ANTI-FGFR2 ANTIBODIES

Abstract

Monoclonal antibodies that bind and inhibit biological activities of human FGFR2 are disclosed. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation or overexpression of FGFR2.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 61/333,590, filed May 11, 2010; the content of which is incorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] The field of the invention is molecular biology, immunology and oncology. More particularly, the field is antibodies that bind human FGFR2.

BACKGROUND

[0003] Fibroblast Growth Factor Receptor 2 (FGFR2), also known as BEK, BFR-1, CD332, CEK3, CFD1, ECT1, F1198662, JWS, KGFR (also known as FGFR2(IIIb)), K-SAM, TK14, and TK25, is one of four highly conserved receptor tyrosine kinases (FGFR1, FGFR2, FGFR3 and FGFR4) that mediate fibroblast growth factor (FGF) signaling by binding FGFs. The FGF receptors are characterized by two or three extracellular immunoglobulin-like domains (IgD1, IgD2 and IgD3), a single-pass transmembrane domain, and a cytoplasmic tyrosine kinase domain. FGF ligand binding induces FGF receptor dimerization and tyrosine autophosphorylation, resulting in cell proliferation, differentiation and migration (Turner et al. (2010) NATURE REVIEWS CANCER 10:116-129; Beenken et al. (2009) NATURE REVIEWS DRUG DISCOVERY 8:235-254; Gomez-Roman et al. (2005) CLIN. CANCER RES. 11:459-65; Chang et al. (2005) BLOOD 106:353-6; Eswarakumar et al. (2005) CYTOKINE GROWTH FACTOR REV. 16:139-49).

[0004] Alternative splicing in the IgD3 domain yields either the Mb or Mc isoform of FGFR1, FGFR2 and FGFR3. The FGFR4 gene is expressed only as the Mc isoform. The different isoforms of FGF receptors exhibit tissue-specific expression, and they respond to a different spectrum of 18 mammalian FGFs (Beenken et al., supra). Binding of FGFs to FGFRs in the presence of heparan sulfate proteoglycans induces autophosphorylation of FGFRs at specific intracellular tyrosine residues. This causes phosphorylation of adaptor molecules, such as FGFR substrate 2 α (FRS2α), which recruits other proteins to activate various signaling cascades, including the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)/Akt pathway (Beenken et al., supra; Eswarakumar et al., supra; Turner et al., supra).

[0005] It has been suggested that the dysregulated FGF signaling can directly drive the proliferation of cancer cells, promote the survival of cancer stem cells, and support tumor angiogenesis (Turner et al., supra). FGFR2 signaling appears to play a role in cancer. Missense mutations in the FGFR2 gene occur in various cancers, including endometrial cancer (Pollock et al., 2007, ONCOGENE 26:7158-7162; Dutt et al., 2008, PROC. NATL. ACAD. SCI. USA 105:8713-8717), ovarian cancer, breast cancer, lung cancer (Greenman et al., 2007, Nature 446:153-158; Ding et al., 2008, NATURE 455:1069-1075; Davies et al., 2005, CANCER RES. 65:7591-7595) and gastric cancer (Tang et al., 2001, CANCER RES. 61:3541-3543). Some of these activating mutations also have been reported in patients with skeletal disorders (Dutt et al., supra). Two independent genome-wide association studies have linked specific single nucleotide polymorphisms (SNPs) in the FGFR2 gene to increased susceptibility to breast cancer (Easton et al., 2007, NATURE 447:1087-1093; Hunter et al., 2007, NAT. GENET. 39:870-874). These cancer-associated SNPs appear to elevate FGFR2 gene expression (Meyer et al., 2008, PLOS BIOL. 6:e108). The FGFR2 gene, located at human chromosome 10q26, is amplified in a subset of breast cancers (Adnane et al., 1991, ONCOGENE 6:659-663; Turner et al., 2010, ONCOGENE 29:2013-2023) and gastric cancer (Hara et al., 1998, LAB. INVEST. 78:1143-1153; Mor et al., 1993, CANCER GENET. CYTOGENET. 65:111-114).

[0006] Naturally occurring antibodies are multimeric proteins that contain four polypeptide chains (FIG. 1). Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (V_L in FIG. 1) and one constant region (C_L in FIG. 1). The heavy chain consists of one variable region (V_H 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. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies.

[0007] Each variable region contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The three CDRs, referred to as CDR₁, CDR₂, and CDR₃, contribute to the antibody binding specificity.

[0008] Inhibitory antibodies specific against human FGFR2 have been difficult to generate because of the high homology between mouse and human FGFR2. In particular, the ligand binding domain of the mouse and human FGFR2 shares approximately 98% sequence identity (Wei et al., 2006, HYBRIDOMA 25:115-124). Thus, there is a need for improved FGFR2 antibodies that can be used as therapeutic agents.

SUMMARY OF THE INVENTION

[0009] The invention is based on the discovery of a family of antibodies that specifically bind human FGFR2. The antibodies contain FGFR2 binding sites based on the CDRs of an antibody that specifically binds FGFR2. When used as therapeutic agents, the antibodies are engineered, e.g., humanized, to reduce or eliminate an immune response when administered to a human patient.

[0010] The antibodies of the invention prevent or inhibit the activation of (i.e., neutralize) human FGFR2. The antibodies of the invention can be used to inhibit the proliferation of tumor cells in vitro or in vivo. When administered to a human cancer patient (or an animal model), the antibodies inhibit or reduce tumor growth in the human patient (or animal model).

[0011] These and other aspects and advantages of the invention are illustrated by the following figures, detailed description and claims. As used herein, "including" means without limitation, and examples cited are non-limiting.

DESCRIPTION OF THE DRAWINGS

[0012] The invention can be more completely understood with reference to the following drawings.

[0013] FIG. 1 (prior art) is a schematic representation of a typical antibody.

[0014] FIG. 2 is a graph summarizing results from an experiment to measure stimulation of proliferation of FGFR2-IIIb-expressing FDCP-1 cells by FGF2 ( ), FGF7 (∇), FGF9 (quadrature) and FGF10 (x).

[0015] FIG. 3 is a graph summarizing results from an experiment to measure stimulation of proliferation of FGFR2-IIIc-expressing FDCP-1 cells by FGF2 ( ), FGF7 (∇), FGF9 (quadrature) and FGF10 (x).

[0016] FIG. 4 is a graph summarizing results from an experiment to measure inhibition of proliferation of FDCP-1 cells expressing wild type FGFR2-IIIb (quadrature), wild type FGFR2-IIIc (∇), or truncated FGFR2-IIIb (*), by treatment with antibody 4B9.

[0017] FIG. 5 is a graph summarizing results from an experiment to measure inhibition of proliferation of FDCP-1 cells expressing wild type FGFR2-IIIb (quadrature), FGFR2-IIIb S252W (.box-solid.), or FGFR2-IIIb N550K (.tangle-solidup.), by treatment with antibody 4B9.

[0018] FIG. 6 is a graph summarizing results from an experiment to measure inhibition of growth of SNU-16 xenograft tumors by treatment with antibody 4B9 at 2 mg/kg (also referred to herein as "mpk") (◯), 5 mpk (Δ), 10 mpk (x) or 20 mpk (*), with mIgG at 20 mpk (.diamond-solid.) serving as a negative control.

[0019] FIG. 7 is a graph summarizing results from an experiment to measure the effect of antibody 4B9 (◯) on the in vivo growth of FGFR2-amplified breast cancer cell line MFM-223 (murine IgG (.diamond-solid.)).

[0020] FIG. 8 is a schematic diagram showing the amino acid sequences of the complete murine immunoglobulin heavy chain variable region of 4B9 (SEQ ID NO: 2) and the complete humanized heavy chain variable regions denoted as Hu4B9-65 (SEQ ID NO: 35) and Hu4B9-82, -83 (SEQ ID NO: 37). The amino acid sequences for each heavy chain variable region are aligned against one another, and Complementary Determining Sequences (CDR) (Kabat definition), CDR₁, CDR₂, and CDR₃, are identified in boxes. The unboxed sequences represent framework (FR) sequences.

[0021] FIG. 9 is a schematic diagram showing the CDR₁, CDR₂, and CDR₃ sequences (Kabat definition) for each of the variable region sequences shown in FIG. 8.

[0022] FIG. 10 is a schematic diagram showing the amino acid sequences of the complete murine immunoglobulin light chain variable region of 4B9 (SEQ ID NO: 4) and the complete humanized light chain variable regions denoted as Hu4B9-65 (SEQ ID NO: 40), Hu4B9-82 (SEQ ID NO: 44), and Hu4B9-83 (SEQ ID NO: 46). The amino acid sequences for each light chain variable region are aligned against one another, and CDR₁, CDR₂, and CDR₃ sequences (Kabat definition) are identified in boxes. The unboxed sequences represent framework (FR) sequences.

[0023] FIG. 11 is a schematic diagram showing the CDR₁, CDR₂, and CDR₃ sequences (Kabat definition) for each of the variable region sequences shown in FIG. 10.

[0024] FIG. 12 is a graph summarizing results from an experiment to measure inhibition of proliferation of FDCP-1 cells expressing wild type FGFR2-IIIb by treatment with antibody 4B9 (quadrature), Hu4B9-65 (.tangle-solidup.), Hu4B9-82 () and Hu4B9-83 (.diamond-solid.).

DETAILED DESCRIPTION

[0025] The FGFR2 antibodies of the invention are based on the antigen binding sites of a monoclonal antibody selected on the basis of neutralizing the biological activity of human FGFR2 polypeptides. The antibodies contain immunoglobulin variable region CDR sequences that define a binding site for human FGFR2.

[0026] Because of the neutralizing activity of these antibodies, they are useful for inhibiting the growth and/or proliferation of certain cancer cells and tumors. The antibodies can be engineered to minimize or eliminate an immune response when administered to a human patient. Various features and aspects of the invention are discussed in more detail below.

[0027] As used herein, unless otherwise indicated, the term "antibody" means an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody or antigen-binding fragment that has been modified, engineered or chemically conjugated. Examples of antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')₂, Fv, single chain antibodies (e.g., scFv) and diabodies. An antibody conjugated to a toxin moiety is an example of a chemically conjugated antibody.

Antibodies that Bind Human FGFR2

[0028] Antibodies of the invention comprise: (a) an immunoglobulin heavy chain variable region comprising the structure CDR_H1-CDR_H2-CDR_H3 and (b) an immunoglobulin light chain variable region comprising the structure CDR_L1-CDR_L2-CDR_L3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR2.

[0029] As disclosed herein, an antibody may comprise: (a) an immunoglobulin heavy chain variable region comprising the structure CDR_H1-CDR_H2-CDR_H3 and (b) immunoglobulin light chain variable region, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR₂. A CDR_H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 (4B9; Hu4B9-65; Hu4B9-82, -83), SEQ ID NO: 7 (4B9; Hu4B9-65), and SEQ ID NO: 47 (Hu4B9-82, -83); a CDR_H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (4B9; Hu4B9-65) and SEQ ID NO: 38 (Hu4B9-82, -83); and a CDR_H3 comprises an amino acid sequence selected from the group consisting of amino acid sequence FDY (4B9; Hu4B9-65; Hu4B9-82, -83) and SEQ ID NO: 11 (4B9; Hu4B9-65; Hu4B9-82, -83). Throughout the specification a particular SEQ ID NO. is followed in parentheses by the antibody that was the origin of that sequence. For example, "SEQ ID NO: 47 (Hu4B9-82, -83)" means that SEQ ID NO: 47 comes from the humanized antibody 4B9 denoted Hu4B9-82, -83.

[0030] In some embodiments, the heavy chain variable region comprises a CDR_H1 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7 (4B9; Hu4B9-65; Hu4B9-82, -83), a CDR_H2 comprising the amino acid sequence of SEQ ID NO: 6 (4B9; Hu4B9-65), and a CDR_H3 comprising the amino acid sequence of SEQ ID NO: 11 (4B9; Hu4B9-65; Hu4B9-82, -83).

[0031] In some embodiments, the heavy chain variable region comprises a CDR_H1 comprising the amino acid sequence of SEQ ID NO: 5 (4B9; Hu4B9-65; Hu4B9-82, -83) or SEQ ID NO: 47 (Hu4B9-82, -83), a CDR_H2 comprising the amino acid sequence of SEQ ID NO: 38 (Hu4B9-82, -83), and a CDR_H3 comprising the amino acid sequence of SEQ ID NO: 11 (4B9; Hu4B9-65; Hu4B9-82, -83).

[0032] Preferably, the CDR_H1, CDR_H2, and CDR_H3 sequences are interposed between human or humanized immunoglobulin FRs. The antibody can be an intact antibody or an antigen-binding antibody fragment.

[0033] In other embodiments, the antibody comprises (a) an immunoglobulin light chain variable region comprising the structure CDR_L1-CDR_L2-CDR_L3, and (b) an immunoglobulin heavy chain variable region, wherein the IgG light chain variable region and the IgG heavy chain variable region together define a single binding site for binding human FGFR₂. A CDR_L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12 (4B9) and SEQ ID NO: 41 (Hu4B9-65; Hu4B9-82; Hu4B9-83); a CDR_L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13 (4B9) and SEQ ID NO: 42 (Hu4B9-65; Hu4B9-82; Hu4B9-83); and a CDR_L3 comprises an amino acid sequence of SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).

[0034] In some embodiments, the light chain variable region comprises a CDR_L1 comprising the amino acid sequence of SEQ ID NO: 12 (4B9); a CDR_L2 comprising the amino acid sequence of SEQ ID NO: 13 (4B9); and a CDR_L3 comprising the amino acid sequence of SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).

[0035] In some embodiments, the light chain variable region comprises a CDR_L1 comprising the amino acid sequence of SEQ ID NO: 41 (Hu4B9-65; Hu4B9-82; Hu4B9-83); a CDR_L2 comprising the amino acid sequence of SEQ ID NO: 42 (Hu4B9-65; Hu4B9-82; Hu4B9-83); and a CDR_L3 comprising the amino acid sequence of SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).

[0036] Preferably, the CDR_L1, CDR_L2, and CDR_L3 sequences are interposed between human or humanized immunoglobulin FRs. The antibody can be an intact antibody or an antigen-binding antibody fragment.

[0037] In some embodiments, the antibody comprises: (a) an immunoglobulin heavy chain variable region comprising the structure CDR_H1-CDR_H2-CDR_H3 and (b) an immunoglobulin light chain variable region comprising the structure CDR_L1-CDR_L2-CDR_L3, wherein the heavy chain variable region and the light chain variable region together define a single binding site for binding human FGFR₂. The CDR_H1 is an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or SEQ ID NO: 7 (4B9; Hu4B9-65; Hu4B9-82, -83); the CDR_H2 is an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (4B9; Hu4B9-65) and SEQ ID NO: 38 (Hu4B9-82, -83); and the CDR_H3 is an amino acid sequence selected from the group consisting of amino acid sequence FDY and SEQ ID NO: 11 (4B9; Hu4B9-65; Hu4B9-82, -83). The CDR_L1 is an amino acid sequence selected from the group consisting of SEQ ID NO: 12 (4B9) and SEQ ID NO: 41 (Hu4B9-65; Hu4B9-82; Hu4B9-83); the CDR_L2 is an amino acid sequence selected from the group consisting of SEQ ID NO: 13 (4B9) and SEQ ID NO: 42 (Hu4B9-65; Hu4B9-82; Hu4B9-83); and the CDR_L3 comprises the amino acid sequence of SEQ ID NO: 14 (4B9; Hu4B9-65; Hu4B9-82; Hu4B9-83).

[0038] In another embodiment, the antibody comprises an immunoglobulin heavy chain variable region selected from the group consisting of SEQ ID NO: 2 (4B9), SEQ ID NO: 35 (Hu4B9-65), and SEQ ID NO: 37 (Hu4B9-82, -83), and an immunoglobulin light chain variable region selected from the group consisting of SEQ ID NO: 4 (4B9), SEQ ID NO: 40 (Hu4B9-65), SEQ ID NO: 44 (Hu4B9-82) and SEQ ID NO: 46 (Hu4B9-83).

[0039] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 (4B9), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 (4B9).

[0040] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35 (Hu4B9-65), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 40 (Hu4B9-65).

[0041] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 (Hu4B9-82, -83), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 44 (Hu4B9-82).

[0042] In some embodiments, the antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 (Hu4B9-82, -83), and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 46 (Hu4B9-83).

[0043] In other embodiments, the antibody comprises (i) an immunoglobulin heavy chain selected from the group consisting of SEQ ID NO: 21 (4B9), SEQ ID NO: 54 (Hu4B9-65), and SEQ ID NO: 56 (Hu4B9-82, -83), and (ii) an immunoglobulin light chain selected from the group consisting of SEQ ID NO: 23 (4B9), SEQ ID NO: 58 (Hu4B9-65), SEQ ID NO: 60 (Hu4B9-82) and SEQ ID NO: 62 (Hu4B9-83).

[0044] In certain embodiments, the antibody comprises (i) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 21 (4B9), and (ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 23 (4B9).

[0045] In certain embodiments, the antibody comprises (i) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 54 (Hu4B9-65), and (ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 58 (Hu4B9-65).

[0046] In certain embodiments, the antibody comprises (i) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 56 (Hu4B9-82, -83), and (ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 60 (Hu4B9-82).

[0047] In certain embodiments, the antibody comprises (i) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 56 (Hu4B9-82, -83), and (ii) an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 62 (Hu4B9-83).

[0048] In other embodiments, an isolated antibody that binds human FGFR2 comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the framework region sequence of SEQ ID NO: 2 (4B9), SEQ ID NO: 35 (Hu4B9-65), and SEQ ID NO: 37 (Hu4B9-82, -83).

[0049] In other embodiments, an isolated antibody that binds human FGFR2 comprises an immunoglobulin light chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire variable region or the framework region sequence of SEQ ID NO: 4 (4B9), SEQ ID NO: 40 (Hu4B9-65), SEQ ID NO: 44 (Hu4B9-82) and SEQ ID NO: 46 (Hu4B9-83).

[0050] Homology or identity may be determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87, 2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25, 3389-3402, incorporated by reference) are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases see Altschul et al., (1994) NATURE GENETICS 6, 119-129 which is fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89, 10915-10919, fully incorporated by reference). Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink^th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings may be Q=9; R=2; wink=1; and gapw=32. Searches may also be conducted using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameter (e.g.: -G, Cost to open gap [Integer]: default=5 for nucleotides/11 for proteins; -E, Cost to extend gap [Integer]: default=2 for nucleotides/1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default=-3; -r, reward for nucleotide match [Integer]: default=1; -e, expect value [Real]: default=10; -W, wordsize [Integer]: default=11 for nucleotides/28 for megablast/3 for proteins; -y, Dropoff (X) for blast extensions in bits: default=20 for blastn/7 for others; -X, X dropoff value for gapped alignment (in bits): default=15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty=10 and Gape Extenstion Penalty=0.1). A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.

[0051] In each of the foregoing embodiments, it is contemplated herein that immunoglobulin heavy chain variable region sequences and/or light chain variable region sequences that together bind human FGFR2 may contain amino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the heavy and/or light chain variable regions.

[0052] In some embodiments, an isolated antibody binds human FGFR2 with a K_D of 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 950 μM, 900 μM, 850 μM, 800 μM, 750 μM, 700 μM, 650 μM, 600 μM, 550 μM, 500 μM, 450 μM, 400 μM, 350 μM, 300 μM, 250 μM, 200 μM, 150 μM, 100 μM, 50 μM or lower. Unless otherwise specified, K_D values are determined by surface plasmon resonance methods under the conditions described, for example, in Examples 5 and 9.

Production of Antibodies

[0053] Methods for producing antibodies of the invention are known in the art. For example, DNA molecules encoding light chain variable regions and heavy chain variable regions can be chemically synthesized using the sequence information provided herein. 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 antibody. 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 polymerase chain reaction (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.

[0054] Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are 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 IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light or heavy chain variable regions.

[0055] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art.

[0056] If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, IgG enhancers, and various introns. This expression vector optionally contains 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 introduced into eukaryotic host cells using convention techniques. The host cells express V_L or V_H fragments, V_L-V_H heterodimers, V_H-V_L or V_L-V_H single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a moiety having another function (e.g., cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing an entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In other embodiments, a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In still other embodiments, a host cell is co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising an entire, or part of, a heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide comprising an entire, or part of, a light chain or light chain variable region).

[0057] A polypeptide comprising an immunoglobulin heavy chain variable region or a light chain variable region can be produced by growing a host cell transfected with an expression vector encoding such variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified using techniques well known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) and histidine tags.

[0058] A monoclonal antibody that binds human FGFR2, or an antigen-binding fragment of the antibody, can be produced by growing a host cell transfected with: (a) an expression vector that encodes a complete or partial immunoglobulin heavy chain, and a separate expression vector that encodes a complete or partial light chain; or (b) a single expression vector that encodes both chains (e.g., complete or partial heavy and light chains) under conditions that permit expression of both chains. The intact antibody (or the antigen-binding fragment of the antibody) can be harvested and purified using techniques well known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags. It is within ordinary skill in the art to express the heavy chain and the light chain from a single expression vector or from two separate expression vectors.

Modifications to the Antibodies

[0059] Methods for reducing or eliminating the antigenicity of antibodies and antibody fragments are known in the art. When the antibodies are to be administered to a human, the antibodies preferably are "humanized" to reduce or eliminate antigenicity in humans. Preferably, the humanized antibodies have the same, or substantially the same, affinity for the antigen as the non-humanized mouse antibody from which it was derived.

[0060] In one humanization approach, chimeric proteins are created in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, e.g., Morrison et al., 1984, PROC. NAT. ACAD. SCI. 81:6851-6855, Neuberger et al., 1984, NATURE 312:604-608; U.S. Pat. Nos. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).

[0061] In an approach known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted into frameworks from another species. For example, murine CDRs can be grafted into human FRs. In some embodiments of the invention, the CDRs of the light and heavy chain variable regions of an anti-FGFR2 antibody are grafted into human FRs or consensus human FRs. 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 in U.S. Pat. Nos. 7,022,500 (Queen); 6,982,321 (Winter); 6,180,370 (Queen); 6,054,297 (Carter); 5,693,762 (Queen); 5,859,205 (Adair); 5,693,761 (Queen); 5,565,332 (Hoogenboom); 5,585,089 (Queen); 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.

[0062] In an approach called "SUPERHUMANIZATION®," human CDR sequences are chosen from human germline genes, based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. See, e.g., U.S. Pat. No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL 169:1119-1125.

[0063] Other methods to reduce immunogenicity include "reshaping," "hyperchimerization," and "veneering/resurfacing." 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, e.g., in U.S. Pat. No. 5,639,641 (Pedersen).

[0064] Another approach for converting a mouse antibody into a form suitable for medical use in humans is known as ACTIVMAB® 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 IgG heavy and light chains are said to be produced. See, e.g., U.S. Pat. Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer).

[0065] Another 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.

[0066] Another approach for modifying a mouse antibody into a form suitable for medical use in humans is HUMAN ENGINEERING® technology, which is practiced commercially by XOMA (US) LLC. See, e.g., PCT Publication No. WO 93/11794 and U.S. Pat. Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619.

[0067] Any suitable approach, including any of the above approaches, can be used to reduce or eliminate human immunogenicity of an antibody disclosed herein.

[0068] If the antibody is for use as a therapeutic agent, it can be conjugated to an effector moiety such as a small molecule toxin or a radionuclide using standard in vitro conjugation chemistries. If the effector moiety is a polypeptide, the antibody can be chemically conjugated to the effector or joined to the effector as a fusion protein. Construction of fusion proteins is within ordinary skill in the art.

Use of Antibodies

[0069] Antibodies disclosed herein can be used to treat various forms of cancer, e.g., breast, ovarian, prostate, cervical, colorectal, lung, pancreatic, gastric, and head and neck cancers. The cancer cells are exposed to a therapeutically effective amount of the antibody so as to inhibit or reduce proliferation of the cancer cells. In some embodiments, the antibodies inhibit cancer cell proliferation by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%.

[0070] In some embodiments, the disclosed antibodies can be used in a method to inhibit tumor growth in a human patient. The method comprises administering to the patient a therapeutically effective amount of the antibody. Cancers associated with FGFR2 overexpression and/or activation include breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer, some forms of brain cancer, melanomas, and gastrointestinal cancers (e.g., colorectal, pancreatic, gastric, head and neck).

[0071] As used herein, "treating" a disease means: (a) reducing symptoms of the disease; (b) inhibiting progression of the disease; (c) causing regression of the disease; or (d) curing the disease.

[0072] Generally, a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 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 of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. 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 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 a monoclonal antibody is lyophilized and reconstituted in buffered saline at the time of administration.

[0073] For therapeutic use, an antibody preferably is combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means buffers, carriers, and excipients 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 include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0074] Pharmaceutical compositions containing antibodies of the invention can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration. A preferred route of administration for monoclonal antibodies is IV infusion. Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include 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 EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0075] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. 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.

[0076] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

EXAMPLES

[0077] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Example 1

Cell Lines and Reagents

[0078] KATO III, HEC-1-A, AN3 CA, SNU-16, and human lung cancer cell lines were acquired from the American Type Culture Collection (Rockville, Md.). FDCP-1 and Ba/F3, MFM-223, MFE-296, MFE-280, MFE-319 and ESS-1 cells were obtained from the German Collection of Microorganisms and Cell Cultures. All human cell lines were cultured according to the instructions specified by the suppliers, at 37° C., in an atmosphere containing 5% CO₂. All FGFs were purchased from R&D Systems, Inc. (Minneapolis, Minn.).

[0079] To establish cell-based assays to screen for functional FGFR2 antibodies, we first engineered Ba/F3 and FDCP-1 cells to express wild type FGFR2 and cancer-associated mutants or variants of FGFR2. FGFR-driven FDCP cells and Ba/F3 cells were obtained by the following methods. FDCP-1 cells were transfected by electroporation with plasmids encoding the Mb, Mc isoform or C-terminally truncated variant of human FGFR2 as well as cancer-associated FGFR2-IIIb S252W, or FGFR2-IIIb N550K mutants. Following selection with G418 (600 μg/ml), single clones were isolated and tested for their FGF1-dependent proliferation in the absence of IL3 by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma-Aldrich, St. Louis, Mo.). MTT reagent (10 μl) was added to the cells and the reaction was stopped with 100 μl of 10% SDS with 2N HCL after four hours. The plates were analyzed the following day. The clones that exhibited robust FGF-1-dependent proliferation in the absence of IL3 were used for subsequent studies. To generate retroviruses expressing FGFR2, cDNAs encoding various human FGFR2 variants were each inserted into a retroviral vector. Retroviruses were produced by transfecting Phoenix cells using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Supernatants containing the retroviruses were used to infect Ba/F3 cells by centrifugation at 2500 rpm for 90 minutes, in the presence of 8 μg/ml of polybrene (Sigma-Aldrich). Individual clones were isolated by limiting dilution, and cell surface receptor expression was verified by flow cytometry.

[0080] Cancer cell lines with FGFR amplification were identified as follows. The CGP copy number database at the Wellcome Trust Sanger Institute (www.sanger.ac.uk) was queried for FGFR2 amplification (gene copy number>7). The copy number of the cell lines with potential FGFR2 amplification was analyzed by quantitative PCR (qPCR) using FGFR2 specific primers (5'-ACTTGGGCTGGAGTGATTTG-3' (SEQ ID NO: 24) and 5'-AATCCCATCTGCACACTTCC-3' (SEQ ID NO: 25)) and reference gene (transketolase) primers (5'-CAAAAACATGGCTGAGCAGA-3' (SEQ ID NO: 26) and 5'-GAAACAGGCCCCACTTTGTA-3' (SEQ ID NO: 27)). The FGFR2 gene copy number was calculated essentially as described in Toyokawa et al., 2009, ONCOL. REP. 21:875-880.

[0081] FGFR gene expression analysis was performed as follows. Total RNA was isolated by the RNeasy® mini kit (Qiagen, Valencia, Calif.). Quantitative RT-PCR (qRT-PCR) was performed using a QuantiTect® SYBR Green RT-PCR kit (Qiagen), with primers specific for FGFR2, FGFR2-IIIb, FGFR2-IIIc, and HPRT. The expression levels were normalized to HPRT.

[0082] Previous studies have demonstrated that ectopic expression of FGFRs in murine pro-B Ba/F3 or bone marrow FDCP-1 cells confers FGF1-dependent proliferation in the absence of IL-3 (Tannheimer et al., 2000, BREAST CANCER RES. 2:311-320; Ornitz et al., 1996, J. BIOL. CHEM. 271:15292-15297). As expected, there was no noticeable proliferation of FDCP-1 cells stably expressing wild-type FGFR2 in the absence of IL-3 and FGF1. It was known that FGF1, 3, 7, 10 and 22 transduce signals through FGFR2-IIIb, and that FGFR2-IIIc responds to a broader panel of ligands including FGF1, 2, 4, 6, 9, 16, 17, 18 and 20 (Tannheimer et al., supra; Ornitz et al., supra; Zhang et al., 2006, J. BIOL. CHEM. 281:15964-15700). The proliferation of FDCP-1 cells expressing the Mb isoform of FGFR2 was stimulated by FGF7 and FGF10, but not by FGF2 and FGF9 (FIG. 2). The proliferation of cells expressing the Mc isoform was enhanced by FGF2 and FGF9 specifically (FIG. 3).

Example 2

Production of Anti-FGFR2Monoclonal Antibodies

[0083] Mice were immunized with a 1:1 mixture of human FGFR21gD2-IgD3 (Mb) and human FGFR2 IgD2-IgD3 (Mc) fused with a human Fc moiety at their C-termini. Mouse immunizations and cell fusions were performed by a commercial vendor (Precision Antibody, Columbia, Md.).

[0084] In a primary screen, hybridoma supernatants were screened to detect binding to human FGFR2 IgD2-IgD3, using an ELISA format. Antibodies that passed the primary screen were subjected to a secondary screen, which was a cell-based proliferation assay described in Example 3 (below).

[0085] The primary screen was performed using the supernatants of the murine hybridoma clones yielded from the splenic fusion of the mice immunized with the extracellular domain of human FGFR2. Assay plates were coated with 100 ng/well of recombinant soluble FGFR2 extracellular domain and then blocked with 5% milk in PBS for one hour at room temperature. Then 50 μl of hybridoma supernatant was added to each well to allow antibody binding for one hour at room temperature. Plates were washed three times with wash buffer (PBS with 0.1% Tween 20) followed by incubation with a HRP-conjugated goat anti-mouse IgG heavy and light chain secondary antibody. The assay was developed using TMB (tetramethylbenzene) as a substrate, and absorbance was read at 620 nm.

Example 3

Identification of FGFR2Antagonist Antibodies

[0086] To screen for FGFR2 antagonist antibodies, hybridoma supernatants containing FGFR2 antibodies were added to FDCP cells ectopically expressing one of the following five forms of FGFR2: (1) wild type FGFR2-IIIb; (2) wild type FGFR2-IIIc; (3) FGFR2-III(b) S252W; (4) FGFR2-III(b) N550K; and (5) FGFR2-III(b) with C-terminal truncation. The supernatants were added to the FGFR2-expressing cells at a 1:1 ratio (volume) in a flat-bottomed 96-well plate (70,000 cells/well) with heparin (5 μg/ml)±FGF1 (8 ng/ml). After incubation at 37° C. for 2 days, MTT assays were conducted as described above.

[0087] The supernatant of clone 4B9 demonstrated potent and selective inhibition of the FDCP-1 proliferation driven by the IIIb-isoform of FGFR2. Antibody 4B9 (also referred to as antibody GP369), produced by clone 4B9, was purified by conventional techniques for further characterization. Surface plasmon resonance analysis indicated that antibody 4B9 exhibited strong affinity towards human FGFR2-IIIb and showed no detectable binding to the human FGFR2-IIIc. No binding of antibody 4B9 to human FGFR1-IIIc or FGFR3-IIIb was detected.

Example 4

Sequence Analysis

[0088] The light chain isotype and heavy chain isotype of antibody 4B9 in Example 1 was determined using the IsoStrip® Mouse Monoclonal Antibody Isotyping Kit according to the manufacturer's instructions (Roche Applied Science, Indianapolis, Ind.). The antibody was determined to be Kappa light chain and IgG1 heavy chain.

[0089] The heavy and light chain variable regions of antibody 4B9 were sequenced using 5' RACE (Rapid Amplification of cDNA Ends). Total RNA was extracted from the 4B9 monoclonal hybridoma cell line using the RNeasy® Miniprep kit according to the vendor's instructions (Qiagen, Valencia, Calif.). Full-length first strand cDNA containing 5' ends was generated using SMARTer® RACE cDNA Amplification Kit (Clontech, Palo Alto, Calif.) according to the manufacturer's instructions using random primers for 5' RACE.

[0090] The variable regions of the kappa and heavy IgG1 chains were amplified by PCR, using KOD Hot Start® Polymerase (EMD Chemicals, Gibbstown, N.J.) according to the manufacturer's instructions. For amplification of 5' cDNA ends in conjunction with the SMARTer® RACE cDNA Amplification Kit, the Universal Primer Mix A primer (Clontech), a mix of 5'CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3' (SEQ ID NO: 28) and 5' CTAATACGACTCACTATAGGGC 3' (SEQ ID NO: 29), was used as a 5' primer. The heavy chain variable region was amplified using the above 5' primers and a 3' IgG 1 constant region specific primer, 5' TATGCAAGGCTTACAACCACA 3' (SEQ ID NO: 30). The kappa chain variable region was amplified with the above 5' primers and a 3' kappa constant region specific primer, CGACTGAGGCACCTCCAGATGTT 3' (SEQ ID NO: 31).

[0091] Individual PCR products were isolated 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 pCR4Blunt plasmid using the Zero Blunt TOPO® PCR Cloning Kit according to the manufacturer's instructions (Invitrogen) and transformed into DH5-α bacteria (Invitrogen) through standard molecular biology techniques. Plasmid DNA isolated from transformed bacterial clones was sequenced using M13 Forward (5' GTAAAACGACGGCCAGT 3') (SEQ ID NO: 32) and M13 Reverse primers (5' CAGGAAACAGCTATGACC 3') (SEQ ID NO: 33) by Beckman Genomics (Danvers, Mass.), using standard dideoxy DNA sequencing methods to identify the sequence of the variable region sequences. The sequences were analyzed using Vector NTI software (Invitrogen) and the IMGT/V-Quest web server to identify and confirm variable region sequences.

[0092] The nucleic acid sequences encoding and the protein sequences defining variable regions of antibody 4B9 are summarized below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are shown in bold/underlined in the amino acid sequences.

[0093] Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of Antibody 4B9 (SEQ ID NO: 1)

TABLE-US-00001 1 gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 61 tcctgcaaga cttctggcta cacatttacc agctactgga tgcactgggt aaaacagagg 121 cctggacagg gtctggaatg gataggggct atttatcctg gaaatagtga tactgactac 181 agccagaagt tcaagggcaa ggccacactg actgcagtca catccgccac cactgcctac 241 atggaactca gcagcctgac aaatgaggac tctgcggtct attactgttc aaagtttgac 301 tactggggcc aaggcaccac tctcacagtc tcctca

[0094] Protein Sequence Defining the Heavy Chain Variable Region of Antibody 4B9 (SEQ ID NO: 2)

TABLE-US-00002 1 evqlqqsgtv larpgasvkm scktsgytft sywmhwvkqr pgqglewiga iypgnsdtdy 61 sqkfkgkatl tavtsattay melssltned savyycskfd ywgqgttltv ss

[0095] Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of Antibody 4B9 (SEQ ID NO: 3)

TABLE-US-00003 1 caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 61 atgacctgca gtgccagctc aagtgtaaat tacatgtact ggtaccagca gaagccaaga 121 tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 181 ttcagtggca gggggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 241 gatgctgcca cttattactg ccagcagtgg agtagtaacc cgtacacgtt cggagggggg 301 accaagctgg aaataaaa

[0096] Protein Sequence Defining the Kappa Chain Variable Region of Antibody 4B9 (SEQ ID NO: 4)

TABLE-US-00004 1 qivltqspal msaspgekvt mtcsasssvn ymywyqqkpr sspkpwiylt snlasgvpar 61 fsgrgsgtsy sltissmeae daatyycqqw ssnpytfggg tkleik

[0097] Table 1 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.

TABLE-US-00005 TABLE 1 SEQ. ID NO. Antibody 4B9 Nucleic Acid or Protein 1 Heavy Chain Variable Region--nucleic acid 2 Heavy Chain Variable Region--protein 3 Light (kappa) Chain Variable Region--nucleic acid 4 Light (kappa) Chain Variable Region--protein 5 Heavy Chain CDR₁ (Kabat definition) 6 Heavy Chain CDR₂ (Kabat definition) 11 Heavy Chain CDR₃ (IGMT definition) 12 Light (kappa) Chain CDR₁ (Kabat definition) 13 Light (kappa) Chain CDR₂ (Kabat definition) 14 Light (kappa) Chain CDR₃ (Kabat definition)

[0098] Mouse monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 2.

TABLE-US-00006 TABLE 2 CDR1 CDR2 CDR3 Kabat 4B9 SYWMH AIYPGNSDTDYSQKFKG FDY (SEQ ID (SEQ ID NO: 6) NO: 5) Chothia 4B9 GYTFTSY YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 7) IMGT 4B9 GYTFTSYW IYPGNSDT SKFDY (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: 11) NO: 9)

[0099] Mouse monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 3.

TABLE-US-00007 TABLE 3 CDR1 CDR2 CDR3 Kabat/Chothia 4B9 SASSSVNYMY LTSNLAS QQWSSNPYT (SEQ ID (SEQ ID (SEQ ID NO: 14) NO: 12) NO: 13) IMGT 4B9 SSVNY LTS QQWSSNPYT (SEQ ID (SEQ ID NO: 14) NO: 15)

[0100] To create the complete heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective constant region. For example, a complete heavy chain comprises the heavy variable sequence followed by the murine IgG1 heavy chain constant sequence and the complete kappa chain comprises a kappa variable sequence followed by the murine kappa light chain constant sequence.

[0101] Nucleic Acid Sequence Encoding the Murine IgG1 Heavy Chain Constant Region (SEQ ID NO: 16)

TABLE-US-00008 1 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 61 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 121 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 181 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag ccagaccgtc 241 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 301 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 361 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 421 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 481 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 541 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 601 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 661 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 721 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 781 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 841 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 901 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 961 tctcctggta aa

[0102] Protein Sequence Defining the Murine IgG1 Heavy Chain Constant Region (SEQ ID NO: 17)

TABLE-US-00009 1 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd 61 lytlsssvtv psstwpsqtv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif 121 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv 181 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv 241 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf 301 tcsvlheglh nhhtekslsh spgk

[0103] Nucleic Acid Sequence Encoding the Murine Kappa Light Chain Constant Region (SEQ ID NO: 18)

TABLE-US-00010 1 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 61 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccagagacat caatgtcaag 121 tggaagattg atggcagtga acgacaaaat ggtgtcctga acagttggac tgatcaggac 181 agcaaagaca gcacctacag catgagcagc accctcacat tgaccaagga cgagtatgaa 241 cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 301 agcttcaaca ggaatgagtg t

[0104] Protein Sequence Defining the Murine Kappa Light Chain Constant Region (SEQ ID NO: 19)

TABLE-US-00011 1 radaaptvsi fppsseqlts ggasvvcfln nfyprdinvk wkidgserqn gvlnswtdqd 61 skdstysmss tltltkdeye rhnsytceat hktstspivk sfnrnec

[0105] The following sequences represent the actual or contemplated full length heavy and light chain sequences (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. Signal sequences for proper secretion of the antibodies are also included at the 5' end of the DNA sequences or the amino terminal end of the protein sequences. The variable region sequences can be ligated to other constant region sequences, to produce active full length IgG heavy and light chains.

[0106] Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgG1 Constant Region) of 4B9 (SEQ ID NO: 20)

TABLE-US-00012 1 atggaatgta actggatact tccttttatt ctgtcggtaa cttcaggggt ctactcagag 61 gttcagctcc agcagtctgg gactgtgctg gcaaggcctg gggcttcagt gaagatgtcc 121 tgcaagactt ctggctacac atttaccagc tactggatgc actgggtaaa acagaggcct 181 ggacagggtc tggaatggat aggggctatt tatcctggaa atagtgatac tgactacagc 241 cagaagttca agggcaaggc cacactgact gcagtcacat ccgccaccac tgcctacatg 301 gaactcagca gcctgacaaa tgaggactct gcggtctatt actgttcaaa gtttgactac 361 tggggccaag gcaccactct cacagtctcc tcagccaaaa cgacaccccc atctgtctat 421 ccactggccc ctggatctgc tgcccaaact aactccatgg tgaccctggg atgcctggtc 481 aagggctatt tccctgagcc agtgacagtg acctggaact ctggatccct gtccagcggt 541 gtgcacacct tcccagctgt cctgcagtct gacctctaca ctctgagcag ctcagtgact 601 gtcccctcca gcacctggcc cagccagacc gtcacctgca acgttgccca cccggccagc 661 agcaccaagg tggacaagaa aattgtgccc agggattgtg gttgtaagcc ttgcatatgt 721 acagtcccag aagtatcatc tgtcttcatc ttccccccaa agcccaagga tgtgctcacc 781 attactctga ctcctaaggt cacgtgtgtt gtggtagaca tcagcaagga tgatcccgag 841 gtccagttca gctggtttgt agatgatgtg gaggtgcaca cagctcagac gcaaccccgg 901 gaggagcagt tcaacagcac tttccgctca gtcagtgaac ttcccatcat gcaccaggac 961 tggctcaatg gcaaggagtt caaatgcagg gtcaacagtg cagctttccc tgcccccatc 1021 gagaaaacca tctccaaaac caaaggcaga ccgaaggctc cacaggtgta caccattcca 1081 cctcccaagg agcagatggc caaggataaa gtcagtctga cctgcatgat aacagacttc 1141 ttccctgaag acattactgt ggagtggcag tggaatgggc agccagcgga gaactacaag 1201 aacactcagc ccatcatgga cacagatggc tcttacttcg tctacagcaa gctcaatgtg 1261 cagaagagca actgggaggc aggaaatact ttcacctgct ctgtgttaca tgagggcctg 1321 cacaaccacc atactgagaa gagcctctcc cactctcctg gtaaa

[0107] Protein Sequence Defining the Full Length Heavy Chain Sequence (Heavy Chain Variable Region and IgG1 Constant Region) of 4B9 (SEQ ID NO: 21)

TABLE-US-00013 1 mecnwilpfi lsvtsgvyse vqlqqsgtvl arpgasvkms cktsgytfts ywmhwvkqrp 61 gqglewigai ypgnsdtdys qkfkgkatlt avtsattaym elssltneds avyycskfdy 121 wgqgttltvs sakttppsvy plapgsaaqt nsmvtlgclv kgyfpepvtv twnsgslssg 181 vhtfpavlqs dlytlsssvt vpsstwpsqt vtcnvahpas stkvdkkivp rdcgckpcic 241 tvpevssvfi fppkpkdvlt itltpkvtcv vvdiskddpe vqfswfvddv evhtaqtqpr 301 eeqfnstfrs vselpimhqd wlngkefkcr vnsaafpapi ektisktkgr pkapqvytip 361 ppkeqmakdk vsltcmitdf fpeditvewq wngqpaenyk ntqpimdtdg syfvysklnv 421 qksnweagnt ftcsvlhegl hnhhteksls hspgk

[0108] Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 4B9 (SEQ ID NO: 22)

TABLE-US-00014 1 atggattttc aagtgcagat tttcagcttc ctgctaatga gtgcctcagt cataatgtcc 61 aggggacaaa ttgttctcac ccagtctcca gcactcatgt ctgcatctcc aggggagaag 121 gtcaccatga cctgcagtgc cagctcaagt gtaaattaca tgtactggta ccagcagaag 181 ccaagatcct cccccaaacc ctggatttat ctcacatcca acctggcttc tggagtccct 241 gctcgcttca gtggcagggg gtctgggacc tcttactctc tcacaatcag cagcatggag 301 gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccgta cacgttcgga 361 ggggggacca agctggaaat aaaacgggct gatgctgcac caactgtatc catcttccca 421 ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 481 taccccagag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggtgtc 541 ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc 601 acattgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag 661 acatcaactt cacccattgt caagagcttc aacaggaatg agtgt

[0109] Protein Sequence Defining the Full Length Light Chain Sequence (Kappa Chain Variable Region and Constant Region) of 4B9 (SEQ ID NO: 23)

TABLE-US-00015 1 mdfqvqifsf llmsasvims rgqivltqsp almsaspgek vtmtcsasss vnymywyqqk 61 prsspkpwiy ltsnlasgvp arfsgrgsgt sysltissme aedaatyycq qwssnpytfg 121 ggtkleikra daaptvsifp psseqltsgg asvvcflnnf yprdinvkwk idgserqngv 181 lnswtdqdsk dstysmsstl tltkdeyerh nsytceathk tstspivksf nrnec

[0110] Table 4 shows the correspondence between the full length sequences of the antibodies discussed in this Example with those presented in the Sequence Listing.

TABLE-US-00016 TABLE 4 SEQ ID NO. Antibody 4B9 Nucleic Acid or Protein 20 Heavy Variable + IgG1 Constant--nucleic acid 21 Heavy Variable + IgG1 Constant--protein 22 Kappa Variable + Constant--nucleic acid 23 Kappa Variable + Constant--protein

Example 5

Binding Affinities

[0111] The binding affinities and binding kinetics of monoclonal antibody 4B9 were measured with respect to the following proteins (R&D Systems, Inc., Minneapolis, Minn.): recombinant human FGFR1 beta (IIIb)/Fc Chimera (rhFGFR1β-IIIc-Fc), recombinant human FGFR2 beta (IIIb)/Fc Chimera (rhFGFR2β-IIIb-Fc), recombinant human FGFR2 beta (IIIc)/Fc Chimera (rhFGFR2β-IIIc-Fc), recombinant human FGFR3 beta (IIIb)/Fc Chimera (rhFGFR3β-IIIb-Fc), and a version of recombinant human FGFR2 beta (IIIb)/Fc (in which the Fc region was removed enzymatically). Binding affinities and binding kinetics were measured by surface plasmon resonance using a Biacore T100 instrument (GE Healthcare, Piscataway, N.J.).

[0112] Rabbit anti-mouse IgGs (GE Healthcare) were immobilized on carboxymethylated dextran CM4 sensor chips (GE Healthcare) by amine coupling, using a standard coupling protocol, according to the vendor's instructions (GE Healthcare). The analyses were performed at 25° C. and 37° C., using PBS containing 0.05% surfactant P20 (GE Healthcare) as running buffer.

[0113] The antibodies were captured in individual flow cells at a flow rate of 10 μl/min. Injection time was varied for each antibody to yield an Rmax between 30 and 60 RU. Buffer and FGFR proteins diluted in running buffer were injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 240 seconds at 60 μl/min. The dissociation phase was monitored for up to 900 seconds. The surface was then regenerated with two 60-second injections of 10 mM Glycine-HCl (pH 1.7), at a flow rate of 60 μl/minute. The FGFR protein concentration range tested was 50 to 3.125 nM (two-fold dilutions).

[0114] Kinetic parameters were determined using the kinetic function of the BlAevalutation software (GE Healthcare) with double reference subtraction. Kinetic parameters for each antibody, k_a (association rate constant), k_d (dissociation rate constant) and K_D (equilibrium dissociation constant) were determined. Kinetic values of the monoclonal antibodies on FGFR proteins at 25° C. and 37° C. are summarized in Table 5.

TABLE-US-00017 TABLE 5 Anti- Temp body Target (° C.) k_a (M^-1 s^-1) k_d (s^-1) K_D (M) 4B9 rhFGFR1β-IIIb-Fc 25 no binding no binding no binding 4B9 rhFGFR2β-IIIb-Fc 25 9.4E+04 4.6E-05 6.1E-10 4B9 rhFGFR2β-IIIb-Fc 37 3.44E+04 3.16E-05 2.96E-09 4B9 rhFGFR2β-IIIb- 25 5.5E+04 8.1E-05 4.2E-09 cleaved 4B9 rhFGFR2β-IIIb- 37 2.54E+05 2.23E-04 1.20E-09 cleaved 4B9 rhFGFR2β-IIIc-Fc 25 no binding no binding no binding 4B9 rhFGFR3β-IIIb-Fc 25 no binding no binding no binding

[0115] The results in Table 5 demonstrate that antibody 4B9 binds rhFGFR2β-IIIb with a K_D of about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 750 pM, 650 pM, 610 pM or less. The results also demonstrate that antibody 4B9 does not bind rhFGFR1β-IIIb, rhFGFR2β-IIIc, and rhFGFR3β-IIIb.

Example 6

Anti-Proliferative Activity

[0116] To assess the potency of antibody 4B9 quantitatively, we carried out dose-response studies, using FDCP-1 cells expressing FGFR2-IIIb or FGFR2-IIIc. FDCP-1 cells expressing FGFR2-IIIb or FGFR2-IIIc were seeded in a 96-well plate in the absence of IL3. Varied amounts of FGFs and heparin were added. MTT assays were carried out after 2-3 days. Varied amounts of antibody 4B9-containing supernatants were added to FDCP-1 cells expressing FGFR2-IIIb, FGFR2-IIIc, or C-terminally truncated FGFR2-IIIb, in the presence of FGF1 and heparin. MTT assays were carried out after 2 days. Varied amounts of purified antibody 4B9 were added to FDCP-1 cells expressing FGFR2-IIIb S252W or FGFR2-IIIb N550K in the presence of FGF1 and heparin. MTT assays were carried out after 2 days.

[0117] Antibody 4B9 potently inhibited FGF1-induced proliferation of FDCP-1 cells driven by FGFR2-IIIb, in a dose-dependent manner, while 4B9 had no significant effect on the FGF1-induced proliferation of FDCP cells expressing the FGFR2-IIIc (FIG. 4). C-terminally truncated FGFR2-IIIb, which causes constitutive phosphorylation of FRS2 adaptor molecule and activation of downstream signaling, is found in gastric and breast cancer cell lines (Itoh et al., 1994, CANCER RES. 54:3237-3241; Moffa et al., 2004, MOL. CANCER RES. 2:643-652). Antibody 4B9 potently inhibited the proliferation of FDCP-1 cells driven by the C-terminally truncated FGFR2-IIIb (FIG. 4).

[0118] FGFR2 mutations have been reported in approximately 12% of endometrial tumor sample (Pollock et al., supra; Dutt et al., supra). Somatic activating mutations in FGFR2 cluster within the linker region between IgD2 and IgD3, the extracellular juxtamembrane domain, or the kinase domain. Two of the most common mutations in endometrial tumors are the S252W mutation (which alters ligand specificity and increases affinity of ligand binding) and the N550K mutation in the kinase domain (which enhances kinase activity). Purified antibody 4B9 potently inhibited cell proliferation driven by the wild type FGFR2-IIIb, as well as FGFR2-IIIb S252W and FGFR2-IIIb N550K, with IC₅₀ values of 0.3 nM, 3.0 nM and 8.1 nM, respectively (FIG. 5).

Example 7

Inhibition of FGFR2-Activated Signaling Pathways

[0119] We investigated the effect of antibody 4B9 on FGFR2-activated signaling pathways. To examine the effect of antibody 4B9 on tyrosine phosphorylation of FGFR2, SNU-16 cells were treated with antibodies at a dose of 5 μg/ml for 1 hour at 37° C., followed by stimulation with heparin alone (20 μg/ml) or heparin-plus-FGF7 (30 ng/ml) for 15 minutes. The cells were lysed in NP-40 lysis buffer containing 1% NP-40, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA and supplemented with protease inhibitors (Roche Applied Science) and Halt phosphatase inhibitors (Thermo Scientific).

[0120] The lysates were analyzed by Western blot with anti-FGFR (Y653/Y654) (R&D Systems, Inc., Minneapolis, Minn.), anti-FGFR2 (sc-122) (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-phospho-ERK1/2 and anti-ERK1/2 (Cell Signaling Technology, Danvers, Mass.), anti-β-tubulin, clone AA2 (Millipore Corporation; Billerica, Mass.) antibodies. The immunoblots were detected by chemiluminescent substrate (ECL Plus®, Amersham Pharmacia Biotech, Piscataway, N.J.). Human Phospho-RTK and MAPK kinase arrays (R&D systems) were carried out according to manufacturer's instructions (R&D systems). For phospho-RTK arrays, the cells were lysed in NP-40 lysis buffer. The arrays were blocked in Array Buffer 1 at room temperature for one hour prior to the addition of cell lysates diluted in Array Buffer 1 and were then incubated at 4° C. overnight. The arrays were visualized by chemiluminescence. For phospho-MAPK arrays, the cells were lysed in Lysis Buffer 6. The diluted cell lysates were added to arrays. After incubation at 4° C. overnight, the arrays were mixed with anti-phospho-MAPK antibody for two hours at room temperature and visualized as described above.

[0121] FGF7 induced tyrosine phosphorylation of FGFR2 and subsequent activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in Ba/F3 cells overexpressing FGFR2, and in FGFR2-amplified SNU-16 cells. Antibody 4B9 effectively suppressed the ligand-induced tyrosine phosphorylation of FGFR2 and activation of ERK1/2 in these cells. In addition, antibody 4B9 downregulated the FGFR2 protein level in SNU-16 cells. A slight decrease in the FGFR2 protein level was observed as early as two hours after exposure to the antibody. A dramatic reduction in the protein level was seen at the six-hour time point.

[0122] We investigated activation of downstream signaling pathways in these cell lines, using a phospho-MAPK array, which measures phosphorylation of ERKs, c-Jun NH₂-Terminal Kinases (JNKs), p38 MAPKs, AKTs, and their downstream effector molecules. We found little phosphorylation of ERK1/2 in the absence of ligand stimulation. Stimulation of SNU-16 cells with FGF7 significantly increased the phosphorylation of ERK1/2. We observed an increase in the phosphorylation of mitogen- and stress-activated kinase 2 (MSK2), p38α MAPK, 90-kD ribosomal protein kinase 1 (RSK1), Akt1, and p70S6 kinase (p70S6K). Antibody 4B9 effectively blocked the phosphorylation of all the downstream signaling proteins activated by FGF7.

Example 8

Inhibition of Tumor Xenograft Growth

[0123] To assess the activity of antibody 4B9 in vivo, we tested the effect of antibody 4B9 on the growth of human cancer xenografts harboring amplification of the FGFR2 gene. Out of the four FGFR2-amplified cell lines that were tested, only SNU-16 and MFM-223 yielded tumors in mice. Therefore, we tested the efficacy of antibody 4B9 against SNU-16 and MFM-223 xenograft tumors.

[0124] All mice were treated in accordance with the OLAW Public Health Service Policy on Human Care and Use of Laboratory Animals and the ILAR Guide for the Care and Use of Laboratory Animals. All in vivo studies were conducted following the protocols approved by the AVEO Institutional Animal Care and Use Committee. For the SNU-16 in vivo studies, 10 week old female C.B-17 SCID mice (Taconic, Germantown, N.Y.) were inoculated subcutaneously into the right flank with 5×10⁶ cells in 1:1 RPMI 1640 (Invitrogen, Carlsbad, Calif.)/Matrigel (BD Biosciences, San Jose Calif.). Tumor measurements were taken twice weekly, using vernier calipers. Tumor volume was calculated using the formula: V=0.5×width×width×length. When tumors approached a volume of 200 mm³, mice were randomized into five groups of ten animals. The next day, mice were treated with 20 mg/kg mIgG (BioXCell; West Lebanon, N.H.), 2 mg/kg 4B9, 5 mg/kg 4B9, 10 mg/kg 4B9, or 20 mg/kg 4B9 by intraperitoneal injection. Mice were dosed twice weekly for the duration of the study. Seventy-two hours after the final dose tumor volumes were measured again for calculation of tumor growth inhibition. All statistical analysis was done using GraphPad PRISM® Version 4.00. Final tumor volumes were analyzed using with a one-way analysis of variance and Tukey multiple comparison test.

[0125] SNU-16 xenograft tumors were treated with a control murine IgG at 20 mg/kg or antibody 4B9 at 2, 5, 10 or 20 mg/kg. As shown in FIG. 6, each 4B9 treatment group showed significant tumor growth inhibition, as compared to mIgG treated controls (70, 72, 77, and 82%, respectively p<0.001) at day 43, which was the last day for the control group to remain in the study. All treatments were well-tolerated with no significant body weight loss. The tumor lysates were also analyzed. Concomitant with inhibition of tyrosine phosphorylation of FGFR2, antibody 4B9 downregulated the total amount of FGFR2 protein in tumors. No significant difference in the total ERK1/2 or phospho-ERK1/2 was detected in the tumors samples treated with control IgG or 4B9 from tumors collected at the end of study. In contrast to the phospho-receptor tyrosine kinase (RTK) profile of SNU-16 cells in vitro, RTK array analysis of SNU-16 xenografts revealed that FGFR2 was the predominant RTK that was tyrosine phosphorylated in vivo, and 4B9 significantly inhibited FGFR2 tyrosine phosphorylation in two of the 4B9-treated SNU-16 tumors tested. In vitro, the proliferation of SNU-16 cells was not sensitive to the treatment of 4B9. Tyrosine phosphorylation of FGFR2 in SNU-16 cells in vivo suggests that the dependence of SNU-16 xenografts on activated FGFR2 signaling in vivo explains their sensitivity to treatment with antibody 4B9.

[0126] The effect of antibody 4B9 was also investigated on the in vivo growth of FGFR2-amplified breast cancer cell line MFM-223. For these studies, 5-week old female NCr nude mice (Taconic; Germantown, N.Y.) were implanted subcutaneously on the left flank with 0.72 mg 90-day release 17-β estradiol pellets (Innovative Research; Sarasota, Fla.) and inoculated subcutaneously into the right flank with 10×10⁶ MFM-223 cells in 1:1 EMEM (ATCC; Manassas, Va.)/Matrigel. When tumors approached a volume of 200 mm³, mice were randomized into two groups of ten animals and treated IP with 20 mg/kg mIgG (BioXCell; West Lebanon, N.H.) or 20 mg/kg 4B9. Mice were dosed twice weekly for the duration of the study. All statistical analysis was done using GraphPad PRISM® Version 4.00. Since there were only two groups in this study final tumor volumes and weights (Day 27, 48 hours after final dose) were analyzed with an unpaired two tailed t-test.

[0127] On day 25, in the MFM-223 xenografts, there was greater than 66% inhibition of tumor volumes (p=0.0015; FIG. 7) and final tumor weights (p=0.0188) in 4B9 treated mice, as compared to mIgG-treated controls. All treatments were well-tolerated, with no significant body weight loss. Similar to what was observed in SNU-16 xenografts, 4B9 strongly down-regulated the total FGFR2 protein in tumors, concomitant with inhibition of tyrosine phosphorylation of FGFR2. No significant difference in the total or phosphor-ERK1/2 was detected in the tumors samples either treated with the control IgG or 4B9 from tumors collected at the end of study.

Example 9

Humanization of Anti-FGFR2Antibodies

[0128] A. Construction of Humanized FGFR2Antibodies

[0129] This Example describes the humanization of the murine antibody designated 4B9, and the characterization of the resulting humanized antibodies. The humanized anti-FGFR2Mb antibodies were designed using methods well-known in the art. The designed amino acid sequences were converted to codon-optimized DNA sequences and synthesized by DNA2.0, Inc. to include (in the following order): 5' HindIII restriction site, Kozak consensus sequence, amino terminal signal sequence, humanized variable region, human IgG 1 or Kappa constant region, stop codon, and a 3' EcoRI restriction site.

[0130] The humanized heavy chains were subcloned into pEE6.4 (Lonza, Basel, Switzerland) via HindIII and EcoRI sites using In-Fusion® PCR cloning (Clontech, Mountain View, Calif.). The humanized Kappa light chains were subcloned into pEE14.4 (Lonza) via HindIII and EcoRI sites using In-Fusion® PCR cloning.

[0131] Humanized antibody chains were transiently transfected into 293T cells to produce antibody. Antibody was purified for subsequent in vitro analysis. Binding of the humanized antibodies to human FGFR2Mb was measured as described below. The results are summarized in Tables 12 and 13.

[0132] Each of the possible combinations of the humanized immunoglobulin heavy chain and immunoglobulin light chain variable regions are set forth below in Table 6.

TABLE-US-00018 TABLE 6 Light Chain Variable Region Heavy Chain Variable Region Hu4B9-65 Kappa (SEQ ID NO: 40) Hu4B9-65 Heavy (SEQ ID NO: 35) Hu4B9-65 Kappa (SEQ ID NO: 40) Hu4B9-82, -83 Heavy (SEQ ID NO: 37) Hu4B9-82 Kappa (SEQ ID NO: 44) Hu4B9-65 Heavy (SEQ ID NO: 35) Hu4B9-82 Kappa (SEQ ID NO: 44) Hu4B9-82, -83 Heavy (SEQ ID NO: 37) Hu4B9-83 Kappa (SEQ ID NO: 46) Hu4B9-65 Heavy (SEQ ID NO: 35) Hu4B9-83 Kappa (SEQ ID NO: 46) Hu4B9-82, -83 Heavy (SEQ ID NO: 37)

[0133] The nucleic acid sequences encoding and the protein sequences defining variable regions of the humanized 4B9 antibodies are summarized below (amino terminal signal peptide sequences are not shown). CDR sequences (Kabat definition) are shown in bold and are underlined in the amino acid sequences.

[0134] Nucleic Acid Sequence Encoding the Hu4B9-65 Heavy Chain Variable Region (SEQ ID NO: 34)

TABLE-US-00019 1 caagtgcagc tcgtccaatc gggagccgaa gtgaagaagc ctggttcctc ggtaaaagta 61 agctgtaagg cgtccggtta cacgtttacc tcatattgga tgcactgggt cagacaggca 121 cccggacagg gactcgagtg gatgggagcg atctacccgg gcaattcgga cactgattac 181 agccagaaat tcaaggggag ggtcacgatc acggcagatg agagcacatc aacagcctat 241 atggagctgt cgtcgcttcg gagcgaggac acggcggtct actactgctc caaattcgac 301 tattgggggc aggggacctt ggtgaccgtg tcatcc

[0135] Protein Sequence Defining the Hu4B9-65 Heavy Chain Variable Region (SEQ ID NO: 35)

TABLE-US-00020 1 qvqlvqsgae vkkpgssvkv sckasgytft sywmhwvrqa pgqglewmga iypgnsdtdy 61 sqkfkgrvti tadeststay melsslrsed tavyycskfd ywgqgtlvtv ss

[0136] Nucleic Acid Sequence Encoding the Hu4B9-82, -83 Heavy Chain Variable Region (SEQ ID NO: 36)

TABLE-US-00021 1 caagtgcagc tcgtccaatc gggagccgaa gtgaagaagc ctggttcctc ggtaaaagta 61 agctgtaagg cgtccggtta cacgttttcc tcatattgga tgcactgggt cagacaggca 121 cccggacagg gactcgagtg gatgggagcg atctacccgg gcaattcgga cactgattac 181 agccagaaat tccaggggag ggtcacgatc acggcagatg agagcacatc aacagcctat 241 atggagctgt cgtcgcttcg gagcgaggac acggcggtct actactgctc caaattcgac 301 tattgggggc aggggacctt ggtgaccgtg tcatcc

[0137] Protein Sequence Defining the Hu4B9-82, -83 Heavy Chain Variable Region (SEQ ID NO: 37)

TABLE-US-00022 1 qvqlvqsgae vkkpgssvkv sckasgytfs sywmhwvrqa pgqglewmga iypgnsdtdy 61 sqkfqgrvti tadeststay melsslrsed tavyycskfd ywgqgtlvtv ss

[0138] Nucleic Acid Sequence Encoding the Hu4B9-65 Kappa Chain Variable Region (SEQ ID NO: 39)

TABLE-US-00023 1 gaaattgtgc tgacccagag cccggcgacc ctgagcctga gcccgggcga acgcgcgacc 61 ctgagctgcc gcgcgagcag cagcgtgaac tatatgtatt ggtatcagca gaaaccgggc 121 caggcgccgc gcccgtggat ttatctgacc agcaaccgcg cgaccggcgt gccggcgcgc 181 tttagcggca gcggcagcgg caccgattat accctgacca ttagcagcct ggaaccggaa 241 gattttgcgg tgtattattg ccagcagtgg agcagcaacc cgtatacctt tggccagggc 301 accaaactgg aaattaaa

[0139] Protein Sequence Defining the Hu4B9-65 Kappa Chain Variable Region (SEQ ID NO: 40)

TABLE-US-00024 1 eivltqspat lslspgerat lscrasssvn ymywyqqkpg qaprpwiylt snratgvpar 61 fsgsgsgtdy tltisslepe dfavyycqqw ssnpytfgqg tkleik

[0140] Nucleic Acid Sequence Encoding the Hu4B9-82 Kappa Chain Variable Region (SEQ ID NO: 43)

TABLE-US-00025 1 gaaatcgtac ttactcagag ccctgccaca ttgtcattgt cacccgggga acgcgccaca 61 ctgtcgtgcc gggcttcatc gagcgtgaac tacatgtatt ggtatcaaca gaaaccaggc 121 caagcaccgc gaccttggat ctacttgacg agcaatcgag ccacgggtat ccccgcgagg 181 ttctccggtt cggggtcggg aactgattac acactgacaa tttcctcgct ggagcccgag 241 gacttcgcgg tgtactattg tcagcagtgg tcatccaacc cgtacacgtt tggacagggg 301 acgaagctcg agatcaag

[0141] Protein Sequence Defining the Hu4B9-82 Kappa Chain Variable Region (SEQ ID NO: 44)

TABLE-US-00026 1 eivltqspat lslspgerat lscrasssvn ymywyqqkpg qaprpwiylt snratgipar 61 fsgsgsgtdy tltisslepe dfavyycqqw ssnpytfgqg tkleik

[0142] Nucleic Acid Sequence Encoding the Hu4B9-83 Kappa Chain Variable Region (SEQ ID NO: 45)

TABLE-US-00027 1 gaaatcgtac ttactcagag ccctgccaca ttgtcattgt cacccgggga acgcgccaca 61 ctgtcgtgcc gggcttcatc gagcgtgaac tacatgtatt ggtatcaaca gaaaccaggc 121 caagcaccgc gaccttggat ctacttgacg agcaatcgag ccacgggtat ccccgcgagg 181 ttctccggtt cggggtcggg aactgatttc acactgacaa tttcctcgct ggagcccgag 241 gacttcgcgg tgtactattg tcagcagtgg tcatccaacc cgtacacgtt tggacagggg 301 acgaagctcg agatcaag

[0143] Protein Sequence Defining the Hu4B9-83 Kappa Chain Variable Region (SEQ ID NO: 46)

TABLE-US-00028 1 eivltqspat lslspgerat lscrasssvn ymywyqqkpg qaprpwiylt snratgipar 61 fsgsgsgtdf tltisslepe dfavyycqqw ssnpytfgqg tkleik

[0144] The amino acid sequences defining the immunoglobulin heavy chain variable regions for the antibodies produced in Example 9 are aligned in FIG. 8. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR₁, CDR₂, and CDR₃ (Kabat definition) are identified by boxes (See FIG. 9).

[0145] The amino acid sequences defining the immunoglobulin light chain variable regions for the antibodies in Example 9 are aligned in FIG. 10. Amino terminal signal peptide sequences (for proper expression/secretion) are not shown. CDR₁, CDR₂ and CDR₃ (Kabat definition) are identified by boxes (See FIG. 11).

[0146] Table 7 is a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.

TABLE-US-00029 TABLE 7 SEQ. ID NO. Nucleic Acid or Protein 34 Hu4B9-65 Heavy Chain Variable Region--nucleic acid 35 Hu4B9-65 Heavy Chain Variable Region--protein 5 Hu4B9-65 Heavy Chain CDR₁ (Kabat definition) 6 Hu4B9-65 Heavy Chain CDR₂ (Kabat definition) 11 Hu4B9-65 Heavy Chain CDR₃ (IGMT definition) 36 Hu4B9-82, -83 Heavy Chain Variable Region--nucleic acid 37 Hu4B9-82, -83 Heavy Chain Variable Region--protein 5 Hu4B9-82, -83 Heavy Chain CDR₁ (Kabat definition) 38 Hu4B9-82, -83 Heavy Chain CDR₂ (Kabat definition) 11 Hu4B9-82, -83 Heavy Chain CDR₃ (IGMT definition) 39 Hu4B9-65 Light (kappa) Chain Variable Region--nucleic acid 40 Hu4B9-65 Light (kappa) Chain Variable Region--protein 41 Hu4B9-65 Light (kappa) Chain CDR₁ (Kabat definition) 42 Hu4B9-65 Light (kappa) Chain CDR₂ (Kabat definition) 14 Hu4B9-65 Light (kappa) Chain CDR₃ (Kabat definition) 43 Hu4B9-82 Light (kappa) Chain Variable Region--nucleic acid 44 Hu4B9-82 Light (kappa) Chain Variable Region--protein 41 Hu4B9-82 Light (kappa) Chain CDR₁ (Kabat definition) 42 Hu4B9-82 Light (kappa) Chain CDR₂ (Kabat definition) 14 Hu4B9-82 Light (kappa) Chain CDR₃ (Kabat definition) 45 Hu4B9-83 Light (kappa) Chain Variable Region--nucleic acid 46 Hu4B9-83 Light (kappa) Chain Variable Region--protein 41 Hu4B9-83 Light (kappa) Chain CDR₁ (Kabat definition) 42 Hu4B9-83 Light (kappa) Chain CDR₂ (Kabat definition) 14 Hu4B9-83 Light (kappa) Chain CDR₃ (Kabat definition)

[0147] Murine and humanized monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 8.

TABLE-US-00030 TABLE 8 CDR1 CDR2 CDR3 Kabat 4B9 SYWMH AIYPGNSDTDYSQ FDY (SEQ ID KFKG NO: 5) (SEQ ID NO: 6) Hu4B9-65 SYWMH AIYPGNSDTDYSQ FDY (SEQ ID KFKG NO: 5) (SEQ ID NO: 6) Hu4B9-82, -83 SYWMH AIYPGNSDTDYSQ FDY (SEQ ID KFQG NO: 5) (SEQ ID NO: 38) CHOTHIA 4B9 GYTFTSY YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 7) Hu4B9-65 GYTFTSY YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 7) Hu4B9-82, -83 GYTFSSY YPGNSD FDY (SEQ ID (SEQ ID NO: 8) NO: 47) IMGT 4B9 GYTFTSYW IYPGNSDT SKFDY (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: 9) NO: 11) Hu4B9-65 GYTFTSYW IYPGNSDT SKFDY (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: 9) NO: 11) Hu4B9-82, -83 GYTFSSYW IYPGNSDT SKFDY (SEQ ID (SEQ ID NO: 10) (SEQ ID NO: 48) NO: 11)

[0148] Murine and humanized monoclonal antibody Kappa light chain CDR sequences (Kabat, Chothia, and IMGT definitions) are shown in Table 9.

TABLE-US-00031 TABLE 9 CDR1 CDR2 CDR3 Kabat/Chothia 4B9 SASSSVNYMY LTSNLAS QQWSSNPYT (SEQ ID NO: 12) (SEQ ID (SEQ ID NO: 13) NO: 14) Hu4B9- RASSSVNYMY LTSNRAT QQWSSNPYT 65 (SEQ ID NO: 41) (SEQ ID (SEQ ID NO: 42) NO: 14) Hu4B9- RASSSVNYMY LTSNRAT QQWSSNPYT 82 (SEQ ID NO: 41) (SEQ ID (SEQ ID NO: 42) NO: 14) Hu4B9- RASSSVNYMY LTSNRAT QQWSSNPYT 83 (SEQ ID NO: 41) (SEQ ID (SEQ ID NO: 42) NO: 14) IGMT 4B9 SSVNY QQWSSNPYT (SEQ ID NO: 15) LTS (SEQ ID NO: 14) Hu4B9- SSVNY QQWSSNPYT 65 (SEQ ID NO: 15) LTS (SEQ ID NO: 14) Hu4B9- SSVNY QQWSSNPYT 82 (SEQ ID NO: 15) LTS (SEQ ID NO: 14) Hu4B9- SSVNY QQWSSNPYT 83 (SEQ ID NO: 15) LTS (SEQ ID NO: 14)

[0149] To create the complete humanized heavy or kappa chain antibody sequences, each variable sequence above is combined with its respective human constant region. For example, a complete heavy chain comprises a heavy variable sequence followed by a human IgG1 heavy chain constant sequence. A complete kappa chain comprises a kappa variable sequence followed by the human kappa light chain constant sequence.

[0150] Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain Constant Region (SEQ ID NO: 49)

TABLE-US-00032 1 gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 61 ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 121 tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 181 ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 241 tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 301 aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 361 cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 421 gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 481 tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 541 agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 601 gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 661 aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 721 atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 781 gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 841 ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 901 cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 961 cagaagtcac tgagcctgag cccagggaag

[0151] Protein Sequence Defining the Human IgG1 Heavy Chain Constant Region (SEQ ID NO: 50)

TABLE-US-00033 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 mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301 qqgnvfscsv mhealhnhyt qkslslspgk

[0152] Nucleic Acid Sequence Encoding the Human Kappa Light Chain Constant Region (SEQ ID NO: 51)

TABLE-US-00034 1 cgcacagttg ctgcccccag cgtgttcatt ttcccaccta gcgatgagca gctgaaaagc 61 ggtactgcct ctgtcgtatg cttgctcaac aacttttacc cacgtgaggc taaggtgcag 121 tggaaagtgg ataatgcact tcaatctgga aacagtcaag agtccgtgac agaacaggac 181 agcaaagact caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa 241 aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag 301 tccttcaata ggggcgaatg t

[0153] Protein Sequence Defining the Human Kappa Light Chain Constant Region (SEQ ID NO: 52)

TABLE-US-00035 1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd 61 skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

[0154] The following sequences represent the actual or contemplated full length heavy and light chain sequences (i.e., containing both the variable and constant regions sequences) for each antibody described in this Example. Signal sequences for proper secretion of the antibodies are also included at the 5' end of the DNA sequences or the amino terminal end of the protein sequences. It is also contemplated herein that the variable region sequences can be ligated to other constant region sequences to produce active full length IgG heavy and light chains.

[0155] Nucleic Acid Sequence Encoding the Full Length Humanized Hu4B9-65 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region) (SEQ ID NO: 53)

TABLE-US-00036 1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61 aggtgccaag tgcagctcgt ccaatcggga gccgaagtga agaagcctgg ttcctcggta 121 aaagtaagct gtaaggcgtc cggttacacg tttacctcat attggatgca ctgggtcaga 181 caggcacccg gacagggact cgagtggatg ggagcgatct acccgggcaa ttcggacact 241 gattacagcc agaaattcaa ggggagggtc acgatcacgg cagatgagag cacatcaaca 301 gcctatatgg agctgtcgtc gcttcggagc gaggacacgg cggtctacta ctgctccaaa 361 ttcgactatt gggggcaggg gaccttggtg accgtgtcat ccgcctcaac aaaaggacca 421 agtgtgttcc cactcgcccc tagcagcaag agtacatccg ggggcactgc agcactcggc 481 tgcctcgtca aggattattt tccagagcca gtaaccgtga gctggaacag tggagcactc 541 acttctggtg tccatacttt tcctgctgtc ctgcaaagct ctggcctgta ctcactcagc 601 tccgtcgtga ccgtgccatc ttcatctctg ggcactcaga cctacatctg taatgtaaac 661 cacaagccta gcaatactaa ggtcgataag cgggtggaac ccaagagctg cgacaagact 721 cacacttgtc ccccatgccc tgcccctgaa cttctgggcg gtcccagcgt ctttttgttc 781 ccaccaaagc ctaaagatac tctgatgata agtagaacac ccgaggtgac atgtgttgtt 841 gtagacgttt cccacgagga cccagaggtt aagttcaact ggtacgttga tggagtcgaa 901 gtacataatg ctaagaccaa gcctagagag gagcagtata atagtacata ccgtgtagtc 961 agtgttctca cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg 1021 tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa ggggcagcct 1081 cgtgaaccac aggtgtacac tctgccaccc agtagagagg aaatgacaaa gaaccaagtc 1141 tcattgacct gcctggtgaa aggcttctac cccagcgaca tcgccgttga gtgggagagt 1201 aacggtcagc ctgagaacaa ttacaagaca acccccccag tgctggatag tgacgggtct 1261 ttctttctgt acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc 1321 agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc actgagcctg 1381 agcccaggga ag

[0156] Protein Sequence Defining the Full Length Humanized Hu4B9-65 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region) (SEQ ID NO: 54)

TABLE-US-00037 1 mdmrvpaqll gllllwlrga rcqvqlvqsg aevkkpgssv kvsckasgyt ftsywmhwvr 61 qapgqglewm gaiypgnsdt dysqkfkgrv titadestst aymelsslrs edtavyycsk 121 fdywgqgtlv tvssastkgp svfplapssk stsggtaalg clvkdyfpep vtvswnsgal 181 tsgvhtfpav lqssglysls svvtvpsssl gtqtyicnvn hkpsntkvdk rvepkscdkt 241 htcppcpape llggpsvflf ppkpkdtlmi srtpevtcvv vdvshedpev kfnwyvdgve 301 vhnaktkpre eqynstyrvv svltvlhqdw lngkeykckv snkalpapie ktiskakgqp 361 repqvytlpp sreemtknqv sltclvkgfy psdiavewes ngqpennykt tppvldsdgs 421 fflyskltvd ksrwqqgnvf scsvmhealh nhytqkslsl spgk

[0157] Nucleic Acid Sequence Encoding the Full Length Humanized Hu4B9-82, -83 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region) (SEQ ID NO: 55)

TABLE-US-00038 1 atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 61 aggtgccaag tgcagctcgt ccaatcggga gccgaagtga agaagcctgg ttcctcggta 121 aaagtaagct gtaaggcgtc cggttacacg ttttcctcat attggatgca ctgggtcaga 181 caggcacccg gacagggact cgagtggatg ggagcgatct acccgggcaa ttcggacact 241 gattacagcc agaaattcca ggggagggtc acgatcacgg cagatgagag cacatcaaca 301 gcctatatgg agctgtcgtc gcttcggagc gaggacacgg cggtctacta ctgctccaaa 361 ttcgactatt gggggcaggg gaccttggtg accgtgtcat ccgcctcaac aaaaggacca 421 agtgtgttcc cactcgcccc tagcagcaag agtacatccg ggggcactgc agcactcggc 481 tgcctcgtca aggattattt tccagagcca gtaaccgtga gctggaacag tggagcactc 541 acttctggtg tccatacttt tcctgctgtc ctgcaaagct ctggcctgta ctcactcagc 601 tccgtcgtga ccgtgccatc ttcatctctg ggcactcaga cctacatctg taatgtaaac 661 cacaagccta gcaatactaa ggtcgataag cgggtggaac ccaagagctg cgacaagact 721 cacacttgtc ccccatgccc tgcccctgaa cttctgggcg gtcccagcgt ctttttgttc 781 ccaccaaagc ctaaagatac tctgatgata agtagaacac ccgaggtgac atgtgttgtt 841 gtagacgttt cccacgagga cccagaggtt aagttcaact ggtacgttga tggagtcgaa 901 gtacataatg ctaagaccaa gcctagagag gagcagtata atagtacata ccgtgtagtc 961 agtgttctca cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg 1021 tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa ggggcagcct 1081 cgtgaaccac aggtgtacac tctgccaccc agtagagagg aaatgacaaa gaaccaagtc 1141 tcattgacct gcctggtgaa aggcttctac cccagcgaca tcgccgttga gtgggagagt 1201 aacggtcagc ctgagaacaa ttacaagaca acccccccag tgctggatag tgacgggtct 1261 ttctttctgt acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc 1321 agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc actgagcctg 1381 agcccaggga ag

[0158] Protein Sequence Defining the Full Length Humanized Hu4B9-82, -83 Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1 Constant Region) (SEQ ID NO: 56)

TABLE-US-00039 1 mdmrvpaqll gllllwlrga rcqvqlvqsg aevkkpgssv kvsckasgyt fssywmhwvr 61 qapgqglewm gaiypgnsdt dysqkfqgrv titadestst aymelsslrs edtavyycsk 121 fdywgqgtlv tvssastkgp svfplapssk stsggtaalg clvkdyfpep vtvswnsgal 181 tsgvhtfpav lqssglysls svvtvpsssl gtqtyicnvn hkpsntkvdk rvepkscdkt 241 htcppcpape llggpsvflf ppkpkdtlmi srtpevtcvv vdvshedpev kfnwyvdgve 301 vhnaktkpre eqynstyrvv svltvlhqdw lngkeykckv snkalpapie ktiskakgqp 361 repqvytlpp sreemtknqv sltclvkgfy psdiavewes ngqpennykt tppvldsdgs 421 fflyskltvd ksrwqqgnvf scsvmhealh nhytqkslsl spgk

[0159] Nucleic Acid Sequence Encoding the Full Length Humanized Hu4B9-65 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 57)

TABLE-US-00040 1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61 cgttgcgaaa ttgtgctgac ccagagcccg gcgaccctga gcctgagccc gggcgaacgc 121 gcgaccctga gctgccgcgc gagcagcagc gtgaactata tgtattggta tcagcagaaa 181 ccgggccagg cgccgcgccc gtggatttat ctgaccagca accgcgcgac cggcgtgccg 241 gcgcgcttta gcggcagcgg cagcggcacc gattataccc tgaccattag cagcctggaa 301 ccggaagatt ttgcggtgta ttattgccag cagtggagca gcaacccgta tacctttggc 361 cagggcacca aactggaaat taaacgcaca gttgctgccc ccagcgtgtt cattttccca 421 cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct caacaacttt 481 tacccacgtg aggctaaggt gcagtggaaa gtggataatg cacttcaatc tggaaacagt 541 caagagtccg tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 601 actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 661 ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt

[0160] Protein Sequence Defining the Full Length Humanized Hu4B9-65 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 58)

TABLE-US-00041 1 mdmrvpaqll gllllwlrga rceivltqsp atlslspger atlscrasss vnymywyqqk 61 pgqaprpwiy ltsnratgvp arfsgsgsgt dytltissle pedfavyycq qwssnpytfg 121 qgtkleikrt vaapsvfifp psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns 181 qesvteqdsk dstyslsstl tlskadyekh kvyacevthq glsspvtksf nrgec

[0161] Nucleic Acid Sequence Encoding the Full Length Humanized Hu4B9-82 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 59)

TABLE-US-00042 1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61 cgttgcgaaa tcgtacttac tcagagccct gccacattgt cattgtcacc cggggaacgc 121 gccacactgt cgtgccgggc ttcatcgagc gtgaactaca tgtattggta tcaacagaaa 181 ccaggccaag caccgcgacc ttggatctac ttgacgagca atcgagccac gggtatcccc 241 gcgaggttct ccggttcggg gtcgggaact gattacacac tgacaatttc ctcgctggag 301 cccgaggact tcgcggtgta ctattgtcag cagtggtcat ccaacccgta cacgtttgga 361 caggggacga agctcgagat caagcgcaca gttgctgccc ccagcgtgtt cattttccca 421 cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct caacaacttt 481 tacccacgtg aggctaaggt gcagtggaaa gtggataatg cacttcaatc tggaaacagt 541 caagagtccg tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 601 actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 661 ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt

[0162] Protein Sequence Defining the Full Length Humanized Hu4B9-82 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 60)

TABLE-US-00043 1 mdmrvpaqll gllllwlrga rceivltqsp atlslspger atlscrasss vnymywyqqk 61 pgqaprpwiy ltsnratgip arfsgsgsgt dytltissle pedfavyycq qwssnpytfg 121 qgtkleikrt vaapsvfifp psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns 181 qesvteqdsk dstyslsstl tlskadyekh kvyacevthq glsspvtksf nrgec

[0163] Nucleic Acid Sequence Encoding the Full Length Humanized Hu4B9-83 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 61)

TABLE-US-00044 1 atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 61 cgttgcgaaa tcgtacttac tcagagccct gccacattgt cattgtcacc cggggaacgc 121 gccacactgt cgtgccgggc ttcatcgagc gtgaactaca tgtattggta tcaacagaaa 181 ccaggccaag caccgcgacc ttggatctac ttgacgagca atcgagccac gggtatcccc 241 gcgaggttct ccggttcggg gtcgggaact gatttcacac tgacaatttc ctcgctggag 301 cccgaggact tcgcggtgta ctattgtcag cagtggtcat ccaacccgta cacgtttgga 361 caggggacga agctcgagat caagcgcaca gttgctgccc ccagcgtgtt cattttccca 421 cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct caacaacttt 481 tacccacgtg aggctaaggt gcagtggaaa gtggataatg cacttcaatc tggaaacagt 541 caagagtccg tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 601 actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 661 ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt

[0164] Protein Sequence Defining the Full Length Humanized Hu4B9-83 Light Chain (Humanized Kappa Chain Variable Region and Human Constant Region) (SEQ ID NO: 62)

TABLE-US-00045 1 mdmrvpaqll gllllwlrga rceivltqsp atlslspger atlscrasss vnymywyqqk 61 pgqaprpwiy ltsnratgip arfsgsgsgt dftltissle pedfavyycq qwssnpytfg 121 qgtkleikrt vaapsvfifp psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns 181 qesvteqdsk dstyslsstl tlskadyekh kvyacevthq glsspvtksf nrgec

[0165] For convenience, Table 10 provides a concordance chart showing the SEQ ID NO. of each sequence discussed in this Example.

TABLE-US-00046 TABLE 10 SEQ ID NO. Nucleic Acid or Protein 49 Human IgG1 constant--nucleic acid 50 Human IgG1 constant--protein 51 Human Kappa constant--nucleic acid 52 Human Kappa constant--protein 53 Humanized Hu4B9-65 Heavy Human Variable + Human IgG1 constant--nucleic acid 54 Humanized Hu4B9-65 Heavy Human Variable + Human IgG1 constant--protein 55 Humanized Hu4B9-82, -83 Heavy Human Variable + Human IgG1 constant--nucleic acid 56 Humanized Hu4B9-82,-83 Heavy Human Variable + Human IgG1 constant--protein 57 Humanized Hu4B9-65 Human Variable + Human Kappa constant--nucleic acid 58 Humanized Hu4B9-65 Human Variable + Human Kappa constant--protein 59 Humanized Hu4B9-82 Human Variable + Human Kappa constant--nucleic acid 60 Humanized Hu4B9-82 Human Variable + Human Kappa constant--protein 61 Humanized Hu4B9-83 Human Variable + Human Kappa constant--nucleic acid 62 Humanized Hu4B9-83 Human Variable + Human Kappa constant--protein

[0166] Table 11 below shows antibodies containing each of the possible combinations of the full-length humanized immunoglobulin heavy and light chains.

TABLE-US-00047 TABLE 11 Antibody Name Light Chain Heavy Chain Hu4B9-65 Hu4B9-65 Kappa Hu4B9-65 Heavy (SEQ ID NO: 58) (SEQ ID NO: 54) Hu4B9-84 Hu4B9-65 Kappa Hu4B9-82, -83 Heavy (SEQ ID NO: 58) (SEQ ID NO: 56) Hu4B9-85 Hu4B9-82 Kappa Hu4B9-65 Heavy (SEQ ID NO: 60) (SEQ ID NO: 54) Hu4B9-82 Hu4B9-82 Kappa Hu4B9-82, -83 Heavy (SEQ ID NO: 60) (SEQ ID NO: 56) Hu4B9-86 Hu4B9-83 Kappa Hu4B9-65 Heavy (SEQ ID NO: 62) (SEQ ID NO: 54) Hu4B9-83 Hu4B9-83 Kappa Hu4B9-82, -83 Heavy (SEQ ID NO: 62) (SEQ ID NO: 56)

[0167] Three of the possible antibody constructs containing the full length immunoglobulin heavy and light chains containing humanized variable regions are designated below:

[0168] Hu4B9-65=Humanized Hu4B9-65 Heavy Chain Variable Region and Human IgG1 Constant Region (SEQ ID NO: 54) plus Hu4B9-65 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 58)

[0169] Hu4B9-82=Humanized Hu4B9-82, -83 Heavy Chain Variable Region and Human IgG1 Constant Region (SEQ ID NO: 56) plus Hu4B9-82 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 60)

[0170] Hu4B9-83=Humanized Hu4B9-82, -83 Heavy Chain Variable Region and Human IgG1 Constant Region (SEQ ID NO: 56) plus Hu4B9-83 Light Chain Variable Region and Human Kappa Constant Region (SEQ ID NO: 62)

[0171] B. Binding Affinities of Humanized Anti-FGFR2Monoclonal Antibodies

[0172] The binding affinities and kinetics of interaction of monoclonal antibodies produced in Example 9 against monomeric recombinant human FGFR2 beta Mb (rhFGFR2β-IIIb-cleaved) were measured by surface plasmon resonance using a Biacore T100 (Biacore (GE Healthcare), Piscataway, N.J.) instrument.

[0173] Goat anti-human IgG Fc (Jackson ImmunoResearch, Catalog No. 109-005-098) was immobilized on carboxymethylated dextran CM4 sensor chips (Biacore) by amine coupling (Biacore) using a standard coupling protocol according to the vendor's instructions. The analyses were performed at 25° C. and 37° C. using PBS (Invitrogen) containing 0.05% surfactant P20 (Biacore) as running buffer.

[0174] Purified antibodies were captured in individual flow cells at a flow rate of 10 μl/minute. Injection time was varied for each antibody to yield an R_max between 30 and 90 RU. Buffer or rhFGFR2β-IIIb-cleaved diluted in running buffer was injected sequentially over a reference surface (no antibody captured) and the active surface (antibody to be tested) for 240 seconds at 60 μl/minute. The dissociation phase was monitored for up to 900 seconds. The surface was then regenerated with two 60 second injections of glycine pH 2.25 (made from glycine pH 2.0 (Biacore) and pH 2.5 (Biacore)) at 30 μl/minute. Experiments were conducted using concentrations of rhFGFR2β-IIIb-cleaved between 20 and 1.25 nM (a two-fold serial dilution).

[0175] Kinetic parameters were determined using the kinetic function of the BlAevaluation software (Biacore) with double reference subtraction. Kinetic parameters for each antibody, k_a (association rate constant), k_d (dissociation rate constant) and K_D (equilibrium dissociation constant) were determined. The kinetic values of certain purified monoclonal antibodies (i.e., Hu4B9-65, Hu4B9-82, and Hu4B9-83) on rhFGFR2β-IIIb-cleaved at 25° C. are summarized in Table 12.

TABLE-US-00048 TABLE 12 ka KD Antibody (1/Ms) kd (1/s) (M) n hu4B9-65 2.4E+05 6.5E-05 2.6E-10 4 hu4B9-82 1.9E+05 9.4E-05 4.9E-10 2 hu4B9-83 2.6E+05 8.9E-05 3.5E-10 3

[0176] The results in Table 12 demonstrate the purified antibodies have affinities ranging from about 260 pM to about 490 pM when tested at 25° C.

[0177] The kinetic values of certain purified monoclonal antibodies (i.e., Hu4B9-65, Hu4B9-82, and Hu4B9-83) on rhFGFR2β-IIIb-cleaved at 37° C. are summarized in Table 13.

TABLE-US-00049 TABLE 13 ka KD Antibody (1/Ms) kd (1/s) (M) n hu4B9-65 3.7E+05 2.8E-04 8.9E-10 7 hu4B9-82 4.0E+05 3.6E-04 9.3E-10 3 hu4B9-83 3.2E+05 2.9E-04 9.2E-10 3

[0178] The results in Table 13 demonstrate the purified antibodies have affinities ranging from about 890 pM to about 930 pM when tested at 37° C.

Example 10

Anti-Proliferative Activity of Humanized Anti-FGFR2Monoclonal Antibodies

[0179] The potency of humanized anti-FGFR2 antibodies was assessed in a cell-based proliferation assay. FDCP-1 cells expressing FGFR2-IIIb were seeded in a 96-well plate in IL-3 free medium containing 8 ng/ml of FGF1 and 5 μg/ml of heparin. Serial dilutions of the antibodies were prepared and added to the plate. After two days of incubation, cell proliferation was examined by a MTT assay as described above in Example 1.

[0180] As shown in FIG. 12, humanized antibodies (Hu4B9-65, Hu4B9-82, and Hu4B9-83) demonstrated dose-dependent inhibition of FGF1-induced FDCP-FGFR2-IIIb cell proliferation. The average IC50s of the 4B9, Hu4B9-65, Hu4B9-82 and Hu4B9-83 from three independent experiments are 1.4, 4.9, 5.7 and 4.7 nM, respectively.

INCORPORATION BY REFERENCE

[0181] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0182] 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 the range of equivalency of the claims are intended to be embraced therein.

Sequence CWU 1

1

621336DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 1gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 60tcctgcaaga cttctggcta cacatttacc agctactgga tgcactgggt aaaacagagg 120cctggacagg gtctggaatg gataggggct atttatcctg gaaatagtga tactgactac 180agccagaagt tcaagggcaa ggccacactg actgcagtca catccgccac cactgcctac 240atggaactca gcagcctgac aaatgaggac tctgcggtct attactgttc aaagtttgac 300tactggggcc aaggcaccac tctcacagtc tcctca 3362112PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 2Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 100 105 110 3318DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 3caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 60atgacctgca gtgccagctc aagtgtaaat tacatgtact ggtaccagca gaagccaaga 120tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca gggggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240gatgctgcca cttattactg ccagcagtgg agtagtaacc cgtacacgtt cggagggggg 300accaagctgg aaataaaa 3184106PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 4Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 55PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 5Ser Tyr Trp Met His 1 5 617PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 6Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly 77PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 7Gly Tyr Thr Phe Thr Ser Tyr 1 5 86PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 8Tyr Pro Gly Asn Ser Asp 1 5 98PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 9Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 108PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 10Ile Tyr Pro Gly Asn Ser Asp Thr 1 5 115PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 11Ser Lys Phe Asp Tyr 1 5 1210PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 12Ser Ala Ser Ser Ser Val Asn Tyr Met Tyr 1 5 10 137PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 13Leu Thr Ser Asn Leu Ala Ser 1 5 149PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 14Gln Gln Trp Ser Ser Asn Pro Tyr Thr 1 5 155PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 15Ser Ser Val Asn Tyr 1 5 16972DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 16gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag ccagaccgtc 240acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960tctcctggta aa 97217324PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 17Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 1 5 10 15 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 100 105 110 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 115 120 125 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 130 135 140 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 145 150 155 160 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 165 170 175 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 180 185 190 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215 220 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 225 230 235 240 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 245 250 255 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 260 265 270 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 275 280 285 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 290 295 300 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 305 310 315 320 Ser Pro Gly Lys 18321DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 18cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccagagacat caatgtcaag 120tggaagattg atggcagtga acgacaaaat ggtgtcctga acagttggac tgatcaggac 180agcaaagaca gcacctacag catgagcagc accctcacat tgaccaagga cgagtatgaa 240cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300agcttcaaca ggaatgagtg t 32119107PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 19Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 201365DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 20atggaatgta actggatact tccttttatt ctgtcggtaa cttcaggggt ctactcagag 60gttcagctcc agcagtctgg gactgtgctg gcaaggcctg gggcttcagt gaagatgtcc 120tgcaagactt ctggctacac atttaccagc tactggatgc actgggtaaa acagaggcct 180ggacagggtc tggaatggat aggggctatt tatcctggaa atagtgatac tgactacagc 240cagaagttca agggcaaggc cacactgact gcagtcacat ccgccaccac tgcctacatg 300gaactcagca gcctgacaaa tgaggactct gcggtctatt actgttcaaa gtttgactac 360tggggccaag gcaccactct cacagtctcc tcagccaaaa cgacaccccc atctgtctat 420ccactggccc ctggatctgc tgcccaaact aactccatgg tgaccctggg atgcctggtc 480aagggctatt tccctgagcc agtgacagtg acctggaact ctggatccct gtccagcggt 540gtgcacacct tcccagctgt cctgcagtct gacctctaca ctctgagcag ctcagtgact 600gtcccctcca gcacctggcc cagccagacc gtcacctgca acgttgccca cccggccagc 660agcaccaagg tggacaagaa aattgtgccc agggattgtg gttgtaagcc ttgcatatgt 720acagtcccag aagtatcatc tgtcttcatc ttccccccaa agcccaagga tgtgctcacc 780attactctga ctcctaaggt cacgtgtgtt gtggtagaca tcagcaagga tgatcccgag 840gtccagttca gctggtttgt agatgatgtg gaggtgcaca cagctcagac gcaaccccgg 900gaggagcagt tcaacagcac tttccgctca gtcagtgaac ttcccatcat gcaccaggac 960tggctcaatg gcaaggagtt caaatgcagg gtcaacagtg cagctttccc tgcccccatc 1020gagaaaacca tctccaaaac caaaggcaga ccgaaggctc cacaggtgta caccattcca 1080cctcccaagg agcagatggc caaggataaa gtcagtctga cctgcatgat aacagacttc 1140ttccctgaag acattactgt ggagtggcag tggaatgggc agccagcgga gaactacaag 1200aacactcagc ccatcatgga cacagatggc tcttacttcg tctacagcaa gctcaatgtg 1260cagaagagca actgggaggc aggaaatact ttcacctgct ctgtgttaca tgagggcctg 1320cacaaccacc atactgagaa gagcctctcc cactctcctg gtaaa 136521455PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 21Met Glu Cys Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Thr Ser Gly 1 5 10 15 Val Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Thr 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 115 120 125 Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 130 135 140 Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val 145 150 155 160 Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser 165 170 175 Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu 180 185 190 Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 195 200 205 Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val 210 215 220 Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys 225 230 235 240 Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 245 250 255 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 260 265 270 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 275 280 285 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 340 345 350 Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 355 360 365 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 370 375 380 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 385 390 395 400 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser 405 410 415 Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 420 425 430 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 435 440 445 Leu Ser His Ser Pro Gly Lys 450 455 22705DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 22atggattttc aagtgcagat tttcagcttc ctgctaatga gtgcctcagt cataatgtcc 60aggggacaaa ttgttctcac ccagtctcca gcactcatgt ctgcatctcc aggggagaag 120gtcaccatga cctgcagtgc cagctcaagt gtaaattaca tgtactggta ccagcagaag 180ccaagatcct cccccaaacc ctggatttat ctcacatcca acctggcttc tggagtccct 240gctcgcttca gtggcagggg gtctgggacc tcttactctc tcacaatcag cagcatggag 300gctgaagatg ctgccactta ttactgccag cagtggagta gtaacccgta cacgttcgga 360ggggggacca agctggaaat aaaacgggct gatgctgcac caactgtatc catcttccca 420ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 480taccccagag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggtgtc 540ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc 600acattgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag 660acatcaactt cacccattgt caagagcttc aacaggaatg agtgt 70523235PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 23Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Met Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Leu 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Leu Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Arg Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 130 135 140 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 165 170 175 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 195

200 205 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 210 215 220 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 2420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24acttgggctg gagtgatttg 202520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25aatcccatct gcacacttcc 202620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26caaaaacatg gctgagcaga 202720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27gaaacaggcc ccactttgta 202845DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 452922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29ctaatacgac tcactatagg gc 223021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30tatgcaaggc ttacaaccac a 213123DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31cgactgaggc acctccagat gtt 233217DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32gtaaaacgac ggccagt 173318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33caggaaacag ctatgacc 1834336DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 34caagtgcagc tcgtccaatc gggagccgaa gtgaagaagc ctggttcctc ggtaaaagta 60agctgtaagg cgtccggtta cacgtttacc tcatattgga tgcactgggt cagacaggca 120cccggacagg gactcgagtg gatgggagcg atctacccgg gcaattcgga cactgattac 180agccagaaat tcaaggggag ggtcacgatc acggcagatg agagcacatc aacagcctat 240atggagctgt cgtcgcttcg gagcgaggac acggcggtct actactgctc caaattcgac 300tattgggggc aggggacctt ggtgaccgtg tcatcc 33635112PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 35Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 36336DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 36caagtgcagc tcgtccaatc gggagccgaa gtgaagaagc ctggttcctc ggtaaaagta 60agctgtaagg cgtccggtta cacgttttcc tcatattgga tgcactgggt cagacaggca 120cccggacagg gactcgagtg gatgggagcg atctacccgg gcaattcgga cactgattac 180agccagaaat tccaggggag ggtcacgatc acggcagatg agagcacatc aacagcctat 240atggagctgt cgtcgcttcg gagcgaggac acggcggtct actactgctc caaattcgac 300tattgggggc aggggacctt ggtgaccgtg tcatcc 33637112PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 37Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 3817PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 38Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asp Tyr Ser Gln Lys Phe Gln 1 5 10 15 Gly 39318DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 39gaaattgtgc tgacccagag cccggcgacc ctgagcctga gcccgggcga acgcgcgacc 60ctgagctgcc gcgcgagcag cagcgtgaac tatatgtatt ggtatcagca gaaaccgggc 120caggcgccgc gcccgtggat ttatctgacc agcaaccgcg cgaccggcgt gccggcgcgc 180tttagcggca gcggcagcgg caccgattat accctgacca ttagcagcct ggaaccggaa 240gattttgcgg tgtattattg ccagcagtgg agcagcaacc cgtatacctt tggccagggc 300accaaactgg aaattaaa 31840106PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 40Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 4110PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 41Arg Ala Ser Ser Ser Val Asn Tyr Met Tyr 1 5 10 427PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 42Leu Thr Ser Asn Arg Ala Thr 1 5 43318DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 43gaaatcgtac ttactcagag ccctgccaca ttgtcattgt cacccgggga acgcgccaca 60ctgtcgtgcc gggcttcatc gagcgtgaac tacatgtatt ggtatcaaca gaaaccaggc 120caagcaccgc gaccttggat ctacttgacg agcaatcgag ccacgggtat ccccgcgagg 180ttctccggtt cggggtcggg aactgattac acactgacaa tttcctcgct ggagcccgag 240gacttcgcgg tgtactattg tcagcagtgg tcatccaacc cgtacacgtt tggacagggg 300acgaagctcg agatcaag 31844106PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 44Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 45318DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 45gaaatcgtac ttactcagag ccctgccaca ttgtcattgt cacccgggga acgcgccaca 60ctgtcgtgcc gggcttcatc gagcgtgaac tacatgtatt ggtatcaaca gaaaccaggc 120caagcaccgc gaccttggat ctacttgacg agcaatcgag ccacgggtat ccccgcgagg 180ttctccggtt cggggtcggg aactgatttc acactgacaa tttcctcgct ggagcccgag 240gacttcgcgg tgtactattg tcagcagtgg tcatccaacc cgtacacgtt tggacagggg 300acgaagctcg agatcaag 31846106PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 46Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 477PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 47Gly Tyr Thr Phe Ser Ser Tyr 1 5 488PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 48Gly Tyr Thr Phe Ser Ser Tyr Trp 1 5 49990DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 49gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 60ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 120tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 180ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 240tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 300aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 360cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 420gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 480tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 540agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 600gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 660aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 720atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 780gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 840ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 900cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 960cagaagtcac tgagcctgag cccagggaag 99050330PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 50Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 51321DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 51cgcacagttg ctgcccccag cgtgttcatt ttcccaccta gcgatgagca gctgaaaagc 60ggtactgcct ctgtcgtatg cttgctcaac aacttttacc cacgtgaggc taaggtgcag 120tggaaagtgg ataatgcact tcaatctgga aacagtcaag agtccgtgac agaacaggac 180agcaaagact caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa 240aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag 300tccttcaata ggggcgaatg t 32152107PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 52Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 531392DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 53atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 60aggtgccaag tgcagctcgt ccaatcggga gccgaagtga agaagcctgg ttcctcggta 120aaagtaagct gtaaggcgtc cggttacacg tttacctcat attggatgca ctgggtcaga 180caggcacccg gacagggact cgagtggatg ggagcgatct acccgggcaa ttcggacact 240gattacagcc agaaattcaa ggggagggtc acgatcacgg cagatgagag cacatcaaca 300gcctatatgg agctgtcgtc gcttcggagc gaggacacgg cggtctacta ctgctccaaa 360ttcgactatt gggggcaggg gaccttggtg accgtgtcat ccgcctcaac aaaaggacca 420agtgtgttcc cactcgcccc tagcagcaag agtacatccg ggggcactgc agcactcggc 480tgcctcgtca aggattattt tccagagcca gtaaccgtga gctggaacag tggagcactc 540acttctggtg tccatacttt tcctgctgtc ctgcaaagct ctggcctgta ctcactcagc 600tccgtcgtga ccgtgccatc ttcatctctg ggcactcaga cctacatctg taatgtaaac 660cacaagccta gcaatactaa ggtcgataag cgggtggaac ccaagagctg cgacaagact 720cacacttgtc ccccatgccc tgcccctgaa cttctgggcg gtcccagcgt ctttttgttc 780ccaccaaagc ctaaagatac tctgatgata agtagaacac ccgaggtgac atgtgttgtt 840gtagacgttt cccacgagga cccagaggtt aagttcaact ggtacgttga tggagtcgaa 900gtacataatg ctaagaccaa gcctagagag gagcagtata atagtacata ccgtgtagtc 960agtgttctca cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg 1020tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa ggggcagcct 1080cgtgaaccac aggtgtacac tctgccaccc agtagagagg aaatgacaaa gaaccaagtc 1140tcattgacct gcctggtgaa aggcttctac cccagcgaca tcgccgttga gtgggagagt 1200aacggtcagc ctgagaacaa ttacaagaca acccccccag tgctggatag tgacgggtct 1260ttctttctgt acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc 1320agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc actgagcctg 1380agcccaggga ag 139254464PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 54Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr 65

70 75 80 Asp Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu 85 90 95 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 225 230 235 240 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 245 250 255 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295 300 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 305 310 315 320 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 370 375 380 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 385 390 395 400 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410 415 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 420 425 430 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 551392DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 55atggacatga gagttcctgc tcagctgctc gggttgctgt tgctttggct ccggggtgct 60aggtgccaag tgcagctcgt ccaatcggga gccgaagtga agaagcctgg ttcctcggta 120aaagtaagct gtaaggcgtc cggttacacg ttttcctcat attggatgca ctgggtcaga 180caggcacccg gacagggact cgagtggatg ggagcgatct acccgggcaa ttcggacact 240gattacagcc agaaattcca ggggagggtc acgatcacgg cagatgagag cacatcaaca 300gcctatatgg agctgtcgtc gcttcggagc gaggacacgg cggtctacta ctgctccaaa 360ttcgactatt gggggcaggg gaccttggtg accgtgtcat ccgcctcaac aaaaggacca 420agtgtgttcc cactcgcccc tagcagcaag agtacatccg ggggcactgc agcactcggc 480tgcctcgtca aggattattt tccagagcca gtaaccgtga gctggaacag tggagcactc 540acttctggtg tccatacttt tcctgctgtc ctgcaaagct ctggcctgta ctcactcagc 600tccgtcgtga ccgtgccatc ttcatctctg ggcactcaga cctacatctg taatgtaaac 660cacaagccta gcaatactaa ggtcgataag cgggtggaac ccaagagctg cgacaagact 720cacacttgtc ccccatgccc tgcccctgaa cttctgggcg gtcccagcgt ctttttgttc 780ccaccaaagc ctaaagatac tctgatgata agtagaacac ccgaggtgac atgtgttgtt 840gtagacgttt cccacgagga cccagaggtt aagttcaact ggtacgttga tggagtcgaa 900gtacataatg ctaagaccaa gcctagagag gagcagtata atagtacata ccgtgtagtc 960agtgttctca cagtgctgca ccaagactgg ctcaacggca aagaatacaa atgcaaagtg 1020tccaacaaag cactcccagc ccctatcgag aagactatta gtaaggcaaa ggggcagcct 1080cgtgaaccac aggtgtacac tctgccaccc agtagagagg aaatgacaaa gaaccaagtc 1140tcattgacct gcctggtgaa aggcttctac cccagcgaca tcgccgttga gtgggagagt 1200aacggtcagc ctgagaacaa ttacaagaca acccccccag tgctggatag tgacgggtct 1260ttctttctgt acagtaagct gactgtggac aagtcccgct ggcagcaggg taacgtcttc 1320agctgttccg tgatgcacga ggcattgcac aaccactaca cccagaagtc actgagcctg 1380agcccaggga ag 139256464PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 56Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu 20 25 30 Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly 50 55 60 Gln Gly Leu Glu Trp Met Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr 65 70 75 80 Asp Tyr Ser Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu 85 90 95 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ser Lys Phe Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 130 135 140 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 210 215 220 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 225 230 235 240 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 245 250 255 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 275 280 285 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295 300 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 305 310 315 320 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 370 375 380 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 385 390 395 400 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410 415 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 420 425 430 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 57705DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 57atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 60cgttgcgaaa ttgtgctgac ccagagcccg gcgaccctga gcctgagccc gggcgaacgc 120gcgaccctga gctgccgcgc gagcagcagc gtgaactata tgtattggta tcagcagaaa 180ccgggccagg cgccgcgccc gtggatttat ctgaccagca accgcgcgac cggcgtgccg 240gcgcgcttta gcggcagcgg cagcggcacc gattataccc tgaccattag cagcctggaa 300ccggaagatt ttgcggtgta ttattgccag cagtggagca gcaacccgta tacctttggc 360cagggcacca aactggaaat taaacgcaca gttgctgccc ccagcgtgtt cattttccca 420cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct caacaacttt 480tacccacgtg aggctaaggt gcagtggaaa gtggataatg cacttcaatc tggaaacagt 540caagagtccg tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 600actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 660ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt 70558235PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 58Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Arg Pro Trp Ile Tyr Leu Thr Ser Asn Arg Ala Thr Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 59705DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 59atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 60cgttgcgaaa tcgtacttac tcagagccct gccacattgt cattgtcacc cggggaacgc 120gccacactgt cgtgccgggc ttcatcgagc gtgaactaca tgtattggta tcaacagaaa 180ccaggccaag caccgcgacc ttggatctac ttgacgagca atcgagccac gggtatcccc 240gcgaggttct ccggttcggg gtcgggaact gattacacac tgacaatttc ctcgctggag 300cccgaggact tcgcggtgta ctattgtcag cagtggtcat ccaacccgta cacgtttgga 360caggggacga agctcgagat caagcgcaca gttgctgccc ccagcgtgtt cattttccca 420cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct caacaacttt 480tacccacgtg aggctaaggt gcagtggaaa gtggataatg cacttcaatc tggaaacagt 540caagagtccg tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 600actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 660ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt 70560235PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 60Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Arg Pro Trp Ile Tyr Leu Thr Ser Asn Arg Ala Thr Gly Ile Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile 85 90 95 Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 61705DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 61atggacatga gggtgcccgc tcaactgctg gggctgctgc tgctgtggct gagaggagct 60cgttgcgaaa tcgtacttac tcagagccct gccacattgt cattgtcacc cggggaacgc 120gccacactgt cgtgccgggc ttcatcgagc gtgaactaca tgtattggta tcaacagaaa 180ccaggccaag caccgcgacc ttggatctac ttgacgagca atcgagccac gggtatcccc 240gcgaggttct ccggttcggg gtcgggaact gatttcacac tgacaatttc ctcgctggag 300cccgaggact tcgcggtgta ctattgtcag cagtggtcat ccaacccgta cacgtttgga 360caggggacga agctcgagat caagcgcaca gttgctgccc ccagcgtgtt cattttccca 420cctagcgatg agcagctgaa aagcggtact gcctctgtcg tatgcttgct caacaacttt 480tacccacgtg aggctaaggt gcagtggaaa gtggataatg cacttcaatc tggaaacagt 540caagagtccg tgacagaaca ggacagcaaa gactcaactt attcactctc ttccaccctg 600actctgtcca aggcagacta tgaaaaacac aaggtatacg cctgcgaggt tacacaccag 660ggtttgtcta gtcctgtcac caagtccttc aataggggcg aatgt 70562235PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 62Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Ile Val Leu Thr Gln Ser Pro Ala Thr 20 25 30 Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser 35 40 45 Ser Ser Val Asn Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Arg Pro Trp Ile Tyr Leu Thr Ser Asn Arg Ala Thr Gly Ile Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235

Claims

1-2. (canceled)

3. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable region comprising a CDR_H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 47, a CDR_H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR_H3 comprising the amino acid sequence of SEQ ID NO: 11.

4. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin light chain variable region comprising a CDR_L1 comprising the amino acid sequence of SEQ ID NO: 41, a CDR_L2 comprising the amino acid sequence of SEQ ID NO: 42, and a CDR_L3 comprising the amino acid sequence of SEQ ID NO: 14.

5. An expression vector containing the nucleic acid of claim 3.

6. An expression vector containing the nucleic acid of claim 4.

7. The expression vector of claim 6, further comprising the nucleic acid of claim 3.

8. A host cell comprising the expression vector of claim 5.

9. A host cell comprising the expression vector of claim 6.

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

11. The host cell of claim 9, further comprising the expression vector of claim 5.

12. A method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region, the method comprising: (a) growing the host cell of claim 8 or 9 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region; and (b) purifying the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region.

13. A method of producing an antibody that binds human FGFR2 or an antigen binding fragment of the antibody, the method comprising: (a) growing the host cell of claim 10 or 11 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain variable region and 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.

14. (canceled)

15. The isolated nucleic acid of claim 3, wherein the nucleotide sequence encodes an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37.

16. The isolated nucleic acid of claim 4, wherein the nucleotide sequence encodes an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 46.

17. An expression vector containing the nucleic acid of claim 15.

18. An expression vector containing the nucleic acid of claim 16.

19. The expression vector of claim 18, further comprising the nucleic acid of claim 15.

20. A host cell comprising the expression vector of claim 17.

21. A host cell comprising the expression vector of claim 18.

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

23. The host cell of claim 21, further comprising the expression vector of claim 17.

24. A method of producing a polypeptide comprising an immunoglobulin heavy chain variable region or an immunoglobulin light chain variable region, the method comprising: (a) growing the host cell of claim 20 or 21 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain variable region of the immunoglobulin light chain variable region; and (b) purifying the polypeptide comprising the immunoglobulin heavy chain variable region or the immunoglobulin light chain variable region.

25. A method of producing an antibody that binds human FGFR2 or an antigen binding fragment of the antibody, the method comprising: (a) growing the host cell of claim 22 or 23 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain variable region and 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.

26. (canceled)

27. The isolated nucleic acid of claim 3, wherein the nucleotide sequence encodes an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 56.

28. The isolated nucleic acid of claim 4, wherein the nucleotide sequence encodes an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 62.

29. An expression vector containing the nucleic acid of claim 27.

30. An expression vector containing the nucleic acid of claim 28.

31. The expression vector of claim 30, further comprising the nucleic acid of claim 27.

32. A host cell comprising the expression vector of claim 29.

33. A host cell comprising the expression vector of claim 30.

34. A host cell comprising the expression vector of claim 31.

35. The host cell of claim 33, further comprising the expression vector of claim 29.

36. A method of producing a polypeptide comprising an immunoglobulin heavy chain, the method comprising: (a) growing the host cell of claim 32 or 33 under conditions so that the host cell expresses the polypeptide comprising the immunoglobulin heavy chain or the immunoglobulin light chain; and (b) purifying the polypeptide comprising the immunoglobulin heavy chain or the immunoglobulin light chain.

37. A method of producing an antibody that binds human FGFR2 or an antigen binding fragment of the antibody, the method comprising: (a) growing the host cell of claim 34 of 35 under conditions so that the host cell expresses a polypeptide comprising the immunoglobulin heavy chain and the immunoglobulin light chain, thereby producing the antibody or antigen-binding fragment of the antibody; and (b) purifying the antibody or the antigen-binding fragment of the antibody.

38-43. (canceled)

44. The isolated nucleic acid of claim 3, wherein the immunoglobulin heavy chain variable region comprises a CDR_H1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR_H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR_H3 comprising the amino acid sequence of SEQ ID NO: 11.

45. The isolated nucleic acid of claim 3, wherein the immunoglobulin heavy chain variable region comprises a CDR_H1 comprising the amino acid sequence of SEQ ID NO: 47, a CDR_H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR_H3 comprising the amino acid sequence of SEQ ID NO: 11.

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