcctg


 


481
gtcaagggct atttccctga gccagtgaca gtgacctgga actctggatc cctgtccagc


 


541
ggtgtgcaca ccttcccagc tgtcctgcag tctgacctct acactctgag cagctcagtg


 


601
actgtcccct ccagcacctg gcccagcgag accgtcacct gcaacgttgc ccacccggcc


 


661
agcagcacca aggtggacaa gaaaattgtg cccagggatt gtggttgtaa gccttgcata


 


721
tgtacagtcc cagaagtatc atctgtcttc atcttccccc caaagcccaa ggatgtgctc


 


781
accattactc tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa ggatgatccc


 


841
gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca gacgcaaccc


 


901
cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat catgcaccag


 


961
gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt ccctgccccc


 


1021
atcgagaaaa ccatctccaa aaccaaaggc agaccgaagg ctccacaggt gtacaccatt


 


1081
ccacctccca aggagcagat ggccaaggat aaagtcagtc tgacctgcat gataacagac


 


1141
ttcttccctg aagacattac tgtggagtgg cagtggaatg ggcagccagc ggagaactac


 


1201
aagaacactc agcccatcat ggacacagat ggctcttact tcgtctacag caagctcaat


 


1261
gtgcagaaga gcaactggga ggcaggaaat actttcacct gctctgtgtt acatgagggc


 


1321
ctgcacaacc accatactga gaagagcctc tcccactctc ctggtaaatg a


 








(18) Protein Sequence Defining the Full Length 3D11 Heavy Chain Sequence



(3D11 Heavy Chain Variable Region and IgG1 Constant Region)(without


signal sequence)


(SEQ ID NO. 139)










1
qvqlkesgpg lvapsqslsi tctvsgfslt syslhwvrqp pgkglewlgv iwaggntnyn



 


61
sslmsrltir kdnsksqvfl kmnslqtddt amyycarerf aywgqgtlvt vsaakttpps


 


121
vyplapgsaa qtnsmvtlgc lvkgyfpepv tvtwnsgsls sgvhtfpavl qsdlytlsss


 


181
vtvpsstwps etvtcnvahp asstkvdkki vqrdcgckpc ictvpevssv fifppkpkdv


 


241
ltitltpkvt cvvvdiskdd pevqfswfvd dvevhtaqtq preeqfnstf rsvselpimh


 


301
gdwlngkefk crvnsaafpa piektisktk grpkapqvyt ipppkeqmak dkvsltcmit


 


361
dffpeditve wqwngqpaen ykntqpimdt dgsyfvyskl nvqksnweag ntftcsvlhe


 


421
glhnhhteks lshspgk


 








(19) Nucleic Acid Sequence Encoding the Full Length 3D11 Light Chain Sequence



(3D11 Kappa Variable Region and Constant Region)(signal sequence underlined)


(SEQ ID NO. 140)










1
atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt caaaatatcc



 


61
agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatatcc aggggagaag


 


121
gtcaccatga cctgcagtgc cagctcaagt gtaagttaca tgcactggta ccagcagaag


 


181
tcaggcacct cccccaaaag atggatttat gacacatcca aactggcttc tggagtccct


 


241
gctcgcttca gtggcagtgg gtctgggacc tcttactccc tcacaatcag tagtatggag


 


301
gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccact cacgttcggt


 


361
gctgggacca agctggagct gaaacgggct gatgctgcac caactgtatc catcttccca


 


421
ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc


 


481
taccccaaag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc


 


541
ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc


 


601
acgttgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag


 


661
acatcaactt cacccattgt caagagcttc aacaggaatg agtgttag


 








(20) Protein Sequence Defining the Full Length 3D11 Light Chain Sequence



(3D11 Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 141)










1
qivltqspai msaypgekvt mtcsasssvs ymhwygqksg tspkrwiydt sklasgvpar



 


61
fsgsgsgtsy sltissmeae daatyycgqw ssnpltfgag tklelkrada aptvsifpps


 


121
seqltsggas vvcflnnfyp kdinvkwkid gserqngvln swtdqdskds tysmsstltl


 


181
tkdeyerhns ytceathkts tspivksfnr nec


 








(21) Nucleic Acid Sequence Encoding the Full Lenght 1D3 Heavy Chain Sequence



(1D3 Heavy Chain Variable Region and IgG1 Constant Region)(signal sequence


underlined)


(SEQ ID NO. 142)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtatgaa



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc


 


121
tgtgcagcct ctggattcac tttcagtgac tattacatgt cttgggttcg ccagactcca


 


181
gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca


 


241
gacagtgtga agggtcgatt caccatctcc cgagacaatg ccaagaacac cctgtacctg


 


301
caaatgagca gtctgaagtc tgaggacaca gccatatatt actgtgtgag acaaggggat


 


361
ggttattacg gagactatgc tatggactac tggggtcaag gaacctcagt catcgtctcc


 


421
tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact


 


481
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg


 


541
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct


 


601
gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc


 


661
gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc


 


721
agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc


 


781
ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt


 


841
gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg


 


901
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca


 


961
gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg


 


1021
gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga


 


1081
ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa


 


1141
gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag


 


1201
tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc


 


1261
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact


 


1321
ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc


 


1381
cactctcctg gtaaatga


 








(22) Protein Sequence Defining the Full Length 1D3 Heavy chain sequence (1D3



Heavy Chain Variable Region and IgG1 Constant Region)(without signal sequence)


(SEQ ID NO. 143)










1
evqlvesggg lvqpggslki scaasgftfs dyymswvrqt pekrlewvay issgggstyy



 


61
pdsvkgrfti srdnakntly lqmsslksed taiyycvrqg dgyygdyamd ywgqgtsviv


 


121
ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq


 


181
sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf


 


241
ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr


 


301
syselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd


 


361
kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn


 


421
tftcsvlheg lhnhhteksl shspgk


 








(23) Nucleic Acid Sequence Encoding the Full Length 1D3 Light Chain Sequence



(1D3 Kappa Variable Region and Constant Region)(signal sequence underlined)


(SEQ ID NO. 144)










1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgtcagatgt



 


61
gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc


 


121
atcacatgtc gaacaagtga gaatatttac agtaatttag cgtggtatca gcagaaacag


 


181
ggaaaatctc ctcagctcct aatctatgct gcaacaaact tagcagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag ttttccctca ggatcaacag cctgcagtct


 


301
gaagattttg ggaggtatta ctgtcaacat ttttggggga ctccgtacac gttcggaggg


 


361
gggaccaaac tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca


 


421
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac


 


481
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg


 


541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg


 


601
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca


 


661
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag


 








(24) Protein Sequence Defining the Full Length 1D3 Light Chain Sequence (1D3



Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 145)










1
diqmtqspas lsvsvgetvt itcrtseniy snlawyqqkq gkspqlliya atnladgvps



 


61
rfsgsgsgtq fslrinslqs edfgryycqh fwgtpytfgg gtkleikrad aaptvsifpp


 


121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt


 


181
ltkdeyerhn sytceathkt stspivksfn rnec


 








(25) Nucleic Acid Sequence Encoding the Full Length 1F3 Heavy Chain Sequence



(1F3 Heavy Chain Variable Region and IgG1 Constant Region)(signal sequence


underlined)


(SEQ ID NO. 146)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgag



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagtctg gagggtccct gaaactctcc


 


121
tgtgcggcct ctggattcac tttcagtaac tatttcatgt cttgggttcg ccagactcca


 


181
gagaagaggc tggagtgggt cgcatatatt agtagtggtg gtggtagcac ctactatcca


 


241
gacagtgtga agggtcgatt caccatctct agagacaatg ccaagaacac cctgtacctg


 


301
caaatgagca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggggat


 


361
ggttactacg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc


 


421
tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact


 


481
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg


 


541
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct


 


601
gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc


 


661
gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc


 


721
agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc


 


781
ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt


 


841
gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg


 


901
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca


 


961
gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg


 


1021
gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga


 


1081
ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa


 


1141
gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag


 


1201
tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc


 


1261
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact


 


1321
ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc


 


1381
cactctcctg gtaaatga


 








(26) Protein Sequence Defining the Full Length 1F3 Heavy Chain Sequence (1F3



Heavy Chain Variable Region and IgG1 Constant Region)(without signal sequence)


(SEQ ID NO. 147)










1
evqlvesggg lvqsggslkl scaasgftfs nyfmswvrqt pekrlewvay issgggstyy



 


61
pdsvkgrfti srdnakntly lqmsslksed tamyycvrqg dgyygdyamd ywgqgtsvtv


 


121
ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfpepvt vtwnsgslss gvhtfpavlq


 


181
sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf


 


241
ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr


 


301
syselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd


 


361
kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn


 


421
tftcsvlheg lhnhhteksl shspgk


 








(27) Nucleic Acid Sequence Encoding the Full Length 1F3 Light Chain Sequence



(1F3 Kappa Variable Region and Constant Region)(signal sequence underlined)


(SEQ ID NO. 148)










1
atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt



 


61
gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc


 


121
atcacatgtc gagcaagtga gaatatttac agtaatttag catggtatca gcagaaacag


 


181
gaaaaatctc ctcagctcct ggtctatgat gcaacacact taccagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag ttttccctca agatcaacag cctgcagtct


 


301
gaagattttg ggaattatta ctgtcaacat ttttggggta ctccgtacac gtttggaggg


 


361
gggaccagac tggaaattaa acgggctgat gctgcaccaa ctgtatccat cttcccacca


 


421
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac


 


481
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg


 


541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg


 


601
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca


 


661
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag


 








(28) Protein Sequence Defining the Full Length 1F3 Light Chain Sequence (1F3



Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 149)










1
diqmtqspas lsysvgetvt itcraseniy snlawyqqkq gkspqllvyd athlpdgvps



 


61
rfsgsgsgtq fslkinslqs edfgsyycqh fwgtpytfgg gtrleikrad aaptvsifpp


 


121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt


 


181
ltkdeyerhn sytceathkt stspivksfn rnec


 








(29) Nucleic Acid Sequence Encoding the Full Length 3A12 Heavy Chain Sequence



(3A12 Heavy Chain Variable Region and IgG1 Constant Region)(signal sequence


underlined)


(SEQ ID NO. 150)










1
atgaactttg ggctcagatt gattttcctt gtccttgttt taaaaggtgt gaagtgtgaa



 


61
gtgcagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaaatctcc


 


121
tgtgcagcct ctggatttac tttcagtaac tatttcatgt cttgggttcg ccagactcca


 


181
gagaagaggc tggagtgggt cgcatacatt agtagtggtg gtggtagcac ctactatcca


 


241
gacagtgtga agggtcgatt caccatctcc agagacaatg ccaagaacac cctgtacctg


 


301
caaatgaaca gtctgaagtc tgaggacaca gccatgtatt actgtgtaag acaaggagat


 


361
ggttactatg gggactatgc tatggactac tggggtcaag gaacctcagt caccgtctcc


 


421
tcagccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact


 


481
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg


 


541
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct


 


601
gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc


 


661
gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc


 


721
agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc


 


781
ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt


 


841
gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg


 


901
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca


 


961
gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg


 


1021
gtcaacagta cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga


 


1081
ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa


 


1141
gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag


 


1201
tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc


 


1261
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact


 


1321
ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc


 


1381
cactctcctg gtaaatga


 








(30) Protein Sequence Defining the Full Length 3A12 Heavy Chain Sequence (3A12



Heavy Chain Variable Region and IgG1 Constant Region)(without signal sequence)


(SEQ ID NO. 151)










1
evqlvesggg lvqpggslki scaasgftfs nyfmswvrqt pekrlewvay issgggstyy



 


61
pdsvkgrfti srdnakntly lqmnslksed tamyycvrqg dgyygdyamd ywgqgtsvtv


 


121
ssakttppsv yplapgsaaq tnsmvtlgcl vkgyfepvt vtwnsgslss gvhtfpavlq


 


181
sdlytlsssv tvpsstwpse tvtcnvahpa sstkvdkkiv prdcgckpci ctvpevssvf


 


241
ifppkpkdvl titltpkvtc vvvdiskddp evqfswfvdd vevhtaqtqp reeqfnstfr


 


301
syselpimhq dwlngkefkc rvnsaafpap iektisktkg rpkapqvyti pppkeqmakd


 


361
kvsltcmitd ffpeditvew qwngqpaeny kntqpimdtd gsyfvyskln vqksnweagn


 


421
tftcsvlheg lhnhhteksl shspgk


 








(31) Nucleic Acid Sequence Encoding the Full Length 3A12 Light Chain Sequence



(3A12 Kappa Variable Region and Constant Region)(signal sequence underlined)


(SEQ ID NO. 152)










1
ataagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgt



 


61
gacatccaga tgactcagtc gccagcctcc ctatctgtat ctgtgggaga aactgtcacc


 


121
atcacatgtc gagcaagtga gaatatttac attaatttaa catggtatca gcagaaacag


 


181
ggaaaatctc ctcagctcct ggtccatgct gcaacaaagt tagcagatgg tgtgccatca


 


241
aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct


 


301
gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtacac gttcggaggg


 


361
gggaccaaac tagaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca


 


421
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac


 


481
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg


 


541
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg


 


601
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca


 


661
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag


 








(32) Protein Sequence Defining the Full Length 3A12 Light Chain Sequence (3A12



Kappa Variable Region and Constant Region)(without signal sequence)


(SEQ ID NO. 153)










1
diqmtqspas lsvsvgetvt itcraseniy inlawyqqkq gkspqllvha atkladgvps



 


61
rfsgsgsgtq yslkinslqs edfgsyycqh fwgtpytfgg gtkleikrad aaptvsifpp


 


121
sseqltsgga svvcflnnfy pkdinvkwki dgserqngvl nswtdqdskd stysmsstlt


 


181
ltkdeyerhn sytceathkt stspivksfn rnec






For convenience, Table 2 provides a concordance chart showing the correspondence between the full length sequences of the antibodies discussed in this Example with those presented in the Sequence Listing.








TABLE 2





SEQ. ID NO.
Protein or Nucleic Acid







122
1A3 Heavy Variable + IgG1 constant-nucleic acid


123
1A3 Heavy Variable + IgG1 constant-protein


124
1A3 Light Variable + constant-nucleic acid


125
1A3 Light Variable + constant-protein


126
2B8 Heavy Variable + IgG1 constant-nucleic acid


127
2B8 Heavy Variable + IgG1 constant-protein


128
2B8 Light Variable + constant-nucleic acid


129
2B8 Light Variable + constant-protein


130
2F8 Heavy Variable + IgG1 constant-nucleic acid


131
2F8 Heavy Variable + IgG1 constant-protein


132
2F8 Light Variable + constant-nucleic acid


133
2F8 Light Variable + constant-protein


134
3B6 Heavy Variable + IgG1 constant-nucleic acid


135
3B6 Heavy Variable + IgG1 constant-protein


136
3B6 Light Variable + constant-nucleic acid


137
3B6 Light Variable + constant-protein


138
3D11 Heavy Variable + IgG1 constant-nucleic acid


139
3D11 Heavy Variable + IgG1 constant-protein


140
3D11 Light Variable + constant-nucleic acid


141
3D11 Light Variable + constant-protein


142
1D3 Heavy Variable + IgG1 constant-nucleic acid


143
1D3 Heavy Variable + IgG1 constant-protein


144
1D3 Light Variable + constant-nucleic acid


145
1D3 Light Variable + constant-protein


146
1F3 Heavy Variable + IgG1 constant-nucleic acid


147
1F3 Heavy Variable + IgG1 constant-protein


148
1F3 Light Variable + constant-nucleic acid


149
1F3 Light Variable + constant-protein


150
3A12 Heavy Variable + 1SG1 constant-nucleic acid


151
3A12 Heavy Variable + IgG1 constant-protein


152
3A12 Light Variable + constant-nucleic acid


153
3A12 Light Variable + constant-protein









Example 3
Production of Various Recombinant hHGF Proteins
This Example describes the cloning and expression of a number of recombinant proteins used to characterize the antibodies created in Example 1 and in Example 14. In particular, this Example describes the cloning and expression of recombinant hHGF protein, a recombinant hHGF protein containing a glycine to glutamate substitution at position 555 (G555E), a recombinant hHGF protein containing a cysteine to arginine substitution at position 561 (C561R), a recombinant mouse-human-mouse (mhm) chimeric HGF protein containing the human V495-L585 HGF sequence disposed within mouse HGF sequence, a recombinant mhm chimeric HGF protein containing the human I499-R566 HGF sequence disposed within mouse HGF sequence, and a recombinant mhm chimeric HGF protein containing human W507-L585 HGF sequence disposed within mouse HGF sequence.
The following expression constructs were generated using standard molecular techniques and the resulting cDNA sequences were confirmed by DNA sequencing:
a. hHGF-Fc
In a first round of PCR, two overlapping PCR fragments were generated introducing a Not I site and encoding a 6×His tag between hHGF and hIgFc. The overlapping PCR fragments served as template in a second round to amplify hHGF-his-IgFc. The resulting fragment was digested by NheI and BamHI and cloned into pcDNA5/FRT (Invitrogen, #35-3014). Then, hHGF was amplified from Invitrogen clone ID: IOH29794 (human HGF cDNA). The sequence was found to correspond to the sequence deposited at the NCBI under accession number NM—000601.4.







(1) 5′hHGF NheI Primer


(SEQ ID NO. 102)


ACTGGCTAGCATGTGGGTGACCAAACTCCT


 


(2) 3′ hHGF NotI His Tag Primer


(SEQ ID NO. 103)


GTGATGGTGATGGTGATGGCGGCCGCATGACTGTGGTACCTTATATG


 


5′ HisIgFc Primer


(SEQ ID NO. 104)


ACTGGCGGCCGCCATCACCATCACCATCAC


 


(4) 3′ IgFc BamHI Primer


(SEQ ID NO. 105)


ACTGGGATCCTCACTATTTACCCGGGGACAG






b. hHGF-Fc G555E and hHGF-Fc C561R
hHGF-Fc mutants G555E and C561R were generated by site directed mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions.









(1) hHGF-Fc (G555E) Sense Primer



(SEQ ID NO. 106)



CATGATGTCCACGAAAGAGGAGATGAG



 



(2) hHGF-Fc (G555E) Anti-sense Primer



(SEQ ID NO. 107)



CTCATCTCCTCTTTCGTGGACATCATG



 



(3) hHGF-Fc (C561R) Sense Primer



(SEQ ID NO. 108)



GGAAGAGGAGATGAGAAACGCAAACAGGTTCTCAATG



 



(4) hHGF-Fc (C561R) Anti-sense Primer



(SEQ ID NO. 109)



CATTGAGAACCTGTTTGCGTTTCTCATCTCCTCTTCC






c. Mouse-Human-Mouse Chimera Fc
The mouse-human-mouse chimera IgFc construct contains mHGF alpha chain-hHGF, β-chain amino acids Val 495-Leu 585 of human HGF, and mHGF C-terminal beta chain followed by 6×His tag and IgG-Fc.
Human HGF cDNA encoding amino acids V495-L585 was amplified from Invitrogen clone ID: IOH29794 (human HGF cDNA). The sequence corresponds to the sequence deposited at the NCBI under accession number NM 000601.4. Mouse HGF sequences were amplified by RT-PCR from mouse liver total RNA (Clontech, #636603) using the Super Script One Step RT-PCR kit from Invitrogen (#10928-034) according to manufacturer's instructions. The mHGF cDNA sequence corresponds to the sequence deposited at the NCBI under accession number D10213.1.
Three fragments, referred to as Fragments 1, 2, and 3, were generated using overlapping PCR primers and annealed in consecutive rounds of PCR amplification. The final product was cleaved with NheI and NotI and cloned into pcDNASIFRT IgGFc.







(1) Fragment 1 Primers for mHGF alpha chain 5′NheI


(SEQ ID NO. 110)


5′ATCGGCTAGCATGATGTGGGGGACCAAAC


 


(SEQ ID NO. 111)


3′ GAATCCCATTTACAACCCGCAGTTGTTTTGTTTTGG


 


(2) Fragment 2 Primers for hHGF beta chain aa


V495-L585


(SEQ ID NO. 112)


5′ CCAAAACAAAACAACTGCGGGTTGTAAATGGGATTC


 


(SEQ ID NO. 113)


3′ CAGGATTGCAGGTCGAGCAAGCTTCATTAAAACCAGATCT


 


(3) Fragment 3 Primer for mHGF beta chain C-


terminus 3′NotI


(SEQ ID NO. 114)


5′ AGATCTGGTTTTAATGAAGCTTGCTCGACCTGCAATCCTG


 


(SEQ ID NO. 115)


3′ GTAATTTTGACATACAAGTTGTGCGGCCGCCATCACCATCACCATCAC






d. Construction of hHGF and mhm Chimera
The vectors encoding hHGF and mhm chimera (V495-L585), pcDNA5/FRT hHGF and pcDNAS/FRT-mhm chimera (V495-L585), without Fc-tag were generated by site directed mutagenesis. A stop codon was introduced 3′ of the 6×His tag using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions. The mutagenesis primer included Primer 1: CATCACCATCACCATCACTAAGCGGGTCTGGTGCCACG (SEQ ID NO. 116), and Primer 2: CGTGCACCAGACCCGCITAGTGATGGTGATGOGTGAT (SEQ ID NO. 117).
In addition, two additional mhm chimeras were created from the pcDNA5/FRT-mhm (V495-L585) construct by site directed mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to manufacturer's instructions. One mhm construct contained the region of I499-R556 of hHGF disposed between murine sequences. The other mhm construct contained the region of W507-L585 of hHGF disposed between murine sequences.
For the mhm chimera (I499-R556), the following point mutations were made in order in the template pcDNA5/FRT-mhm chimera (V495-L585) construct: D558E, C561R, V564I, V567I and M583L, using the appropriate oligonucleotide sequences. For the mhm chimera (W507-L585), the following point mutations were introduced in one step in the template pcDNA5/FRT-mhm chimera (V495-L585) construct: Q502R, N504T and 1505V, using the appropriate oligonucleotide sequences.
The resulting nucleotide sequence of the hHGF-Fc protein is set forth as SEQ ID NO. 118, including signal sequence (nucleotides 1-93) and prodomain (nucleotides 94-162). The amino acid sequence of the hHGF-Fc protein is set forth as SEQ ID NO. 119.
The resulting nucleotide sequence encoding the mhm (V495-L585)-Fc chimeric protein is set forth in SEQ ID NO. 120, including signal sequence (nucleotides 1-96) and prodomain (nucleotides 97-165). The amino acid sequence of the mhm (V495-L585)-Fc chimeric protein is set forth in SEQ ID NO. 121.
The resulting nucleotide sequence encoding, and the protein sequence defining, the mhm (V495-L585) construct are set forth in SEQ ID NOS. 211 and 212, respectively. The nucleic acid sequence set forth in SEQ ID NO. 211 includes the signal sequence (nucleotides 1-96) and the prodomain (nucleotides 97-165), and the protein sequence set forth in SEQ ID NO. 212 includes the active protein sequence (without the signal sequence or the prodomain). The resulting nucleotide sequence encoding, and the protein sequence defining, the mhm (1499-R556) construct are set forth in SEQ ID NOS. 213 and 214, respectively. The nucleic acid sequence set forth in SEQ ID NO. 213 includes the signal sequence (nucleotides 1-96) and the prodomain (nucleotides 97-165), and the protein sequence set forth in SEQ ID NO. 214 includes the active protein sequence (without the signal sequence or the prodomain). The resulting nucleotide sequence encoding, and the protein sequence defining, the mhm (W507-L585) are set forth in SEQ ID NOS. 215 and 216, respectively. The nucleic acid sequence set forth in SEQ ID NO. 215 includes the signal sequence (nucleotides 1-96) and the prodomain (nucleotides 97-165), and the protein sequence set forth in SEQ ID NO. 216 includes the active protein sequence (without the signal sequence or the prodomain).
e. Protein Expression
(1) Cell Culture
CHO FlpIn cells (Invitrogen, Catalog No. R758-07)) were grown in F12K media (ATCC, Catalog No. 30-2004), 10% FCS (Invitrogen, Catalog No. 10438026), 1% Penicillin (10000 units/mL)/Streptomycin (10,000 μg/mL) (Invitrogen, Catalog No. 15140-122) at 37° C., 5% CO2, 100 g/mL Zeocin (Invitrogen, Catalog No. R250-01).
(2) Generation of Stable CHO FlpIn Cell Lines
CHO Flpin host cells were transfected with a 9:1 ratio of pOG44:pcDNA5/FRT expression plasmid DNA using lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Catalog No. 11668-027). As controls, cells were transfected with empty pcDNA5/FRT vector/pOG44 and pOG44 plasmid (Invitrogen, Catalog No. 35-3018) alone. Twenty four hours after transfection, the cells were split, and after forty eight hours 0.5 mg/mL Hygromycin B (Sigma, Catalog No. H0654-SPEC) was added to the cells. Polyclonal selection of stable cells was performed in F12K, 10% FCS, 1% Penicillin/Streptomycin, 0.5 mg/mL Hygromycin B.
(3) Protein Expression in Stable CHO FlpIn Cell Lines
Approximately 2×106 cells were seeded in 15 cm plates and grown in F12K (ATCC, Catalog No. 30-2004)/DMEM high glucose (Invitrogen, Catalog No. 11995065) 1:1, 5% ultra low IgG FCS (Invitrogen, #16250-78) at 37° C., 5% CO2 for 5-6 days. Supernatants were harvested and resulting proteins analyzed by ELISA and by surface plasmon resonance.
Example 4
Binding Characteristics of Anti-hHGF Monoclonal Antibodies
The monoclonal antibodies produced in Example I were characterized by their ability to bind hHGF, and certain of the recombinant HGF proteins produced in Example 3.
The antibodies were analyzed by surface-plasmon resonance using a BIAcore T100 instrument to assess their ability to bind HGF and certain of the fusion proteins discussed in Example 3. Each antibody was immobilized on a carboxymethylated dextran CM5 sensor chip (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a standard coupling protocol according to manufacturer's instructions.
Analyses were performed at 25° C. using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No. R-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running buffer. Supernatant containing different HGF fusion proteins or supernatant from cells transfected with empty vector were injected over each antibody at a flow rate of 30 L/min for 3 minutes. The resulting binding was determined as resonance units (RU) over baseline 30 seconds after the end of injection. Binding was compared to human HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer. Non-specific binding was monitored by comparing binding to a control surface where mouse IgG (Rockland, Catalog No. 010-0102) was immobilized using the same amine coupling procedure.
The results are summarized in the Table 3.













TABLE 3






rhHGF (R&D
rmHGF (R&D
mhm chimera





Antibody
Systems)
Systems
(V495-L585)
human HGF
G555E
C561R







1A3
Yes
No
No
Yes
Yes
Yes


1D3
Yes
No
Yes
Yes
Yes
Yes


1F3
Yes
Yes
Yes
Yes
Yes
Yes


2B8
Yes
No
Yes
Yes
Yes
Yes


2F8
Yes
Yes
No
Yes
Yes
Yes


3A12
Yes
No
No
Yes
Yes
Yes


3B6
Yes
No
No
Yes
Yes
Yes


3D11
Yes
No
No
Yes
Yes
Yes









The results in Table 3 demonstrate that each of the antibodies bind rHGF and purified human HGF. Furthermore, all of the antibodies bind hHGF containing point mutations G555E and C561R. In general, all of the antibodies except for 1F3 and 2F8 did not bind murine HGF demonstrating that the antibodies 1A3, 1D3, 2B8, 3A12, 3B6, and 3D11 specifically bind human HGF. Antibodies 1D3, 1F3, and 2B8 bind the mouse-human-mouse chimera whereas the remaining antibodies did not. The results suggest that the antibodies 1D3 and 2B8 at least in part bind to residues 495-585 of human HGF. The antibodies 1A3, 3A12, 3B6, and 3D11 appear to bind portions of human hHGF other than residues 495-585. At present, it is uncertain why 2F8 does not bind the mhm chimera as it appears to bind both hHGF and mHGF.
Example 5
Ability of Anti-hHGF Monoclonal Antibodies to Bind Reduced and Non-Reduced HGF
In this Example, the anti-hHGF monoclonal antibodies produced in Example I were analyzed for their ability to bind reduced and non-reduced HGF.
The reactivity of the anti-HGF sera with the recombinant hHGF was assessed by immunoblotting. Eight μg of recombinant hHGF protein in NuPAGE MOPS SDS running buffer (Invitrogen) with or without NuPAGE sample reducing buffer (Invitrogen) was fractionated on a 4-12% Bis-Tris 1.0 mm×2D well gel (Invitrogen, Carlsbad, Calif.). The fractionated proteins then were transferred onto a nitrocellulose membrane using standard procedures. The nitrocellulose membranes were blocked with 5% nonfat milk powder solution in Tris buffered Saline with 0.1% Tween-20® (TBST), and then mounted onto a Mini Protean II Multi-Screen apparatus (BioRad) for further blocking.
The resulting membranes were probed with the purified antibodies on a Multi-Screen apparatus. The purified antibodies were diluted to 5 μg/mL in blocking buffer. The nitrocellulose membrane then was removed from the apparatus, and incubated with horseradish peroxidase-labeled anti-mouse IgG antibodies. The results are summarized in Table 4, where the numbers reflect the extent of binding with − representing the least (little or no binding) and 3+ representing the most binding.











TABLE 4








Reduced
Non-Reduced



Antibody
(exposure: 3-5 min)
(exposure: 20 sec)









1A3
2+
2+



1D3
2+
2+



1F3
2+
2+



2B8
−
1+



2F8
2+
2+



3A12
−
2+



3B6
3+
2+



3D11
−
3+










The data in Table 4 demonstrate that all the antibodies bind non-reduced rhHGF. In contrast, monoclonal antibodies 1A3, 1D3, 1F3, 2F8, 3B6 bound reduced rhHGF but antibodies 2B8, 3A12, and 3D11 did not bind to reduced rhHGF.
Example 6
Binding Affinities
The binding affinities and kinetics of interaction of each of the antibodies produced in Example 1 against hHGF were measured by surface plasmon resonance.
Rabbit anti-mouse immunoglobulins (BIAcore, Catalog No. BR-1005-14) were immobilized on carboxymethylated dextran CM5 sensor chips (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a standard coupling protocol according to manufacturer's instructions. The analyses were performed at 25° C. using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No. BR-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930), and 10 mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running buffer.
The antibodies were captured in an individual flow cell at a flow rate of 10 μL/min. Injection time was variable for each antibody to yield approximately 20 RU of antibody captured for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 2 minutes at 60 μL/min. The dissociation phase was monitored for 15 or 90 minutes, depending on concentration. The surface then was regenerated with 10 mM Glycine-HCl, pH 1.7 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 μL/min before another cycle was initiated. HGF concentrations tested were 0.46 nM to 7.5 nM.
Kinetic parameters were determined using the kinetic function of the BIAevalutation software with reference subtraction. Kinetic parameters for each antibody, k, (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized in Table 5.













TABLE 5










KD



Antibody
ka (1/Ms)
SE (ka)
kd (1/s)
SE (kd)
(pM)
SD





















1A3
1.7 × 106
7.3 × 104
5.2 × 10−5
8.4 × 10−7
30.1
5.6


1D3
1.7 × 106
3.1 × 104
8.2 × 10−5
1.7 × 10−6
54.2
27.4


1F3
1.5 × 106
5.0 × 104
2.6 × 10−5
6.6 × 10−7
18.1
8.2


2B8
1.6 × 106
2.9 × 104
2.1 × 10−5
1.4 × 10−7
13.5
4.4


3A12
1.6 × 106
3.7 × 104
1.6 × 10−4
1.6 × 10−6
103.0
10.4


3B6
2.0 × 106
6.5 × 104
3.9 × 10−5
3.2 × 10−7
17.0
3.4









The data in Table 5 demonstrate that the antibodies bind hHGF with a KD of about 100 pM or less, about 50 pM or less, or 20 pM or less.
Example 7
Neutralization Activity of Anti-hHGF Antibodies
In this Example, the antibodies produced in Example 1 were characterized for their ability to (a) inhibit the binding of hHGF to c-Met, and (b) inhibit HGF stimulated BrdU incorporation in 4 MBr-5 cells.
a. HGF-Met Binding Inhibition Assay (Neutralization Assay)
The antibodies were tested by ELISA for their ability to inhibit hHGF binding to c-Met.
Specifically, Wallac 96-well DELFIA assay plates (Wallac Inc., Catalog No. AAAND-0001) were coated with 100 μL of 6.25 μg/mL HGF (R&D Systems, Catalog No. 294-HGN-025) in carbonate coating buffer (15 mM Na2CO3 and 34 mM NaHCO3, pH 9.0) for 16 hours at 4° C. The plates then were blocked with 200 μL of 5% non-fat dry milk in PBS for 1 hour at room temperature. The antibodies were prepared in a separate plate by adding increasing concentrations of the antibodies under investigation (0.033-667 nM, 3-fold-serial dilution) to 2 nM c-Met (R&D Systems, Catalog No. 358-MT/CF) in 5% non-fat dry milk in PBS. 100 μL of sample per well was transferred to the assay plate and incubated overnight at 4° C. The assay plates then were washed 3 times with PBS-0.1% Tween 20, and incubated for 2 hours at room temperature with 100 μL/well of 2 μg/mL biotinylated anti-human c-Met antibody (R&D Systems, Catalog No. BAF358) prepared in 5% non-fat dry milk in PBS.
The resulting plates then were washed three times with PBS-0.1% Tween 20, and incubated for 1 hour at room temperature with Eu-labeled Streptavidin (Wallac, Catalog No. 1244-360) diluted 1:1000 in DELFIA assay buffer (Wallac, Catalog No. 4002-0010). The resulting plates were washed 3 times with DELFIA wash solution (Wallac, Catalog No. 4010-0010) and incubated with 100 μL/well DELFIA enhancement solution (Wallac #4001-0010) for 15 minutes at room temperature with agitation.
The plates were read on Victor3V instrument (Perkin Elmer) using the Europium method. The IC50 values were calculated and are summarized in Table 6.











TABLE 6







Antibody
IC50 (nM)
SD









1A3
5.65
0.91



1D3
4.43
2.27



1F3
6.57
0.28



2B8
5.57
1.19



2F8
5.36
0.88



3A12
5.26
2.11



3B6
—
—



3D11
5.66
2.75










The results demonstrate that all the antibodies (i.e., 1D3, 1A3, 2B8, 3A12, 1F3, 3D11, and 2F8) other than 3B6 efficiently neutralize HGF binding to c-Met.
b. Neutralization of HGF Stimulated BrdU Incorporation in 4 MBr-5 Cells
Ten μL of 12.5 nM of hHGF was dispensed into individual wells of a 96-well tissue culture microtiter plate (Costar Catalog No. 3903). Ten μL of serially diluted antibodies at concentrations of 6667, 2222, 740, 247, 82, 27, 9.1, 3.0, 1.0, 0.33 nM were added to each well. The HGF antibody mixture then was incubated at room temperature for 30 minutes. Monkey bronchial epithelial cells 4 MBr-5 (ATCC, CCL208) cultured in F-12K (ATCC, 30-2004), 15% FBS (Gibco 10438-026), 30 ng/mL EGF (Sigma E9644), 1% penicillin/streptomycin (PS, Gibco Catalog No. 15140-122) were dissociated with Trypsin (Gibco Catalog No. 25200-056), resuspended in assay media (F-12K, 2.5% FBS, 1% PS) at 75,000 cells/mL, and 80 μL of the cell suspension was dispensed to the HGF antibody mixture.
The resulting cells were incubated at 37° C., 5% CO2. Forty eight hours later, 10 μL of 100 μM BrdU (Roche Catalog No. 1669915) was added. Seventy two hours later, the media was removed, the plates were dried with a hair dryer and were processed with the BrdU ELISA in accordance with manufacturer's instructions (Roche Catalog No. 1669915).
The luminescent signal was quantified by a Synergy HT plate reader (Bio-Tek). The data were fit to a sigmoidal dose response with variable slope with the equation y=bottom+(top-bottom)/(1+10^(log(EC50−x)*hill slope)) in GraphPad Prism (GraphPad Software). Each experiment was repeated at least 3 times in duplicates, and average EC50 values are presented in Table 7.










TABLE 7







Antibody
IC50 (nM)



















1A3
4.69



1D3
4.99



1F3
1.94



2B8
1.41



2F8
19.24



3A12
30.30



3B6
36.08



3D11
51.12










The results in Table 7 demonstrate that all of the antibodies, 1A3, 1D3, 1F3, 2B8, 2F8, 3A12, 3B6, and 3D11 inhibit HGF induced proliferation in 4 MBr-5 cells.
Example 8
Anti-Scatter Activity of Anti-hHGF Antibodies
This Example describes a characterization of the antibodies produced in Example 1 for their ability to inhibit HGF induced scatter activity. HGF induces “scattering” (motility) of clusters in MDCK cells (ATCC, Manassas, Va., Catalog No. CCL-34).
MDCK cells were seeded in 96-well Costar tissue culture plates (Corning Incorporated, Corning, N.Y., Catalog No. 3595) at a density of 4×103 cells per well in 80 μL MEM (ATCC, Manassas, Va., Catalog No. 30-2003) containing 10% Fetal Bovine Serum (Invitrogen Catalog No. 10438026), and 1% penicillin-streptomycin (Invitrogen Catalog No. 15140122). Each of the antibodies to be investigated was diluted to 6,667 nM in MEM containing 10% Fetal Bovine Serum and 1% penicillin-streptomycin. Each of the different antibody dilutions, as well as MEM containing 10%0/Fetal Bovine Serum and 1% penicillin-streptomycin without antibody, then was separately combined with an equal volume of MEM containing 10% Fetal Bovine Serum and 1% penicillin-streptomycin, and 100 ng/ml HGF (R&D Systems Catalog No. 294-HGN-025). The antibody/HGF dilutions were incubated for 30 minutes at 25° C. Twenty μL of each antibody/HGF dilution was added separately to individual wells, yielding a final antibody concentration of 666.7 nM, and a final HGF concentration of 10 ng/ml. The MDCK cells then were incubated for 24 hours at 37° C. with 5% CO2.
After 24 hours incubation, the MDCK cells were carefully washed once with 100 μL per well of ice-cold PBS (Invitrogen Catalog No. 14190144), and fixed with 100 μL per well of ice-cold methanol while rocking for 10 minutes at 25° C. The plates then were washed carefully once with distilled water. A volume of 100 μL crystal violet solution, consisting of 0.5% crystal violet (Sigma, St. Louis, Mo., Catalog No. C3886) and 50% ethanol in distilled water, was added to each well, and the cells were incubated for 20 minutes at 25° C. while rocking.
Following staining with crystal violet solution, the cells were washed carefully three times with distilled water. Then, PBS was added to each well to prevent drying of samples. The cells were imaged using the Leica DMIRB microscope (Leica Microsystems GmbH, Wetzler, Germany), DC500 camera (Leica Microsystems GmbH, Wetzler, Germany), and MagnaFire 2.1C software (Optronics, Goleta, Calif.), and samples were rated for level of scattering. The results are summarized in Table 8.







TABLE 8







Inhibition of HGF-induced MDCK Cell Scattering











Antibody
Trial 1
Trial 2







1A3
++
+



1D3
++
++



1F3
+
+



2B8
+++
+++



2F8
+
+



3A12
−
−/+



3B6
++
++



3D11
−
−







− No Inhibition



+++ Very strong, nearly complete inhibition



++ Strong inhibition



+ Detectable inhibition






The results in Table 8 demonstrate that antibody 2B8 inhibited HGF-induced scattering more than the other antibodies. Antibodies I D3 and 3B6 displayed an intermediate level of inhibition; antibody 1A3 displayed a low to intermediate level of inhibition: antibodies 1F3 and 2F8 displayed a low level of inhibition; and antibodies 3A12 and 3D11 gave little or no detectable inhibition.
Example 9
Inhibition of HGF-Stimulated c-Met Phosphorylation
This Example describes a characterization of the antibodies produced in Example 1 for their ability to inhibit the HGF-stimulated c-Met phosphorylation in PC-3 cells. HGF induces phosphorylation of Met in PC-3 cells (ATCC No. CRL-1435).
PC-3 cells were seeded into individual wells of 96-well Costar tissue culture plates (Corning Catalog No. 3595) at a density of 4.5×104 cells per well in 100 μL F-12K (ATCC, Manassas, Va., Catalog No. 30-2004) containing 10% Fetal Bovine Serum (Invitrogen Catalog No. 10438026) and 1% penicillin-streptomycin (Invitrogen Catalog No. 15140122). After 24 hours at 37° C. with 5% CO2, the media was removed, and cells were rinsed once with serum-free F-12K containing 1% penicillin-streptomycin. Cells then were incubated for 24 hours in 100 μL serum-free F-12K containing 1% penicillin-streptomycin.
The following 10 different dilutions of each of the antibodies being investigated were prepared in serum-free F-12K containing 1% penicillin-streptomycin: 6667 nM, 2222 nM, 741 nM, 247 nM, 82.3 nM, 27.4 nM, 9.1 nM, 3.0 nM, 1.0 nM, and 0.3 nM. Each antibody dilution, and, serum-free F-12K containing 1% penicillin-streptomycin without antibody, were separately combined with an equal volume of serum-free F-12K containing 1% penicillin-streptomycin and 500 ng/mL HGF (R&D Systems Catalog No. 294-HGN-025). These antibody/HGF dilutions were incubated for 30 minutes at 25° C. This resulted in a final concentration of 1.25 nM HGF.
The PC-3 cells then were rinsed once with serum-free F-12K containing 1% penicillin-streptomycin. Next, 70 μL of serum-free F-12K containing 1% penicillin-streptomycin was added to the cells, followed by 10 μL of 10 mM Na3VO4 (Sigma Catalog No. S6508) in serum-free F-12K containing 1% penicillin-streptomycin. The cells then were incubated for 60 minutes at 37° C. with 5% CO2. Following this incubation, 20 μL of each antibody/HGF dilution was added separately to separate wells, yielding a final HGF concentration of 50 ng/mL, and the following final concentrations of each antibody: 666.7 nM, 222.2 nM, 74.1 nM, 24.7 nM, 8.23 nM, 2.74 nM, 0.91 nM, 0.30 nM, 0.10 nM, 0.03 nM. The cells then were incubated for 10 minutes at 37° C. with 5% CO2, after which point the media/antibody/HGF mixture was removed, the plates were placed on ice. The cells then were rinsed once with 100 μL per well of ice-cold PBS (Invitrogen Catalog No. 14190144) containing 1 mM Na3VO4. The cells then were incubated for 30 minutes at 4° C. in 100 μL per well ice-cold lysis buffer consisting of 1% OmniPur Triton X-100 (MERCK KGaA, Darmstadt, Germany, Catalog No. 9410), 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.3 mM Na3VO4, 1× protease inhibitor cocktail (Sigma Catalog No. P8340), and 1× phosphatase inhibitor cocktail 2 (Sigma Catalog No. 5726).
Biotinylated anti-human HGF-R (c-met) antibody (R&D Systems Catalog No. BAF358) was diluted to a concentration of 2 μg/mL in DELFIA Assay Buffer (PerkinElmer, Turku, Finland, Catalog No. 4002-0010) containing 1% bovine serum albumin (Sigma Catalog No. A2153), and 50 μL of this dilution was added per well of yellow streptavidin microtitration plates (PerkinElmer Catalog No. AAAND-0005). The plates then were incubated with antibody for 30 minutes at 25° C. with rocking. Following incubation, the plates were washed with DELFIA wash solution (PerkinElmer Catalog No. 4010-0010), and 80 μL of each of the different PC-3 cell lysates was added separately to individual wells of the washed streptavidin microtitration plates.
The streptavidin microtitration plates containing PC-3 cell lysates were incubated for 60 minutes at 25° C. with shaking, and then washed with DELFIA wash solution. 100 μL of 600 ng/mL DELFIA Eu-N1 P-Tyr-100 antibody (PerkinElmer Catalog No. AD0159) diluted in DELFIA Assay Buffer containing 1% bovine serum albumin was added to each well of the washed streptavidin microtitration plates previously incubated with PC-3 cell lysates. The plates were incubated for 60 minutes at 25° C., with rocking. The plates were washed a final time with DELFIA wash solution. Then 200 μL of DELFIA Enhancement Solution (PerkinElmer Catalog No. 4001-0010) was added to each well of the washed streptavidin microtitration plates, and the plates were incubated in the dark for 5 minutes at 25° C., with shaking.
Signal then was measured using the Europium protocol on the Victor3V reader (PerkinElmer). EC50 values were calculated using Prism 4 for Windows (GraphPad Software, Inc., San Diego, Calif.) and the sigmoidal dose-response equation.
The results summarized as EC50s in nM are tabulated in Table 9.









TABLE 9






Average of
Standard


Antibody
Two Trials
Deviation

















1A3
0.684
0.242


1D3
0.984
0.129


1F3
1.19
1.01


2B8
0.287
0.104


2F8
1.39
2.12


3A12
2.00
0.553


3B6
1.01
1.11


3D11
2.28
N/A









The data in Table 9 demonstrate that all eight antibodies are potent inhibitors of HGF-induced c-Met phosphorylation in PC-3 cells.
Example 10
Tumor Inhibition in U87MG Xenograft Model
The ability of murine monoclonal antibodies of the invention to inhibit tumor growth was tested in an U87MG xenograft model. U87MG cells (ATCC) were expanded in culture at 37° C. in an atmosphere containing 5% CO2 and 95% air, using a medium comprising Dulbecco's Modified Eagle medium (DMEM) with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin. The cells were subcultured and maintained by detaching the cells from the wall of the culture dish using trypsin-EDTA.
Near-confluent cells were collected by trypsinization and then 5×106 cells in 50% Matrigel (BD Biosciences; catalog no. 356237) were injected subcutaneously into the upper dorsal area between the shoulder blades of 7-week old female ICR SCID mice (Taconic Labs). The long (L) and short (W) diameters (mm) of tumors were measured with a caliper. Tumor volume (vol.) was calculated as: volume (mm3)=L×W2/2. When the tumors grew to approximately 200 mm3, the tumor-bearing mice were randomized into 5 groups of 10 mice each. One group received PBS. Each of the other 4 groups received one of the antibody 1A3, 1D3, 1F3 or 2B8. All antibodies were dosed at 1 mg/kg body weight, twice per week, by intra-peritoneal injections of 5 doses. Tumor volumes and mouse body weights were recorded twice per week. Tumor growth inhibition was analyzed using Student's t-test. The results are summarized in FIG. 6 and Table 10.







TABLE 10





Percent Inhibition



















2B8 vs PBS
93%
p = 0.001



1A3 vs PBS
73%
p = 0.0075



1D3 vs PBS
51%
p = 0.075



1F3 vs PBS
60%
p = 0.027










Partial regression was achieved in 2B8 treated group (FIG. 6). Statistically significant growth inhibition was observed in the 1A3-treated and 1F3-treated groups (Table 10). There was 51% tumor growth inhibition for 1D3 with a p value of 0.075. No significant body weight loss was observed.
Example 11
Tumor Inhibition in U118 Xenograft Model
The ability of the antibodies 1A3, 1D3, 1F3 and 2B8 to inhibit tumor growth was tested in an U118 xenograft model. U118 cells (ATCC) were expanded as described in Example 10 (above) with respect to the U87MG cells.
Subcutaneous tumors were established as described in Example 10 above, except that the mice used were 7 weeks old female NCr nude mice (Taconic), and treatment was started when the tumors grew to approximately 80 mm3. As in the U87MG model, all the antibodies were dosed at 1 mg/kg body weight twice a week by intra-peritoneal injections for 4 doses. Tumor volumes and body weights of the mice were recorded twice per week. Tumor growth inhibition was analyzed using Student's t-test. The results are summarized in FIG. 7 and Table 11.







TABLE 11





Percent Inhibition



















2B8 vs IgG
75%
p = 0.007



1A3 vs IgG
57%
p = 0.01



1D3 vs IgG
47%
p = 0.12



1F3 vs IgG
30%
p = 0.39










Statistically significant tumor growth inhibition was observed in 2B8 and 1A3 treated groups (FIG. 7). There was modest tumor growth inhibition in 1F3 and 1D3 groups with p values less than 0.05, which was defined as statistical significance in this study (Table 11). No significant body weight loss was observed.
Example 12
Humanization of Murine Monoclonal Antibodies
This Example describes the humanization of the murine 2B8 antibody, together with a characterization of the resulting humanized antibodies. The murine 2B8 Heavy and Light Variable Regions were “humanized” by two methods.
A. Humanization Procedure I
In the first method, three humanized heavy chain variable regions and two humanized kappa light chain variable regions were designed based on the “superhumanization” method described in Hwang et al. (2005) METHODS 36:35-42; Tan et al. (2002) J. IMMUNOL. 169:1119-1125; U.S. Pat. No. 6,881,557.
The Chothia canonical structural class was determined for each mouse 2B8 CDR based on CDR length and amino acid composition. Human germline variable regions consisting of the same Chothia canonical structural class light and heavy variable regions were identified based on known human germline variable region reference alleles described at the International Immunogentics Information System (IMGT) website (available on the world wide web at imgt.cines.fr and biochem.unizh.ch/antibody/Sequences/index.htmi). These human germline variable regions of the same structural class were compared to murine 2B8 variable regions by calculating the percent identity or similarity between CDR amino acid residues. Those human germline variable regions with the highest identity and/or similarity with mouse 2B8 CDR residues were chosen for CDR grafting. The framework residues of the human germline variable regions were preserved while the mouse 2B8 CDR residues were used to replace the corresponding human germline variable region residues that were different between mouse 2B8 CDR and human germline CDRs. The human J region that was most similar to the 2B8 mouse J region was then added to the carboxyl terminus of the “superhumanized” variable region. A signal sequence was then added to the amino terminus of the “superhumanized” variable regions and these amino acid sequences were converted into nucleic acid sequences.
The complete variable region nucleic acid sequence was constructed using gene synthesis PCR methods (Young et al. (2004) NUCL. ACIDS RES. 32:e59) and cloned into a mammalian expression vector (based on pcDNA3.2 DEST (Invitrogen)) containing human constant IgG1 (G1m(17,1) allotype) or Kappa (Km(3) allotype (allele 2)) regions (downstream of the variable regions) using standard molecular biology techniques. All four heavy chain IgG1 antibodies (chimeric 2B8 and 3 humanized heavy chains (Hu2B8 Hv1-f.1, Hu2B8 Hv5-a. 1, Hu2B8 Hv5-51.1) were expressed in the possible combinations with all 3 kappa chain antibodies (chimera 2B8 and 2 humanized light chains (Hu2B8 Kv1-39.1 and Hu2B8 Kv3-15.1) creating 12 different antibody proteins. Binding of the chimeric, chimeric/humanized, and humanized antibodies to human HGF was then measured as described below and the results are summarized in FIG. 8. Each of the possible combinations of immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth below in Table 12A.








TABLE 12A





Heavy Chain Variable Region
Light Chain Variable Region







Chimeric 2B8 (SEQ ID NO: 12)
Chimeric 2B8 (SEQ ID NO: 14)


Chimeric 2B8 (SEQ ID NO: 12)
Hu2B8 Kv1-39.1 (SEQ ID NO: 173)


Chimeric 2B8 (SEQ ID NO: 12)
Hu2B8 Kv3-15.1 (SEQ ID NO: 179)


Hu2B8 Hv1-f.1 (SEQ ID NO: 159)
Chimeric 2B8 (SEQ ID NO: 14)


Hu2B8 Hv1-f.1 (SEQ ID NO: 159)
Hu2B8 Kv1-39.1 (SEQ ID NO: 173)


Hu2B8 Hv1-f.1 (SEQ ID NO: 159)
Hu2B8 Kv3-15.1 (SEQ ID NO: 179)


Hu2B8 Hv5-a.1 (SEQ ID NO: 165)
Chimeric 2B8 (SEQ ID NO: 14)


Hu2B8 Hv5-a.1 (SEQ ID NO: 165)
Hu2B8 Kv1-39.1 (SEQ ID NO: 173)


Hu2B8 Hv5-a.1 (SEQ ID NO: 165)
Hu2B8 Kv3-15.1 (SEQ ID NO: 179)


Hu2B8 Hv5-51.1 (SEQ ID NO: 169)
Chimeric 2B8 (SEQ ID NO: 14)


Hu2B8 Hv5-51.1 (SEQ ID NO: 169)
Hu2B8 Kv1-39.1 (SEQ ID NO: 173)


Hu2B8 Hv5-51.1 (SEQ ID NO: 169)
Hu2B8 Kv3-15.1 (SEQ ID NO: 179)









Each of the possible combinations of immunoglobulin heavy chains and immunoglobulin light chains are set forth below in Table 12B.








TABLE 12B





Immunoglobulin Heavy Chain
Immunoglobulin Light Chain







Chimeric 2B8 IgG1 (SEQ ID NO: 155)
Chimeric 2B8 Kappa (Km(3))



(SEQ ID NO: 157)


Chimeric 2B8 IgG1 (SEQ ID NO: 155)
Hu2B8 Kv1-39.1 + Kappa Constant (Km(3)



allotype) (allele 2) (SEQ ID NO: 177)


Chimeric 2B8 IgG1 (SEQ ID NO: 155)
Hu2B8 Kv3-15.1 + Kappa Constant (Km(3)



allotype) (allele 2) (SEQ ID NO: 181)


Hu2B8 Hv1-f.1 + IgG1 Constant
Chimeric 2B8 Kappa (Km(3))


(G1M(17,1)) allotype (SEQ ID NO: 163)
(SEQ ID NO: 157)


Hu2B8 Hv1-f.1 + IgG1 Constant
Hu2B8 Kv1-39.1 + Kappa Constant (Km(3)


(G1M(17,1)) allotype (SEQ ID NO: 163)
allotype) (allele 2) (SEQ ID NO: 177)


Hu2B8 Hv1-f.1 + IgG1 Constant
Hu2B8 Kv3-15.1 + Kappa Constant (Km(3)


(G1M(17,1)) allotype (SEQ ID NO: 163)
allotype) (allele 2) (SEQ ID NO: 181)


Hu2B8 Hv5-a.1 + IgG1 Constant
Chimeric 2B8 Kappa (Km(3))


(G1M(17,1)) allotype (SEQ ID NO: 167)
(SEQ ID NO: 157)


Hu2B8 Hv5-a.1 + IgG1 Constant
Hu2B8 Kv1-39.1 + Kappa Constant (Km(3)


(G1M(17,1)) allotype (SEQ ID NO: 167)
allotype) (allele 2) (SEQ ID NO: 177)


Hu2B8 Hv5-a.1 + IgG1 Constant
Hu2B8 Kv3-15.1 + Kappa Constant (Km(3)


(G1M(17,1)) allotype (SEQ ID NO: 167)
allotype) (allele 2) (SEQ ID NO: 181)


Hu2B8 Hv5-51.1 + IgG1 Constant
Chimeric 2B8 Kappa (Km(3))


(G1M(17,1)) allotype (SEQ ID NO: 171)
(SEQ ID NO: 157)


Hu2B8 Hv5-51.1 + IgG1 Constant
Hu2B8 Kv1-39.1 + Kappa Constant Km(3)


(G1M(17,1)) allotype (SEQ ID NO: 171)
allotype) (allele 2) (SEQ ID NO: 177)


Hu2B8 Hv5-51.1 + IgG1 Constant
Hu2B8 Kv3-15.1 + Kappa Constant (Km(3)


(G1M(17,1)) allotype (SEQ ID NO: 171)
allotype) (allele 2) (SEQ ID NO: 181)









Two of the possible antibody constructs containing the full length immunoglobulin heavy and light chains containing humanized variable regions are designated below:

    
    
        sh2B8-9 (G1m(17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(17,1) allotype) (SEQ ID NO. 171) plus hu2B8 Kv 1-39.1 (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID NO. 177)
        sh2B8-12 (G1m(17,1))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(17,1) allotype)) (SEQ ID NO. 171) plus hu2B8 Kv 3-15.1 (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID NO. 181).
    
    


The nucleic acid sequences encoding and the protein sequences defining each of the humanized antibodies are summarized below. In this section, the last nucleotide of each variable region is the first base of the next codon generated by the variable/constant region junction. This nucleotide is included in the Variable Region because it is part of that exon. Amino acid sequences of Constant Regions listed below include the translation of this junction codon.








(1) Nucleic Acid Sequence Encoding the Full Length Chimeric 2B8 Heavy Chain



(Mouse Variable Region and Human IgG1 Constant Region)(allotype G1m(17,1))


(signal sequence underlined)


(SEQ ID NO. 154)










1
atgggatgga gctatatcat cctctttttg gtagcaacag ctacagatgt ccactcccag



 


61
gtccaactgc agcagcctgg ggctgaactg gtgaagcctg ggacttcagt gaagctgtcc


 


121
tgcaaggctt ctggctacac cttcaccacc tactggatgc actgggtgaa tcagaggcct


 


181
ggacaaggcc ttgagtggat tggagagatt aatcctacca acggtcatac taactacaat


 


241
gagaagttca agagcaaggc cacactgact gtagacaaat cctccagcac agcctacatg


 


301
caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag aaactatgtt


 


361
ggtagcatct ttgactactg gggccaaggc accactctca ccgtctcctc agcctccacc


 


421
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg


 


481
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca


 


541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac


 


601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc


 


661
aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt


 


721
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc


 


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca


 


841
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac


 


901
ggcgtggaqg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac


 


961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag


 


1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa


 


1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag


 


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag


 


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc


 


1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg


 


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


 


1381
ctctccctgt ctccgggtaa atga


 








(2) Protein Sequence Defining the Full Length Chimeric 2B8 Heavy Chain



(Chimeric 2B8 IgG1 (G1m(17,1) allotype)(without signal sequence)


(SEQ ID NO. 155)










1
qvqlqqpgae lvkpgtsvkl sckasgytft tywmhwvnqr pgqglewige inptnghtny



 


61
nekfkskatl tvdkssstay mqlssltsed savyycarny vgsifdywgq gttltvssas


 


121
tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl


 


181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps


 


241
vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst


 


301
yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt


 


361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq


 


421
gnvfscsvmh ealhnhytqk slslspgk


 








(3) Nucleic Acid Sequence Encoding the Full Length Chimeric 2B8 Light Chain



(Mouse Variable Region and Human Constant Region)(Chimeric 2B8 Kappa (Km(3)))


(signal sequence underlined)


(SEQ ID NO. 156)










1
atggaatcac agactctggt cttcatatcc atactgctct ggttatatgg tgctgatggg



 


61
aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gagggtcacc


 


121
ttgagctgca aggccagtga gaatgtggtt tcttatgtat cctggtatca acagaaacca


 


181
gcgcagtctc ctaaactgct gatatacggg gcatccaacc ggaacactgg ggtccccgat


 


241
cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcgggct


 


301
gaagaccttg cagattatca ctgtgggcag agttacaact atccgtacac gttcggaggg


 


361
gggaccaggc tggaaataaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca


 


421
tctgatgagc agttgaaatc tgaaactgcc tctgttgtgt gcctgctgaa taacttctat


 


481
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag


 


541
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg


 


601
ctaagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc


 


661
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga


 








(4) Protein Sequence Defining the Full Length Chimeric 2B8 Light Chain (Chimeric



2B8 Kappa (Km(3)))(without signal sequence)


(SEQ ID NO. 157)










1
nivmtqspks msmsvgervt lsckasenvv syvswyqqkp aqspklliyg asnrntgvpd



 


61
rftgsgsatd ftltissvra edladyhcgq synypytfgg gtrleikrtv aapsvfifpp


 


121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt


 


181
lskadyekhk vyacevthqg lsspvtksfn rgec


 








(5) Nucleic Acid Sequence Encoding Humanized Hu2B8 Hv1-f.1 Heavy Chain.



Variable Region (signal sequence underlined)


(SEQ ID NO. 158)










1
atggactgca cctggaggat cctcctcttg gtggcaccag ctacaggcac ccacgccgag



 


61
gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc


 


121
tgcaaggttt ctggatacac cttcaccacc tactggatgc actgggtgca acaggcccct


 


181
ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat


 


241
gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg


 


301
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt


 


361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag


 








(6) Protein Sequence Defining Humanized Hu2B8 Hv1-f.1 Heavy Chain Variable



Region (without signal sequence)


(SEQ ID NO. 159)










1
evqlvqsgae vkkpgatvki sckvsgytft tywmhwvqqa pgkglewmge inptnghtny



 


61
nekfqgrvti tadtstdtay melsslrsed tavyycatny vgsifdywgq gtlvtvss


 








(7) Nucleic Acid Sequence Encoding Human IgG1 Heavy Chain Constant Region



(G1m(17, 1) allotype)


(SEQ ID NO. 160)










1
cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg



 


61
gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt


 


121
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag


 


181
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct


 


241
acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttgagccca


 


301
aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac


 


361
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg


 


421
aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt


 


481
acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca


 


541
gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg


 


601
agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca


 


661
aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggatgagc


 


721
tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg


 


781
ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc


 


841
tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc


 


901
agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc


 


961
agaagagcct ctccctgtct ccgggtaaat ga


 








(8) Protein Sequence Defining Human IgG1 Heavy Chain Constant Region



(G1m(17, 1) allotype). The first amino acid is derived from translation


of the last nucleotide of variable region and beginning two nucleotides


of the IgG1 Heavy Chain sequence.


(SEQ ID NO. 161)










1
astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss



 


61
glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep kscdkthtcp pcpapellgg


 


121
psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn


 


181
styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsrde


 


241
ltknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw


 


301
qqgnvfscsv mhealhnhyt qkslslspgk


 








(9) Nucleic Acid Sequence Encoding the Full Length Heavy Chain Humanized



Hu2B8 Hv1f.1 Variable Region and Human IgG1 (Glm(17,1) allotype) Heavy


Chain Constant Region (signal seuence underlined)


(SEQ ID NO. 162)










1
atggactgca cctggaggat cctcctcttg gtggcagcag ctacaggcac ccacgccgag



 


61
gtccagctgg tacagtctgg ggctgaggtg aagaagcctg gggctacagt gaaaatctcc


 


121
tgcaaggttt ctggatacac cttcaccacc tactggatac actgggtgca acaggcccct


 


181
ggaaaagggc ttgagtggat gggagagatt aatcctacca acggtcatac taactacaat


 


241
gagaagttcc agggcagagt caccataacc gcggacacgt ctacagacac agcctacatg


 


301
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcaac aaactatgtt


 


361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc


 


421
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg


 


481
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca


 


541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac


 


601
tccctcagca gcgtggtgac catgccctcc agcagcttgg gcacccagac ctacatctgc


 


661
aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt


 


721
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc


 


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca


 


841
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac


 


901
ggcgtagagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac


 


961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag


 


1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa


 


1081
gagcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag


 


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag


 


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc


 


1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg


 


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


 


1381
ctctccctgt ctccgggtaa atga


 








(10) Protein Sequence Defining the Full Length Heavy Chain Humanized Hu2B8



Hv1f.1 Variable Region and Human IgG1 Heavy Chain Constant Region (G1m(17,1)


allotype)(without signal sequence)


(SEQ ID NO. 163)










1
evqlvqsgae vkkpgatvki sckvsgytft tywmhwvqqa pgkglewmge inptnghtny



 


61
nekfqgrvti tadtstdtay melsslrsed tavyycatny vgsifdywgq gtlvtvssas


 


121
tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl


 


181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps


 


241
vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst


 


301
yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt


 


361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq


 


421
gnvfscsvmh ealhnhytqk slslspgk


 








(11) Nucleic Acid Sequence Encoding Humanized Hu2B8 Hv5a.1 Heavy Chain



Variable Region (signal sequence underlined)


(SEQ ID NO. 164)










1
atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa



 


61
gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc


 


121
tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc


 


181
gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat


 


241
ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg


 


301
cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt


 


361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag


 








(12) Protein Sequence Defining Humanized Hu2B8 Hv5a.1 Heavy Chain Variable



Region (without signal sequence)


(SEQ ID NO. 165)










1
evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrqm pgkglewmge inptnghtny



 


61
npsfqghvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss


 








(13) Nucleic Acid Sequence Encoding the Full Length Humanized Hu2B8 Hv5a.1



Heavy Chain Variable Region and human IgG1 (G1m(17,1) allotype) Heavy


Chain Constant Region (signal sequence underlined)


(SEQ ID NO. 166)










1
atagggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa



 


61
gtacagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaggatctcc


 


121
tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc


 


181
gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat


 


241
ccgtccttcc aaggccacgt caccatctca gctgacaagt ccatcagcac tgcctacctg


 


301
cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt


 


361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc


 


421
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg


 


481
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca


 


541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac


 


601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc


 


661
aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt


 


721
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc


 


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca


 


841
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac


 


901
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac


 


961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc taaatggcaa ggagtacaag


 


1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa


 


1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag


 


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag


 


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc


 


1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg


 


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


 


1381
ctctccctgt ctccgggtaa atga


 








(14) Protein Sequence Defining the Full Length Humanized Hu2B8 Hv5a.1 Heavy



Chain Variable Region and Human IgG1 (G1m(17,1) allotype) Heavy Chain


Constant Region (without signal sequence)


(SEQ ID NO. 167)










1
evqlvqsgae vkkpgeslri sckgsgysft tywmhwvrqm pgkglewmge inptnghtny



 


61
npsfqghvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas


 


121
tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl


 


181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps


 


241
vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst


 


301
yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt


 


361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq


 


421
gnvfscsvmh ealhnhytqk slslspgk


 








(15) Nucleic Acid Sequence Encoding Humanized Hu2B8 Hv5-51.1 Heavy Chain



Variable Region (signal sequence underlined)


(SEQ ID NO. 168)










1
atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa



 


61
gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc


 


121
tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc


 


181
gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat


 


241
ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg


 


301
cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt


 


361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc ag


 








(16) Protein Sequence Defining Humanized Hu2B8 Hv5-51.1 Heavy Chain Variable



Sequence (without signal sequence)


(SEQ ID NO. 169)










1
evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny



 


61
npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvss


 








(17) Nucleic Acid Sequence Encoding the Full Length Humanized Hu2B8 Hv5-51.1



Heavy Chain Variable Region and Human IgG1 (G1m(17,1) allotype) Heavy Chain


Constant Region (signal sequence underlined)


(SEQ ID NO. 170)










1
atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa



 


61
gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc


 


121
tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc


 


181
gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat


 


241
ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg


 


301
cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt


 


361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc


 


421
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg


 


481
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca


 


541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac


 


601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc


 


661
aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt


 


721
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc


 


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca


 


841
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac


 


901
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac


 


961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag


 


1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa


 


1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag


 


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag


 


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc


 


1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg


 


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


 


1381
ctctccctgt ctccgggtaa atga


 








(18) Protein Sequence Defining the Full Length Humanized H2B8 Hv.5-51.1 Heavy



Chain Variable Region and Human IgG1 (G1m(17,1) allotype) Heavy Chain Constant


Region (without signal sequence


(SEQ ID NO. 171)










1
evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny



 


61
npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas


 


121
tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl


 


181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkkvepks cdkthtcppc papellggps


 


241
vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst


 


301
yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsrdelt


 


361
knqvsltclv kgfyrsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq


 


421
gnvfscsvmh ealhnhytqk slslspgk


 








(19) Nucleic Acid Sequence Encoding Humanized Hu2B8 Kv1-39.1 Kappa Chain



Variable Region (signal sequence underlined). Two possible start ATGs are


shown in uppercase.


(SEQ ID NO. 172)










1
ATGgacATGa gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc



 


61
agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga


 


121
gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag


 


181
aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc


 


241
ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg


 


301
caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt


 


361
ggccagggga ccaagctgga gatcaaac


 








(20) Protein Sequence Defining Humanized Hu2B8 Kv1-39.1. Kappa Chain Variable



Region (without signal sequence)


(SEQ ID NO. 173)










1
diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp gkapklliyg asnrntgvps



 


61
rfsgsgsgtd ftltisslqp edfatyycgq synypytfgq gtkleik


 








(21) Nucleic Acid Sequence Encoding Human Kappa Chain Constant Region



(Km(3) allotype)(allele 2)


(SEQ ID NO. 174)










1
gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg



 


61
gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt


 


121
ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca


 


181
gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga


 


241
aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga


 


301
gcttcaacag gggagagtgt tga


 








(22) Protein Sequence Defining Human Kappa Chain Constant Region (Km(3)



allotype)(allele 2). The first amino acid is derived from translation of the


last nucleotide of variable region and beginning two nucleotides of the Kappa


Light Chain sequence.


(SEQ ID NO. 175)










1
rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd



 


61
skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec


 








(23) Nucleic Acid Sequence Encoding the Full Length Humanized Hu2B8 Kv1-39.1



Light Chain Variable Region and Human Kappa Chain Constant Region (Km(3)


Allotype)(allele 2)(signal sequence underlined)


(SEQ ID NO. 176)










1
atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc



 


61
agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga


 


121
gtcaccatca cttgcaaggc cagtgagaat gtggtttctt atgtatcctg gtatcagcag


 


181
aaaccaggga aagcccctaa gctcctgatc tatggggcat ccaaccggaa cactggggtc


 


241
ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg


 


301
caacctgaag attttgcaac ttactactgt gggcagagtt acaactatcc gtacacgttt


 


361
ggccagggga ccaagctgga gatcaaacga actgtggctg caccatctgt cttcatcttc


 


421
ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac


 


481
ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac


 


541
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc


 


601
ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat


 


661
cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagaatgttg a


 








(24) Protein Sequence Defining the Full Length Humanized Hu2B8 Kv1-39.1 Light



Chain Variable Region and Human Kappa Chain Constant Region (Km(3) allotype)


(allele 1)


(SEQ ID NO. 177)










1
diqmtqspss lsasvgdrvt itckasenvv syvswyqqkp gkapklliyg asnrntgvps



 


61
rfsgsgsgtd ftltisslqp edfatyycgq synypytfgq gtkleikrtv aapsvfifpp


 


121
sdeqlksgta svvcllnnfy preakvqwkv dhalqsgnsq esvteqdskd styslsstlt


 


181
lskadyekhk vyacevthqg lsspvtksfn rgec


 








(25) Nucleic Acid Sequence Encoding Humanized Hu2B8 Kv3-15.1 Light Chain



Variable Region (signal sequence underlined)


(SEQ ID NO. 178)










1
atggaagccc cagggcagct tctcttcctc ctgctactct ggctcccaga taccactgga



 


61
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc


 


121
ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct


 


181
ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg tatcccagcc


 


241
aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct


 


301
gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag


 


361
gggaccaagc tggagatcaa ac


 








(26) Protein Sequence Defining Humanized Hu2B8 Kv3-15.1 Liqht Chain Variable



Region (without signal sequence)


(SEQ ID NO. 179)










1
eivmtqspat lsvspgerat lsckasenvv syvswyqqkp gqaprlliyg asnrntgipa



 


61
rfsgsgsgte ftltisslqs edfavyycgq synypytfgq gtkleik


 








(27) Nucleic Acid Encoding the Full Length Humanized Hu2B8 Kv3-15.1 Light



Chain Variable Region and Human Kappa Chain Constant Region (Km(3) allotype)


(allele 2)(signal sequence underlined)


(SEQ ID NO. 180)










1
atggaagccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccactgga



 


61
gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc


 


121
ctctcctgca aggccagtga gaatgtggtt tcttatgtat cctggtacca gcagaaacct


 


181
ggccaggctc ccaggctcct catctatggg gcatccaacc ggaacactgg tatcccagcc


 


241
aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct


 


301
gaagattttg cagtttatta ctgtgggcag agttacaact atccgtacac gtttggccag


 


361
gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca


 


421
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat


 


481
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag


 


541
gagagtgtca caaagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg


 


601
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc


 


661
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttga


 








(28) Protein Sequence Defining Humanized Hu2B8 Kv3-15.1 Light Chain Variable



Region and Human Kappa Chain Constant Region (Km(3) allotype)(allele 2)


(without signal sequence)


(SEQ ID NO. 181)










1
eivmtqspat lsvspgerat lsckasenvv syvswyqqkp gqaprlliyg asnrntgipa



 


61
rfsgsgsgte ftltisslqs edfavyycgq synypytfgq gtkleikrtv aapsvfifpp


 


121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt


 


181
lskadyekhk vyacevthqg lsspvtksfn rgec






For convenience, Table 13 provides a concordance chart showing the correspondence between the full length sequences and of the antibodies discussed in this section with those presented in the Sequence Listing.








TABLE 13





SEQ. ID 



NO.
Protein or Nucleic Acid







154
Chimeric 2B8 IgG1 (G1m(17,1)) - nucleic acid


155
Chimeric 2B8 IgG1 (G1m(17,1)) - protein


156
Chimeric 2B8 Kappa (Km(3)) - nucleic acid


157
Chimeric 2B8 Kappa (Km(3)) - protein


158
Hu2B8 Hv1f.1 Heavy Chain Variable Region - nucleic acid


159
Hu2B8 Hv1f.1 Heavy Chain Variable Region - protein


160
Human IgG1 Heavy Chain Constant Region (G1m(17,1))



allotype - nucleic acid


161
Human IgG1 Heavy Chain Constant Region (G1m(17,1)) 



allotype - protein


162
Hu2B8 Hv1f.1 + IgG1 Constant (G1m(17,1) allotype) - 



nucleic acid


163
Hu2B8 Hv1f.1 + IgG1 Constant (G1m(17,1) allotype) - protein


164
Hu2B8 Hv5a.1 Heavy Chain Variable Region - nucleic acid


165
Hu2B8 Hv5a.1 Heavy Chain Variable Region - protein


166
Hu2B8 Hv5a.1 + IgG1 Constant (G1m(17,1) allotype) - 



nucleic acid


167
Hu2B8 Hv5a.1 + IgG1 Constant (G1m(17,1) allotype) - protein


168
Hu2B8 Hv5-51.1 Heavy Chain Variable Region - nucleic acid


169
Hu2B8 Hv5-51.1 Heavy Chain Variable Region - protein


170
Hu2B8 Hv5-51.1 + IgG1 Constant (G1m(17,1 allotype) -



nucleic acid


171
Hu2B8 Hv5-51.1 + IgG1 Constant (G1m(17,1 allotype) - 



protein


172
Hu2B8 Kv1-39.1 Kappa Chain Variable Region - nucleic acid


173
Hu2B8 Kv1-39.1 Kappa Chain Variable Region - protein


174
Human Kappa Chain Constant Region (Km(3) allotype) 



(allele 2) - nucleic acid


175
Human Kappa Chain Constant Region (Km(3) allotype) 



(allele 2) - protein


176
Hu2B8 Kv1-39.1 + Kappa Constant (Km(3) allotype) 



(allele 2) - nucleic acid


177
Hu2B8 Kv1-39.1 + Kappa Constant (Km(3) allotype) (allele 2) - 



protein


178
Hu2B8 Kv3-15.1 Kappa Chain Variable Region - nucleic acid


179
Hu2B8 Kv3-15.1 Kappa Chain Variable Region - protein


180
Hu2B8 Kv3-15.1 + Kappa Constant (Km(3) allotype) (allele 2) -



nucleic acid


181
Hu2B8 Kv3-15.1 + Kappa Constant (Km(3) allotype) (allele 2) -



protein









B. Humanization Procedure 2

The second humanization method employed for reducing immunogenicity of the mouse 2B8 antibody is based on the method described in Studnicka et al. (1994) PROTEIN ENG. 7:805-814. The heavy and kappa human germline variable regions most identical (at the amino acid level) to those of mouse 2B8 were identified. Residues that differed between mouse and human were converted into the human sequence depending on the likely risk that such a change would affect binding or immunogenicity. Low risk residues (i.e., residues that when changed would likely not affect antigen binding and would also reduce potential immunogenicity) were changed to the human amino acid in the heavy variable region (creating LR2B8HC) and the kappa variable region (creating LR2B8LC). Additionally, low risk and medium risk (i.e., residues that when changed are somewhat likely to have an effect on antigen binding residues and would also reduce potential immunogenicity) were changed to the human amino acid in the heavy variable region (creating LRMR2B8HC) and the kappa variable region (creating LRMR2B8LC). The human IgG1 heavy chain constant region (G1m(3) allotype (allele 1)) was added to the carboxyl terminus of the two human engineered heavy variable regions and the human Kappa constant region (Km(3) allotype (allele 1)) was added to the carboxyl terminus of two human engineered light variable regions, thus creating four human engineered antibody chains. Variable region nucleic acid sequences were first synthesized by gene synthesis methods and then added to human constant region sequences. These human engineered antibodies were cloned into mammalian protein expression vectors, and protein was expressed in the four possible combinations of heavy chain plus light chain. Binding of the chimeric, chimeric/humanized, or humanized antibodies to human HGF was measured using conventional techniques, as described below.
The nucleic acid sequences encoding and the protein sequences defining each of the humanized antibodies are summarized below. In this section, the last nucleotide of each variable region is the first base of the next codon generated by the variable/constant region junction. This nucleotide is included in the Variable Region because it is part of that exon. Amino acid sequences of Constant Regions listed below include the translation of this junction codon.








(1) Nucleic Acid Sequence Encoding the Humanized LR2B8HC Heavy Chain



Variable Region (signal sequence underlined)


(SEQ ID NO. 182)










1
atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa



 


61
gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca


 


121
tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc


 


181
ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat


 


241
gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg


 


301
gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta


 


361
ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc ag


 








(2) Protein Sequence Defining Humanized LR2B8HC Heavy Chain Variable Region



(without signal sequence)


(SEQ ID NO. 183)










1
qvqlvqpgae vvkpgtsvkl sckasgytft tywmhwvnqa pgqglewige inptnghtny



 


61
nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdwvgq gtlltvss


 








(3) Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain Constant



Region (G1m(3) allotype)(allele 1)


(SEQ ID NO. 184)










1
ccagcacaaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg



 


61
gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt


 


121
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag


 


181
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct


 


241
acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca


 


301
aatcttgtga caaaactcac acatgtccac cgtgcccagc acctgaactc ctggggggac


 


361
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg


 


421
aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt


 


481
acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca


 


541
gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg


 


601
agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca


 


661
aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga


 


721
tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg


 


781
ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc


 


841
tggactccga cggctccttc ttcctctata gcaagctcac cgtggacaag agcaggtggc


 


901
agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc


 


961
agaagagcct ctccctgtcc ccgggtaaat ga


 








(4) Protein Sequence Defining Human IgG1 Heavy Chain Constant (G1m(3)



allotype)(allele 1 or 2). The first amino acid is derived from translation of


the last nucleotide of variable region and the beginning two nucleotides of


the IgG1 Heavy Chain sequence.


(SEQ ID NO. 185)










1
astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss



 


61
glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg


 


121
psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn


 


181
styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree


 


241
mtknqysltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw


 


301
qqgnvfscsv mhealhnhyt qkslslspgk


 








(5) Nucleic Acid Sequence Encoding the Full Length Heavy Chain Humanized



LR2B8HC Heavy Chain Variable Region and Human IgG1 Heavy Chain Constant Region


(G1m(3) allotype)(allele 1)(signal sequence underlined)


(SEQ ID NO. 186)










1
atgggctggt catatattat tctctttctt gttgctaccg ctaccgatgt gcactctcaa



 


61
gtccaactcg tacaaccagg cgctgaagtc gtaaaacccg gaacatctgt taaactctca


 


121
tgcaaagcct caggatacac tttcacaact tactggatgc attgggtcaa tcaagccccc


 


181
ggacaaggcc tcgaatggat tggcgaaatt aacccaacta acggacatac taattataat


 


241
gaaaaattta agggcaaagc tacactcacc gtcgataaat caacctctac agcttatatg


 


301
gaactttcat ccctgagatc agaagataca gccgtctact attgcgccag aaactacgta


 


361
ggatcaatat tcgattactg gggtcaaggc actctcctca cagtcagctc agccagcaca


 


421
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg


 


481
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca


 


541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac


 


601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc


 


661
aacgtgaatc acaagcccag caacaccaag gtgaacaaga gagttgagcc caaatcttat


 


721
gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc


 


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca


 


841
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac


 


901
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac


 


961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag


 


1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa


 


1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag


 


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag


 


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc


 


1261
gacggctcct tcttcctcta tagcaagctc accgtggaca agaacaggtg gcagcagggg


 


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


 


1381
ctctccctgt ccccgggtaa atga


 








(6) Protein Sequence Defining the Full Length Heavy Chain Humanized LR2B8HC



Heavy Chain Variable Region and Human IgG1 Heavy Chain Constant Region (G1m(3)


allotype)(allele 1)(without signal sequence)


(SEQ ID NO. 187)










1
qvglvgpgae vvkpgtsvkl sckasgytft tywmhwynqa pgqglewige inptnghtny



 


61
nekfkgkatl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvssas


 


121
tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl


 


181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps


 


241
vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst


 


301
yrvvsvltv1 hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt


 


361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq


 


421
gnvfscsvmh ealhnhytqk slslspgk


 








(7) Nucleic Acid Sequence Encoding the Humanized LRMR2B8HC Heavy Chain



Variable Region (signal sequence underlined)


(SEQ. ID NO. 188)










1
atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag



 


61
gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca


 


121
tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc


 


181
ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat


 


241
caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg


 


301
gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta


 


361
ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc ag


 








(8) Protein Sequence Defining Humanized LRMR2B8HC Heavy Chain Variable



Region (without signal sequence)


(SEQ ID NO. 189)










1
qvglvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pgqglewige inptnghtny



 


61
nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvss


 








(9) Nucleic Acid Sequence Encoding the Full Length Heavy Chain Humanized



LRMR2B8HC Heavy Chain Variable Region and Human IgG1 Heavy Chain Constant


Region (G1m(3) allotype)(allele 1)(signal sequence underlined)


(SEQ ID NO. 190)










1
atgggttggt catatattat actctttctc gtagccaccg ccaccgacgt acactctcag



 


61
gttcaactcg tacaacccgg cgccgaagtc aagaaaccag gaacatcagt caaactctca


 


121
tgtaaagcaa gcggatacac ctttactact tattggatgc attgggtaag acaagccccc


 


181
ggacaaggac tcgaatggat aggcgaaata aatcccacta atggacatac aaattataat


 


241
caaaaatttc aaggacgcgc tacactcacc gtcgataaat caacctcaac cgcatacatg


 


301
gaactcagct ccctccgatc cgaagacact gccgtttatt attgtgccag aaactatgta


 


361
ggatctattt tcgattactg gggacaagga acacttctca ccgtaagctc agccagcaca


 


421
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg


 


481
gccctgggct gcctggtcaa gaactacttc cccgaaccgg tgacggtgtc gtggaactca


 


541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac


 


601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc


 


661
aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt


 


721
gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc


 


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca


 


841
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac


 


901
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac


 


961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag


 


1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa


 


1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag


 


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag


 


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc


 


1261
gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg


 


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


 


1381
ctctccctgt ccccgggtaa atga


 








(10) Protein Sequence Defining the Full Length Heavy Chain Humanized



LRMR2B8HC Heavy Chain Variable Region and Human IgG1 Heavy Chain Constant


Region (G1m(3) allotype)(allele 1)(without signal sequence)


(SEQ ID NO. 191)










1
qvqlvqpgae vkkpgtsvkl sckasgytft tywmhwvrqa pggglewige inptnghtny



 


61
nqkfqgratl tvdkststay melsslrsed tavyycarny vgsifdywgq gtlltvssas


 


121
tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl


 


181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps


 


241
vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst


 


301
yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt


 


361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq


 


421
gnvfscsvmh ealhnhytqk slslspgk


 








(11) Nucleic Acid Sequence Encoding the Humanized LR2B8LC Light Chain



Variable Region (signal sequence underlined)


(SEQ ID NO. 192)










1
atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc



 


61
gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc


 


121
cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc


 


181
ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac


 


241
agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca


 


301
gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa


 








(12) Protein Sequence Defining Humanized LR2B8LC Light Chain Variable Region



(without signal sequence)


(SEQ ID NO. 193)










1
divmtqspds mamsvgervt lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd



 


61
rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik


 








(13) Nucleic Acid Sequence Encoding the Human Kappa Chain Constant Region



(Km(3) allotype)(allele 1)


(SEQ ID NO. 194)










1
gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg



 


61
gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt


 


121
ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca


 


181
gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga


 


241
aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga


 


301
gcttcaacag gggagagtgt tag


 








(14) Protein Sequence Defining the Human Kappa Chain Constant Region (Km(3)



allotype)(allele 1). The first amino acid derived from translation of the last


nucleotide of variable region and beginning two nucleotides of the Kappa Light


Chain sequence.


(SEQ ID NO. 195)










1
rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd



 


61
skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec


 








(15) Nucleic Acid Sequence Encoding the Full Length Humanized LR2B8LC Light



Chain Variable Region and the Human Kappa Chain Constant Region (Km(3)


allotyp)(allele 1)


(SEQ ID NO. 196)










1
atggaaagtc agacccttgt attcatctct attcttcttt ggttgtatgg agcagacggc



 


61
gacattgtga tgacccaatc ccccgatagt atggccatga gtgtaggaga aagagtcacc


 


121
cttaattgca aagcctccga aaatgtcgtt tcatatgtgt cttggtatca acaaaaaccc


 


181
ggccaatcac ccaaacttct catatacggc gcttcaaaca gaaacacagg cgttcccgac


 


241
agatttagtg gatccggatc agctacagat ttcaccctta ccatcagttc agttcaagca


 


301
gaagacgttg cagactatca ttgcggacaa tcttataact acccttacac attcggacaa


 


361
ggaaccaaac tcgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca


 


421
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat


 


481
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag


 


541
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg


 


601
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc


 


661
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag


 








(16) Protein Sequence Encoding the Full Length Humanized LR2B8LC Light Chain



Variable Region and the Human Kappa Chain Constant Region (Km(3) allotype)


(allele 1)


(SEQ ID NO. 197)










1
divmtqspds mamsvgervt lnckasenvv syvswyqqkp gqspklliyg asnrntgvpd



 


61
rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleikrtv aapsvfifpp


 


121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt


 


181
lskadyekhk vyacevthqg lsspvtksfn rgec


 








(17) Nucleic Acid Sequence Encoding the Humanized LRMR2B8LC Light Chain



Variable Region (signal sequence underlined)


(SEQ ID NO. 198)










1
atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga



 


61
gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca


 


121
cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct


 


181
ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac


 


241
agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc


 


301
gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa


 


361
ggcactaaac ttgaaattaa ac


 








(18) Protein Sequence Defining the Humanized LRMR2B8LC Light Chain Variable



Region (without signal sequence)


(SEQ ID NO. 199)










1
divmtqspds lamslgervt lnckasenvv syvswyqqkp gqspklliyg asnresgvpd



 


61
rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleik


 








(19) Nucleic Acid Sequence Encoding the Full Length Humanized LRMR2B8LC



Light Chain Variable Region and the Human Kappa Chain Constant Region (Km(3)


allotype)(allele 1)(signal sequence underlined)


(SEQ ID NO. 200)










1
atggaatccc aaacccttgt tttcatctct atccttctct ggctttatgg cgccgacgga



 


61
gacatcgtaa tgacacaatc ccctgactct cttgctatga gcttgggcga acgagtaaca


 


121
cttaactgca aagcatccga aaatgtcgta tcttacgtat cctggtatca gcaaaaacct


 


181
ggtcaaagtc ctaaacttct tatatatggt gcaagtaatc gtgaaagtgg cgtcccagac


 


241
agatttagcg gttcaggttc agcaactgac tttacactta caatttctag cgttcaggcc


 


301
gaagacgttg cagactatca ttgtggacaa tcttataact atccttatac tttcggacaa


 


361
ggcactaaac ttgaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca


 


421
tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat


 


481
cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag


 


541
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg


 


601
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc


 


661
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag


 








(20) Protein Sequence Defining the Full Length Humanized LRMR2B8LC Light



Chain Variable Region and the Human Kappa Chain Constant Region (Km(3)


allotype)(allele 1)


(SEQ ID NO. 201)










1
divmtqspds lamslgervt lnckasenvv syvswyqqkp gqspklliyg asnresgvpd



 


61
rfsgsgsatd ftltissvqa edvadyhcgq synypytfgq gtkleikrtv aapsvfifpp


 


121
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt


 


181
lskadyekhk vyacevthqg lsspvtksfn rgec






For convenience, Table 14 provides a concordance chart showing the correspondence between the full length sequences and of the antibodies discussed in this section with those presented in the Sequence Listing.








TABLE 14





SEQ. ID 



NO.
Protein or Nucleic Acid







182
LR2B8HC Heavy Chain Variable Region - nucleic acid


183
LR2B8HC Heavy Chain Variable Region - protein


184
Human IgG1 Heavy Chain Constant Region (G1m(3) allotype) 



(allele 1) - nucleic acid


185
Human IgG1 Heavy Chain Constant Region (G1m(3) allotype) 



(allele 1) - protein


186
LR2B8HC + IgG1 Constant (G1m(3) allotype) (allele 1) - 



nucleic acid


187
LR2B8HC + IgG1 Constant (G1m(3) allotype) (allele 1) - 



protein


188
LRMR2B8HC Heavy Chain Variable Region - nucleic acid


189
LRMR2B8HC Heavy Chain Variable Region - protein


190
LRMR2B8HC + IgG1 Constant (G1m(3) allotype) (allele 1) - 



nucleic acid


191
LRMR2B8HC + IgG1 Constant (G1m(3) allotype) (allele 1) - 



protein


192
LR2B8LC Light Chain Variable Region - nucleic acid


193
LR2B8LC Light Chain Variable Region - protein


194
Human Kappa Chain Constant Region (Km(3) allotype) 



(allele 1) - nucleic acid


195
Human Kappa Chain Constant Region (Km(3) allotype) 



(allele 1) - protein


196
LR2B8LC + Kappa Constant (Km(3) allotype) (allele 1) - 



nucleic acid


197
LR2B8LC + Kappa Constant (Km(3) allotype) (allele 1) - 



protein 


198 
LRMR2B8LC Light Chain Variable Region - nucleic acid


199
LRMR2B8LC Light Chain Variable Region - protein


200
LRMR2B8LC + Kappa Constant (Km(3) allotype) (allele 1) - 



nucleic acid


201
LRMR2B8LC + Kappa Constant (Km(3) allotype) (allele 1) - 



protein









Table 15 summarizes the heavy chain CDR sequences (Kabat Definition) of the humanized 2B8 antibodies prepared by humanization procedure 1 and by humanization procedure 2 described herein above in this Example.











TABLE 15









Full Length






Heavy Chain






Variable


Antibody
CDR1
CDR2
CDR3
Region







Murine 2138
TYWMH
EINPTNGHTNYNEKFKS
NYVGSIFDY
SEQ ID NO


Heavy
(SEQ ID NO: 15)
(SEQ ID NO: 16)
(SEQ ID NO: 17)
12


 


Hu2B8 
TYWMH
EINPTNGHTNYNEKFQG
NYVGSIFDY
SEQ ID NO


Hv1f.1
(SEQ ID NO: 15)
(SEQ ID NO: 202)
(SEQ ID NO: 17)
159


 


Hu2B8 
TYWMH
EINPTNGHTNYNPSFQG
NYVGSIFDY
SEQ ID NO:


Hv5a.1
(SEQ ID NO: 15)
(SEQ ID NO: 203)
(SEQ ID NO: 17)
165


 


Hu2B8 
TYWMH
EINPTNGHTNYNPSFQG
NYVGSIFDY
SEQ ID NO:


Hv5-51.1
(SEQ ID NO: 15)
(SEQ ID NO: 203)
(SEQ ID NO: 17)
169


 


LR2B8HC
TYWMH
EINPTNGHTNYNEKFKG
NYVGSIFDY
SEQ ID NO:



(SEQ ID NO: 15)
(SEQ ID NO: 204)
(SEQ ID NO: 17)
183


 


LRMR2B8HC
TYWMH
EINPTNGHTNYNQKFQG
NYVGSIFDY
SEQ ID NO:



(SEQ ID NO: 15)
(SEQ ID NO: 205)
(SEQ ID NO: 17)
189









Table 16 summarizes the light chain CDR sequences (Kabat Definition) of the humanized 2B8 antibodies prepared by humanization procedure 1 and by humanization procedure 2 described herein above in this Example.











TABLE 16









Full Length Light






Chain Variable


Antibody
CDR1
CDR2
CDR3
Region







Murine 2B8
KASENVVSYVS
GASNRNT
GQSYNYPYT
SEQ ID NO. 14


Light
(SEQ ID NO: 18)
(SEQ ID NO: 19)
(SEQ ID NO: 20)



 


Hu2B8 Kv1-39.1
KASENVVSYVS
GASNRNT
GQSYNYPYT
SEQ ID NO: 173



(SEQ ID NO: 18)
(SEQ ID NO: 19)
(SEQ ID NO: 20)



 


Hu2B8 Kv3-15.1
KASENVVSYVS
GASNRNT
GQSYNYPYT
SEQ ID NO: 179



(SEQ ID NO: 18)
(SEQ ID NO: 19)
(SEQ ID NO: 20)



 


LR2B8LC
KASENVVSYVS
GASNRNT
GQSYNYPYT
SEQ ID NO: 193



(SEQ ID NO: 18)
(SEQ ID NO: 19)
(SEQ ID NO: 20)



 


LRMR2B8LC
KASENVVSYVS
GASNRES
GQSYNYPYT
SEQ ID NO: 199



(SEQ ID NO: 18)
(SEQ ID NO: 206)
(SEQ ID NO: 20)









C. Binding Affinity of Humanized 2B8 Antibodies

Antigen-binding affinity and kinetics of interaction were assessed by surface plasmon resonance technology using a BIAcore T100 instrument. Mouse anti-human immunoglobulins (Jackson ImmunoResearch Labs, 209-005-098) were immobilized on carboxymethylated dextran CM4 sensor chips (BIAcore, Catalog No. BR-1005-34) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a standard coupling protocol according to manufacturer's recommendations. The analyses were performed at 25° C. using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No. BR-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running buffer.
The antibodies were captured on individual flow cell at a flow rate of 10 μL/min. Injection time was variable for each antibody to yield approximately 20 RU of antibody captured for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 2 minutes at 60 μL/min. The dissociation phase was monitored for 15 or 90 minutes, depending on concentration. The surface then was regenerated with 10 mM Glycine-HCl, pH 2.0 (BIAcore, Catalog No. BR-1003-55) injected for 3 minutes at a flow rate of 60 μL/min before another cycle was initiated. HGF concentrations tested were 1.88, 3.75 and 7.5 nM. Determination of kinetic parameters was achieved using the kinetic function of the BIAevalutation software with reference subtraction. Kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized in FIG. 8.
The results summarized in FIG. 8 show that certain combinations of superhumanized heavy chains (Hu2B8 Hv5a.1, Hu2B8 Hv5-51.1 or Hu2B8 Hv1-f.1) and light chains (Hu2B8 Kv1-39.1 or Hu2B8 Kv3-15.1) retain similar binding affinity (KD) to HGF as chimeric 2B8 (mouse variable regions with human constant regions) and 2B8 (Table 5).
D. Mutually Exclusive Binding Assay
Mutually exclusive binding to HGF was assessed by surface plasmon resonance technology using a BIAcore T100 instrument. Mouse anti-human immunoglobulins (Jackson ImmunoResearch Labs, 209-005-098) were immobilized on carboxymethylated dextran CM5 sensor chips (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a standard coupling protocol according to manufacturer's recommendations. The analyses were performed at 25° C. using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore, #BR-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/ml CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running buffer.
The humanized antibodies were captured on an individual flow cell at a flow rate of 30 μL/min. Injection time was variable for each antibody to yield approximately 150 RU of antibody captured for each cycle. HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer at a final concentration of 7.5 μg/mL was injected for 90 sec at 30 μL/min over the captured humanized antibodies. Binding of HGF was monitored before subsequent injection of mouse 2B8 antibody or polyclonal goat anti-HGF antibody (R & D Systems, AF294) for 3 min at 30 μL/min. The surface then was regenerated with 10 mM Glycine-HCl, pH 2.0 (BIAcore, Catalog No. BR-1003-55) injected for 3 min at a flow rate of 60 μL/min before another antibody was tested. The results are summarized in FIG. 9.
Results summarized in FIG. 9 show that both humanized 2B8 antibodies and chimeric 2B8 antibodies prevent murine 2B8 from binding HGF. These results demonstrate that the humanized antibodies still bind the same HGF epitope as the original 2B8 antibody.
Example 13
Production of Humanized 2B8 Variants
a. Human EngineeredTM Antibodies
Codon- and expression-optimized low risk and low-plus-moderate risk Human Engineered light chain (LR2BSLC and LRMR2B8LC, respectively) and heavy chains (LR2B8HC and LRMR2B8HC, respectively) were cloned in-phase into XOMA's transient antibody expression vectors, which contain human Kappa and Gamma-1 constant regions modules. The four Human Engineered 2B8 variants were produced by transient transfection in HEK293E cells. The following four antibodies were produced:

    
    
        HE2B8-1=LR2B8HC (+IgG1 constant region (G1m(3) allotype (allele 1)) (SEQ ID NO. 187) plus LR2B8LC (+Kappa constant region (Km(3) allotype (allele 1))) (SEQ ID NO. 197)
        HE2B8-2=LR2B8HC (+IgG1 constant region (G1m(3) allotype (allele 1)) (SEQ ID NO. 187) plus LRMR2B8LC (4 Kappa constant region (Km(3) allotype (allele 1))) (SEQ ID NO. 201)
        HE2B8-3=LRMR2B8HC (+IgG1 constant region (G1m(3) allotype (allele 1)) (SEQ ID NO. 191) plus LR2B8LC (+Kappa constant region (Km(3) allotype (allele 1))) (SEQ ID NO. 197)
        HE2B8-4=LRMR2B8HC (+IgG1 constant region (G1m(3) allotype (allele 1)) (SEQ ID NO. 191) plus LRMR2B8LC (+Kappa constant region (Km(3) allotype (allele 1))) (SEQ ID NO. 201)
    
    


The light and heavy chains were co-transfected into XOMA's suspension adapted HEK293E cells grown in IS293 media (Irvine Scientific, Irvine, Calif.) using 2 liter shake flasks. After 24 hours in the shake flasks, 200 mL of transfected cells were centrifuged, resuspended in 40 mL of fresh medium and transferred to Integra flasks (Wilson Wolf Manufacturing Inc., MN) for production. After incubation for seven days, the cell suspensions were removed from the Integra flasks, centrifuged and the culture supernatants retained. Antibodies in the culture supernatants were purified on protein A spin columns (Pro-Chem), dialyzed against PBS, concentrated and sterile filtered.
b. SuperhumanizedTM Antibodies
Full length Hu2B8_Hv5-51.1+human IgG1 constant domain (G1m(3) allotype) cDNA was cloned into pEE6.4 (Lonza Biologics, Berkshire, UK) using HindIII and EcoRI restriction sites. Full length Hu2B8_Kv1-39.1 variable region+human Kappa constant domain cDNA and full length Hu2B8_Kv3-15.1 variable region+human Kappa constant domain cDNA were each cloned into pEE14.4 (Lonza Biologics) using HindIII and EcoRI restriction sites. The hCMV-MIE promoter+full length Hu2B8_Hv5-51.1+human IgG1 constant domain (G1m(3) allotype) cDNA+SV40 poly A fragment (in pEE6.4) was removed by NotI/SalI digestion and inserted into either Kappa chain pEE14.4 vector through NotI/SalI sites, thus creating 2 different expression vectors that each simultaneously express heavy and light chain to make the following antibodies:

    
    
        sh2B8-9 (G1m(3))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(3) allotype) (allele
    
    


2)) (SEQ ID NO. 210) plus hu2B8 Kv 1-39.1 (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID NO: 177)

    
    
        sh2B8-12 (G1m(3))=hu2B8 Hv5-51.1 (+IgG1 constant region (G1m(3) allotype) (allele 2)) (SEQ ID NO. 210) plus hu2B8 Kv 3-15.1 (+Kappa constant region (Km(3) allotype (allele 2))) (SEQ ID No. 181)
    
    


The nucleic acid sequences encoding and the protein sequences defining the human IgG1 Heavy Constant Region GIm(3) allotype (allele 2) and each of the full length heavy chain sequences are set forth below. The light chain sequences were the same as described in Example 12.








(1) Nucleic Acid Sequence Encoding Human IgG1 Heavy Chain Constant Region



(G1m(3) allotype)(allele 2)


(SEQ ID NO. 207)










1
cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg



 


61
gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt


 


121
ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag


 


181
gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct


 


241
acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga gttgagccca


 


301
aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac


 


361
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg


 


421
aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt


 


481
acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca


 


541
gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg


 


601
agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaag accatctcca


 


661
aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga


 


721
tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg


 


781
ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc


 


841
tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc


 


901
agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac cactacacgc


 


961
agaagagcct ctccctgtct ccgggtaaat ga


 








(2) Protein Sequence Defining Human IgG1 Heavy Chain Constant Region (G1m(3)



allotype)(allele 1 or 2). The first amino acid is derived from translation of


the last nucleotide of variable region and the beginning two nucleotides of


the IgG1 Heavy Chain sequence.


(SEQ ID NO. 208)










1
astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss



 


61
glyslssyvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg


 


121
psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn


 


181
styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree


 


241
mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw


 


301
qqgnvfscsv mhealhnhyt qkslslspgk


 








(3) Nucleic Acid Sequence Encoding the Full Length Chain Containing Humanized



Hu2B8 Hv5-51.1 Heavy Chain Variable Region and the Human IgG1 Heavy Chain Constant


Region G1m(3) allotype (allele 2)(signal sequence underlined).


(SEQ ID NO. 209)










1
atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgaa



 


61
gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc


 


121
tgtaagggtt ctggatacag ctttaccacc tactggatgc actgggtgcg ccagatgccc


 


181
gggaaaggcc tggagtggat gggggagatt aatcctacca acggtcatac taactacaat


 


241
ccgtccttcc aaggccaggt caccatctca gctgacaagt ccatcagcac tgcctacctg


 


301
cagtggagca gcctgaaggc ctcggacacc gccatgtatt actgtgcgag aaactatgtt


 


361
ggtagcatct ttgactactg gggccaagga accctggtca ccgtctcctc agcctccacc


 


421
aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg


 


481
gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca


 


541
ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac


 


601
tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc


 


661
aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt


 


721
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc


 


781
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca


 


841
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac


 


901
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac


 


961
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag


 


1021
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga agaccatctc caaagccaaa


 


1081
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag


 


1141
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag


 


1201
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc


 


1261
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg


 


1321
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc


 


1381
ctctccctgt ctccgggtaa atga


 








(4) Protein Sequence Defining the Full Length Heavy Chain Containing Humanized



Hu2B8 Hv5-51.1 and the Human IgG1 Heavy Chain Constant Region G1m(3) allotype


(allele2) (without signal sequence)


(SEQ ID NO. 210)










1
evqlvqsgae vkkpgeslki sckgsgysft tywmhwvrqm pgkglewmge inptnghtny



 


61
npsfqgqvti sadksistay lqwsslkasd tamyycarny vgsifdywgq gtlvtvssas


 


121
tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl


 


181
yslssvvtvp ssslgtqtyi cnvnhkpsnt kvdkrvepks cdkthtcppc papellggps


 


241
vflfppkpkd tlmisrtpev tcvvvdvshe dpevkfnwyv dgvevhnakt kpreeqynst


 


301
yrvvsvltvl hqdwlngkey kckvsnkalp apiektiska kgqprepqvy tlppsreemt


 


361
knqvsltclv kgfypsdiav ewesngqpen nykttppvld sdgsfflysk ltvdksrwqq


 


421
gnvfscsvmh ealhnhytqk slslspgk






Each dual expression vector was transfected into 293T cells for transient expression using DMEM 10% fetal bovine serum. Forty-eight hours after transfection, cells were washed with and then replaced with serum free medium, IS GROTM (Irvine Scientific, Santa Ana, Calif.) containing 4 mM L-Glutamine. Supernatant was harvested daily and replaced with fresh media for 10 days. The culture supernatants were centrifuged, filtered (0.45 μm) and concentrated 10-100 fold. Antibodies were purified on ProSep vA resin (Millipore), dialyzed against PBS, concentrated and sterile filtered.
Example 14
Binding Characteristics of Humanized 2B8 Variants
The humanized antibodies produced in Example 13 were characterized by their ability to bind hHGF and the recombinant HGF proteins produced in Example 3.
The antibodies were analyzed by surface-plasmon resonance using a BIAcore T100 instrument to assess their ability to bind hHGF and the fusion proteins discussed in Example 3. Each antibody was immobilized on a carboxymethylated dextran CM5 sensor chip (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a standard coupling protocol according to manufacturer's instructions.
Analyses were performed at 25° C. using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No. R-1000-54), 2 mg/mL BSA (EMD, Catalog No. 2930) and 10 mg/mL CM-Dextran Sodium salt (Fluka, Catalog No. 86524) as running buffer. Supernatant containing different HGF fusion proteins or supernatant from cells transfected with empty vector were injected over each antibody at a flow rate of 30 μL/min for 3 minutes. The resulting binding was determined as resonance units (RU) over baseline 30 seconds after the end of injection. Binding was compared to human HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer. Non-specific binding was monitored by comparing binding to a control surface. The results are summarized in the Table 17.












TABLE 17






rhHGF 
rmHGF
MHM 
MHM 
MHM 



(R&D
(R&D 
chimera
chimera
chimera


Antibody
Systems)
Systems)
(495-585)
(507-585)
(499-556)







2B8
Yes
No
Yes
Yes
Yes


HE2B8-1
Yes
No
Yes
Yes
Yes


HE2B8-2
Yes
No
Yes
Yes
Yes


HE2B8-3
Yes
No
Yes
Yes
Yes


HE2B8-4
Yes
No
Yes
Yes
Yes


sh2B8-9
Yes
No
Yes
Yes
Yes


(G1m(3))







sh2B8-12
Yes
No
Yes
Yes
Yes


(G1m(3))









The results in Table 17 demonstrate that each of the humanized 2B8-based antibodies bind rhHGF and all three mouse-human-mouse chimeras.
Example 15
Binding Affinities of Humanized 2B8 Variants
The binding affinities and kinetics of interaction of the antibodies listed in Table 15 were measured by surface plasmon resonance.
Mouse anti-human immunoglobulins (Jackson Labs, Catalog No. 209-005) were immobilized on carboxymethylated dextran CM4 sensor chips (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a standard coupling protocol according to manufacturer's instructions. The analyses were performed at 25° C. using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No. BR-1000-54), and 2 mg/mL BSA (EMD, Catalog No. 2930).
The antibodies were captured in an individual flow cell at a flow rate of 10 μL/min. Injection time was variable for each antibody to yield approximately 20 RU of antibody captured for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 2 minutes at 60 μL/min. The dissociation phase was monitored for 15 or 90 minutes, depending on concentration. The surface then was regenerated with 10 mM Glycine-HCl, pH 2.2 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 μL/min before another cycle was initiated. HGF concentrations tested were 0.46 nM to 7.5 nM.
Kinetic parameters were determined using the kinetic function of the BIAevalutationTM software with reference subtraction. Kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized in Table 18.













TABLE 18







Antibody
Ka (l/Ms)
Kd (l/s) 
KD (pM)
SD






















2B8
1.4 × 106
1.0 × 10−5
7.3
—



HE2B8-1
2.2 × 106
1.4 × 10−5
7.1
5.2



HE2B8-2
1.8 × 106
9.6 × 10−6
5.2
2.7



HE2B8-3
2.0 × 106
4.1 × 10−6
2.0
1.1



HE2B8-4
1.7 × 106
1.1 × 10−5
6.5
1.3



sh2B8-9 
2.0 × 106
1.7 × 10−5
8.1
5.3



(G1m(17,1)







sh2B8-12 
1.9 × 106
2.3 × 10−5
12
0.4



(G1m(17,1)










These data show that the humanized antibodies have fast association rates (ka), very slow dissociation rates (kd), and very high affinities (KD). In particular, the antibodies have affinities ranging from 2.0-12 pM.
Example 16
Comparison of Binding Affinities at 25° C. and 37° C.
The binding affinities and kinetics of interaction of antibody HE2B8-4, sh2B8-9, sh2B8-12, and murine 2B8 were measured by surface plasmon resonance under different conditions.
Mouse anti-human immunoglobulins (Jackson Labs, Catalog No. 209-005) or rabbit anti-mouse immunoglobulins (BIAcore, Catalog No. BR-1005-14) were immobilized on carboxymethylated dextran CM4 sensor chips (BIAcore, Catalog No. BR-1006-68) by amine coupling (BIAcore, Catalog No. BR-1000-50) using a standard coupling protocol according to manufacturer's instructions. In the case of 25° C. measurements for sh2b8-9 and sh2B8-12, a CM5 sensor chip (BIAcore, Catalog No. BR-1006-68) was used. The analyses were performed at 25° C. and 37° C. using PBS (GIBCO, Catalog No. 14040-133) containing 0.05% surfactant P20 (BIAcore, Catalog No. BR-1000-54), and 2 mg/mL BSA (EMD, Catalog No. 2930) as running buffer.
The antibodies were captured in an individual flow cell at a flow rate of 10 L/min. Injection time was variable for each antibody to yield approximately 20 RU of antibody captured for each cycle. Buffer or HGF (R&D Systems, Catalog No. 294-HGN-025) diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 2 minutes at 60 UL/min. The dissociation phase was monitored for 15 or 90 minutes, depending on concentration. The surface of mouse anti-human immunoglobulins sensor chips was then regenerated with 10 mM Glycine-HCl, pH 2.2 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 μL/min before another cycle was initiated. The surface of rabbit anti-mouse immunoglobulins sensor chips was regenerated with 10 mM Glycine-HCl, pH 1.7 (BIAcore, Catalog No. BR-1003-54) injected for 3 minutes at a flow rate of 60 μL/min before another cycle was initiated. HGF concentrations tested were 0.46 nM to 7.5 nM.
Kinetic parameters were determined using the kinetic function of the BIAevaluation software with reference subtraction. Kinetic parameters for each antibody, ka (association rate constant), kd (dissociation rate constant) and KD (equilibrium dissociation constant) are summarized below in Table 19.











TABLE 19





Antibody
Temp. (° C.)
ka (l/Ms)
kd (l/s)
KD (pM)



















2B8
25
1.6 × 106
2.1 × 10−5
13.5


2B8
37
2.8 × 106
1.3 × 10−5
4.5


HE2B8-4
25
2.0 × 106
1.2 × 10−5
5.6


HE2B8-4
37
3.1 × 106
1.0 × 10−5
3.3


sh2B8-9 
25
2.0 × 106
1.7 × 10−5
8.1


(G1m(17,1))






sh2B8-9 
37
2.5 × 106
1.4 × 10−5
5.8


(G1m(3))






sh2B8-12 
25
1.9 × 106
2.3 × 10−5
12.0


(G1m(17,1))






sh2B8-12 
37
2.4 × 106
1.1 × 10−5
4.8


(G1m(3))









As expected, the association rate constants increased with an increase in the temperature. Surprisingly, the dissociation constants did not change significantly with a corresponding increase in temperature. Consequently, the overall equilibrium dissociation constants (KD) were approximately 1.4 to 3 times smaller (higher affinity) at physiological temperature (37° C.).
Example 17
Neutralization Activity of Humanized 2B8 Variants
The antibodies described in Example 14 were characterized for their ability to (a) inhibit the binding of hHGF to c-Met, and (b) inhibit HGF stimulated BrdU incorporation in 4 MBr-5 cells.
HGF-Met Binding Inhibition Assay (Neutralization Assay) was performed as described in as follows. The antibodies were tested by ELISA for their ability to inhibit hHGF binding to c-Met. Specifically, Wallac 96-well DELFIA assay plates (Wallac Inc., Catalog No. AAAND-0001) were coated with 100 μL of 6.25 μg/mL HGF (R&D Systems, Catalog No. 294-HGN-025) in carbonate coating buffer (15 mM Na2CO3 and 34 mM NaHCO3, pH 9.0) for 16 hours at 4° C. The plates then were blocked with 200 μL of 5% non-fat dry milk in PBS for 1 hour at room temperature. The antibodies were prepared in a separate plate by adding increasing concentrations of the antibodies under investigation (0.033-250 nM, 2-fold-serial dilution) to 2 nM biotinylated c-Met in 5% non-fat dry milk in PBS. c-Met (R&D Systems, Catalog No. 358-MT/CF) is biotinylated according to manufacturer's instruction at 10:1 biotin to c-Met ratio (Pierce, Catalog No. 21335). 100 μL of sample per well was transferred to the assay plate and incubated for 2 hours at room temperature. The resulting plates were washed three times with PBS-0.1% Tween 20, and incubated for 1 hour at room temperature with Eu-labeled Streptavidin (Wallac, Catalog No. 1244-360) diluted 1:1000 in DELFIA assay buffer (Wallac, Catalog No. 4002-0010). The resulting plates were washed 3 times with DELFIA wash solution (Wallac, Catalog No. 4010-0010) and incubated with 100 μL/well DELFIA enhancement solution (Wallac #4001-0010) for 15 minutes at room temperature with agitation. The plates were read on Victor3V instrument (Perkin Elmer) using the Europium method. The IC50 values were calculated using Prism.
The IC50 values obtained are shown in Table 20.











TABLE 20







Antibody
IC50 (nM)
SD









2B8
9.2
1.2



HE2B8-1
6.0
1.2



HE2B8-2
5.7
1.1



HE2B8-3
5.9
1.1



HE2B8-4
6.5
1.2



sh2B8-9 (G1m(3))
4.2
—



sh2B8-12 (G1m(3)
6.8
—










These results from Table 20 demonstrate that the humanized antibodies tested efficiently neutralize HGF binding to c-Met.
The antibodies in Table 17 were also tested in the cell proliferation assay described in Example 7(b). The results are summarized below in Table 21.











TABLE 21







Antibody
IC50 (nM)
SD









2B8
0.86
0.35



HE2B8-1
0.47
0.15



HE2B8-2
0.66
0.13



HE2B8-3
0.55
0.28



HE2B8-4
0.58
0.26



sh2B8-9 (G1m(3))
0.52
0.11



sh2B8-12 (G1m(3))
0.81
0.22










The results from Table 21 demonstrate that all the humanized antibodies tested inhibit HGF-induced proliferation of 4 MBr-5 cells.
Example 18
Anti-Scatter Activity of Humanized 2B8 Variants
The antibodies in Table 17 were tested in the anti-scatter assay described in Example 8. The results are summarized below in Table 22.







TABLE 22







Inhibition of HGF-induced MDCK Cell Scattering











Antibody
Trial 1
Trial 2







2B8
++
++



HE2B8-1
++
++



HE2B8-2
++
++



HE2B8-3
++
++



HE2B8-4
++
++



sh2B8-9 (G1m(3))
++
++



sh2B8-12 (G1m(3))
++
++







− No Inhibition



+++ Very strong, nearly complete inhibition



++ Strong inhibition



+ Detectable inhibition






The results in Table 22 demonstrate that all the humanized antibodies tested inhibited HGF-induced scattering to the same extent as the murine monoclonal antibody 2B8.
Example 19
Inhibition of HGF-Stimulated c-Met Phosphorylation
The antibodies in Table 17 were tested in the c-Met phosphorylation assay described in Example 9. The results are summarized below in Table 23.











TABLE 23








Average of
Standard



Antibody
Two Trials
Deviation









2B8
0.91
0.02



he2B8-1
0.80
0.04



he2B8-2
0.88
0.15



he2B8-3
0.79
0.05



he2B8-4
0.75
0.14



sh2B8-9 (G1m(3))
0.93
0.03



sh2B8-12 (G1m(3))
0.81
0.07










The results in Table 23 demonstrate that all the humanized antibodies tested are potent inhibitors of HGF-induced c-Met phosphorylation in PC-3 cells.
Example 20
Tumor Inhibition in U87MG Xenograft Model
The ability of the humanized monoclonal antibodies of the invention to inhibit tumor growth was tested in an U87MG xenograft model. U87MG cells (ATCC) were expanded in culture at 37° C. in an atmosphere containing 5% CO2 and 95% air, using a medium comprising Dulbecco's Modified Eagle medium (DMEM) with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin. The cells were subcultured and maintained by detaching the cells from the wall of the culture dish using trypsin-EDTA.
Near-confluent cells were collected by trypsinization and then 5×106 cells in 50% Matrigel (BD Biosciences; catalog no. 356237) were injected subcutaneously into the upper dorsal area between the shoulder blades of 7-week old female ICR SCID mice (Taconic Labs). The long (L) and short (W) diameters (mm) of tumors were measured with a caliper. Tumor volume (vol.) was calculated as: volume (mm3)=L×W2/2. When the tumors grew to approximately 200 mm3, the tumor-bearing mice were randomized into 5 groups of 10 mice each. One group received PBS and one group received human IgG control. Each of the other 4 groups received one of the humanized antibodies (HE2B8-1, HE2B8-2, HE2B8-3, and HE2B8-4). All the antibodies were dosed at 0.25 mg/kg body weight, twice per week, by intra-peritoneal injections of 5 doses. Tumor volumes and mouse body weights were recorded twice per week. Tumor growth inhibition was analyzed using Student's t-test.
The humanized antibodies tested were active in vivo. There was 57% tumor growth inhibition for HE2B8-1 with a p value of 0.02, 61% tumor growth inhibition for HE2B8-2 with a p value of 0.02, 85% tumor growth inhibition for HE2B8-3, with a p value of 0.0004, and 74% tumor growth inhibition for HE2B8-4 with a p value of 0.001. No significant body weight loss was observed.
A subsequent study was performed as described above in female NCR nude mice (Taconic Labs) bearing subcutaneous U87MG tumors inoculated in the flank. Each group (10 mice each) received one of the following treatments at 0.5 mg/kg: PBS vehicle control, huIgG control, HE2B8-4, or sh2B8-9. Treatment was given intra-peritoneal twice weekly for a minimum of 5 weeks. Each treatment group demonstrated similar tumor regression with tumor growth inhibition of 113% for sh2B8-9 and 115% for HE2B8-4, and a minimum tumor growth delay of 30 days. Both treatments were well-tolerated with no significant body weight loss.
Incorporation by Reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

## Claims

1. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, wherein
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 15,
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 204, and
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 17; and

an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, wherein
(i) CDRL1 comprises the amino acid sequence of SEQ ID NO: 18,
(ii) CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and
(iii) CDRL3 comprises the amino acid sequence of SEQ ID NO: 20;

wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together bind human hepatocyte growth factor (HGF).

2. The isolated nucleic acid of claim 1, wherein the complementarity determining sequences (CDRs) are interposed between human or humanized immunoglobulin framework regions.

3. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 183.

4. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 193.

5. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 187.

6. The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 197.

7. An expression vector comprising the nucleic acid of claim 1.

8. A host cell comprising the expression vector of claim 7.

9. A host cell comprising a first expression vector comprising a nucleic acid sequence encoding an immunoglobulin heavy chain variable region comprising the structure CDRH1-CDRH2-CDRH3, wherein
(i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 15,
(ii) CDRH2 comprises the amino acid sequence of SEQ ID NO: 204, and
(iii) CDRH3 comprises the amino acid sequence of SEQ ID NO: 17; and

a second expression vector comprising a nucleic acid sequence encoding an immunoglobulin light chain variable region comprising the structure CDRL1-CDRL2-CDRL3, wherein
(i) CDRL1 comprises the amino acid sequence of SEQ ID NO: 18,
(ii) CDRL2 comprises the amino acid sequence of SEQ ID NO: 19, and
(iii) CDRL3 comprises the amino acid sequence of SEQ ID NO: 20;

wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together bind human hepatocyte growth factor (HGF).

10. A method of producing a monoclonal antibody that binds human hepatocyte growth factor (HGF) or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 8 under conditions that permit the expression of a polypeptide comprising the immunoglobulin heavy chain variable region and a polypeptide comprising the immunoglobulin light chain variable region, thereby producing the antibody or antigen binding fragment of the antibody; and
(b) purifying the antibody or antigen binding fragment of the antibody.

11. A method of producing a monoclonal antibody that binds human hepatocyte growth factor (HGF) or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 9 under conditions that permit the expression of a polypeptide comprising the immunoglobulin heavy chain variable region and a polypeptide comprising the immunoglobulin light chain variable region, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.

12. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 183, and encoding an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 193, wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together bind human hepatocyte growth factor (HGF).

13. An expression vector comprising the nucleic acid of claim 12.

14. A host cell comprising the expression vector of claim 13.

15. A host cell comprising:
a first expression vector comprising a nucleic acid sequence encoding an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 183; and
a second expression vector comprising a nucleic acid sequence encoding an immunoglobulin light chain variable region comprising amino acid sequence of SEQ ID NO: 193;
wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together bind human hepatocyte growth factor (HGF).

16. A method of producing a monoclonal antibody that binds human hepatocyte growth factor (HGF) or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 14 under conditions that permit the expression of a polypeptide comprising the immunoglobulin heavy chain variable region and a polypeptide comprising the immunoglobulin light chain variable region, thereby producing the antibody or antigen binding fragment of the antibody; and
(b) purifying the antibody or antigen binding fragment of the antibody.

17. A method of producing a monoclonal antibody that binds human hepatocyte growth factor (HGF) or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 15 under conditions that permit the expression of a polypeptide comprising the immunoglobulin heavy chain variable region and a polypeptide comprising the immunoglobulin light chain variable region, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.

18. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 187, and encoding an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 197, wherein the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region together bind human hepatocyte growth factor (HGF).

19. An expression vector comprising the nucleic acid of claim 18.

20. A host cell comprising the expression vector of claim 19.

21. A host cell comprising:
a first expression vector comprising a nucleic acid sequence encoding an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 187; and
a second expression vector comprising a nucleic acid sequence encoding an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 197;
wherein the immunoglobulin heavy chain and the immunoglobulin light chain together bind human hepatocyte growth factor (HGF).

22. A method of producing a monoclonal antibody that binds human hepatocyte growth factor (HGF) or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 20 under conditions that permit the expression of a polypeptide comprising the immunoglobulin heavy chain and a polypeptide comprising the immunoglobulin light chain, thereby producing the antibody or antigen binding fragment of the antibody; and
(b) purifying the antibody or antigen binding fragment of the antibody.

23. A method of producing a monoclonal antibody that binds human hepatocyte growth factor (HGF) or an antigen binding fragment of the antibody, the method comprising:
(a) growing the host cell of claim 21 under conditions that permit the expression of a polypeptide comprising the immunoglobulin heavy chain and a polypeptide comprising the immunoglobulin light chain, thereby producing the antibody or the antigen-binding fragment of the antibody; and
(b) purifying the antibody or the antigen-binding fragment of the antibody.