WO2025026333A1

WO2025026333A1 - Antibody against fgfr2b, method for preparing the same, and use thereof

Info

Publication number: WO2025026333A1
Application number: PCT/CN2024/108672
Authority: WO
Inventors: Mengxue YANG; Ruipeng ZHANG; Xiangju Gu; Meijuan HU
Original assignee: Laekna Therapeutics Shanghai Co Ltd
Current assignee: Laekna Therapeutics Shanghai Co Ltd
Priority date: 2023-07-31
Filing date: 2024-07-31
Publication date: 2025-02-06
Anticipated expiration: 2026-01-31
Also published as: TW202506735A

Abstract

Provided is antibodies and antigen-binding fragments thereof that specifically bind to FGFR2b and compositions comprising such antibodies or antigen-binding fragments thereof. In a specific aspect, the antibodies or antigen-binding fragments thereof that specifically bind to FGFR2b inhibit tumor cell proliferation and induce ADCC in various target cells. Methods for treating FGFR2b expressing cancer by administering the antibody or antigen-binding fragment thereof that specifically bind to FGFR2b are also provided.

Description

ANTIBODY AGAINST FGFR2B, METHOD FOR PREPARING THE SAME, AND USE THEREOF

FIELD OF THE INVENTION

This application generally relates to antibodies. More specifically, the application relates to monoclonal antibodies against fibroblast growth factor receptor 2 IIIb isoform (FGFR2IIIb or FGFR2b) , a method for preparing the same, and the use thereof.

BACKGROUND OF THE INVENTION

Fibroblast growth factor receptor 2 (FGFR2) is a transmembrane receptor tyrosine kinase that plays an important role in cell growth and differentiation. Alternative splicing of the FGFR2 gene gives rise to two major isoforms, FGFR2b and FGFR2c, which differ in their extracellular domains and ligand-binding specificities.

FGFR2b is predominantly expressed in epithelial cells and is essential for the development of epithelial tissues such as the lung, skin, and gastrointestinal tract. It is also involved in wound healing and tissue regeneration by promoting cell migration and proliferation. In contrast, FGFR2c is mainly expressed in mesenchymal cells and is involved in bone and cartilage development.

Aberrant activation of FGFR2b has been implicated in the pathogenesis of various types of cancer. In particular, FGFR2b overexpression or high mRNA level have been observed in gastric cancer, squamous lung carcinoma, ovarian cancer, intrahepatic cholangiocarcinoma, and triple-negative breast cancer. FGFR2b promotes tumor growth, angiogenesis, and metastasis through the activation of downstream signaling pathways such as the mitogen-activated protein kinase (MAPK) , phosphatidylinositol 3-kinase (PI3K) /AKT, and signal transducer and activator of transcription (STAT) pathways.

Herein, we describe an anti-FGFR2b antibody and its use in treating cancer. The antibody selectively binds to FGFR2b and inhibits its downstream signaling pathway, resulting in the suppression of tumor growth and metastasis. By targeting the FGFR2b isoform specifically, the antibody may avoid potential side effects associated with targeting other FGFR family members or isoforms. Furthermore, the antibody has demonstrated favorable pharmacokinetic, efficacy and safety profiles in animal models. The anti-FGFR2b antibody has significant potential as a therapeutic agent for cancer patients with FGFR2b (over) expression or mutations.

SUMMARY OF THE INVENTION

These and other objectives are provided for by the present invention which, in a broad sense, is directed to compounds, methods, compositions and articles of manufacture that provide antibodies with improved efficacy. The benefits provided by the present invention are broadly applicable in the field of antibody therapeutics and diagnostics and may be used in conjunction with antibodies that react with a variety of targets.

The present invention provides antibodies (e.g., monoclonal antibodies, preferably humanized monoclonal antibodies) and antigen-binding fragments thereof that bind to FGFR2b (e.g., human FGFR2b) . The anti-FGFR2b antibodies and antigen-binding fragments thereof can, for example, block the binding to FGFR2b to an FGFR2b ligand, such as FGF7, inhibit tumor cell proliferation, and induce ADCC in various target cells. Also provided are isolated nucleic acids (polynucleotides) , such as complementary DNA (cDNA) , encoding anti-FGFR2b antibodies and antigen-binding fragments thereof. Further provided are vectors (e.g., expression vectors) and cells (e.g., host cells) comprising nucleic acids (polynucleotides) encoding anti-FGFR2b antibodies and antigen-binding fragments thereof. It also provides methods of making anti-FGFR2b antibodies and antigen-binding fragments thereof. The invention further provides the methods for validating the function of the anti-FGFR2b antibodies in vitro and in vivo. The antibodies of the invention provide a potent agent for the treatment of multiple diseases comprising cancer.

In some aspects, the present invention provides antibody or antigen-binding fragment thereof that specifically binds to FGFR2b.

In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences selected from the group consisting of: SEQ ID NOs: 1-6, respectively; SEQ ID NOs: 9-14, respectively; SEQ ID NOs: 17-22, respectively; SEQ ID NOs: 25-30, respectively; SEQ ID NOs: 33-38, respectively; SEQ ID NOs: 41-46, respectively; SEQ ID NOs: 49-54, respectively; SEQ ID NOs: 57-62, respectively; SEQ ID NOs: 65-70, respectively; SEQ ID NOs: 73-78, respectively; SEQ ID NOs: 81-86, respectively; SEQ ID NOs: 89-94, respectively; SEQ ID NOs: 97-102, respectively; SEQ ID NOs: 105-110, respectively; SEQ ID NOs: 113-118, respectively; SEQ ID NOs: 121-126, respectively; SEQ ID NOs: 129-134, respectively; SEQ ID NOs: 137-142, respectively; SEQ ID NOs: 1, 242 and 3-6, respectively; SEQ ID NOs: 57-59, 243-244 and 62, respectively; and SEQ ID NOs: 81, 245, 83, 246-247 and 86, respectively.

In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(A) a heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, or 143;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, or 143; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, or 143; and

(B) a light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136 or 144;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136 or 144; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136 or 144.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of: SEQ ID NOs: 7 and 8, respectively; SEQ ID NOs: 15 and 16, respectively; SEQ ID NOs: 23 and 24, respectively; SEQ ID NOs: 31 and 32, respectively; SEQ ID NOs: 39 and 40, respectively; SEQ ID NOs: 47 and 48, respectively; SEQ ID NOs: 55 and 56, respectively; SEQ ID NOs: 63 and 64, respectively; SEQ ID NOs: 71 and 72, respectively; SEQ ID NOs: 79 and 80, respectively; SEQ ID NOs: 87 and 88, respectively; SEQ ID NOs: 95 and 96, respectively; SEQ ID NOs: 103 and 104, respectively; SEQ ID NOs: 111 and 112, respectively; SEQ ID NOs: 119 and 120, respectively; SEQ ID NOs: 127 and 128, respectively; SEQ ID NOs: 135 and 136, respectively; or SEQ ID NOs: 143 and 144, respectively.

(A) a heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 145, 146, 147, 151, 152, 153, 154, 155, 156, 157, 165, 166, 167, 168, 169, 170, 177, 178, 179, 180, 181, 182 or 183;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 145, 146, 147, 151, 152, 153, 154, 155, 156, 157, 165, 166, 167, 168, 169, 170, 177, 178, 179, 180, 181, 182 or 183; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 145, 146, 147, 151, 152, 153, 154, 155, 156, 157, 165, 166, 167, 168, 169, 170, 177, 178, 179, 180, 181, 182 or 183; and

(B) a light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 148, 149, 150, 158, 159, 160, 161, 162, 163, 164, 171, 172, 173, 174, 175, 176, 184, 185, 186, 187, 188, 189 or 190;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 148, 149, 150, 158, 159, 160, 161, 162, 163, 164, 171, 172, 173, 174, 175, 176, 184, 185, 186, 187, 188, 189 or 190; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 148, 149, 150, 158, 159, 160, 161, 162, 163, 164, 171, 172, 173, 174, 175, 176, 184, 185, 186, 187, 188, 189 or 190.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of: SEQ ID NOs: 145 and 148, respectively; SEQ ID NOs: 146 and 149, respectively; SEQ ID NOs: 147 and 150, respectively; SEQ ID NOs: 151 and 158, respectively; SEQ ID NOs: 152 and 159, respectively; SEQ ID NOs: 153 and 160, respectively; SEQ ID NOs: 154 and 161, respectively; SEQ ID NOs: 155 and 162, respectively; SEQ ID NOs: 156 and 163, respectively; SEQ ID NOs: 157 and 164, respectively; SEQ ID NOs: 165 and 171, respectively; SEQ ID NOs: 166 and 172, respectively; SEQ ID NOs: 167 and 173, respectively; SEQ ID NOs: 168 and 174, respectively; SEQ ID NOs: 169 and 175, respectively; SEQ ID NOs: 170 and 176, respectively; SEQ ID NOs: 177 and 184, respectively; SEQ ID NOs: 178 and 185, respectively; SEQ ID NOs: 179 and 186, respectively; SEQ ID NOs: 180 and 187, respectively; SEQ ID NOs: 181 and 188, respectively; SEQ ID NOs: 183 and 190, respectively.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, wherein the heavy chain constant region is selected from the group consisting of human immunoglobulins IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, wherein the heavy chain constant region is human immunoglobulin IgG1 heavy chain constant region, preferably afucosylated IgG1 heavy chain constant region.

In a particular embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 239 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) .

In a preferred embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 239.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine residues in the hinge region are altered (e.g., increased or decreased) as described in, e.g., U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain may be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or antigen-binding fragment thereof.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody or antigen-binding fragment thereof described herein (e.g., CH2 domain (residues 231-340 of human IgG1) and/or CH3 domain (residues 341-447 of human IgG1) and/or the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat) ) to alter one or more functional properties of the antibody or antigen-binding fragment thereof, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity (ADCC) .

In specific embodiment, an IgG1 heavy chain constant region comprises K97R, D239E, and/or L241M variants, and according to EU numbering, these amino acid mutations are numbered K214R, D356E, and L358M. In specific embodiment, an IgG1 heavy chain constant region comprises S239D and I332E mutations (S239D/I332E) . In specific embodiment, an IgG1 heavy chain constant region comprises S239D/A330L/I332E.

In a particular embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 240 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) .

In a preferred embodiment, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 240.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain constant region, wherein the light chain constant region is selected from the group consisting of human immunoglobulins IgGκ and IgGλ light chain constant regions. In some embodiments, the light chain of an antibody described herein is a kappa light chain. In preferred embodiment, the antibody or antigen-binding fragment thereof comprises the light chain constant region of human immunoglobulins IgGκ.

In a particular embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant region having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 241(RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) .

In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 241.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 192, 193, 194, 199, 200, 201, 202, 203, 204, 205, 213, 214, 215, 216, 217, 218, 225, 226, 227, 228, 229 or 230, and the light chain having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 196, 197, 198, 206, 207, 208, 209, 210, 211, 212, 219, 220, 221, 222, 223, 224, 231, 232, 233, 234, 235, 236 or 237.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the amino acid sequences of: SEQ ID NOs: 192 and 196, respectively; SEQ ID NOs: 193 and 197, respectively; SEQ ID NOs: 194 and 198, respectively; SEQ ID NOs: 199 and 206, respectively; SEQ ID NOs: 200 and 207, respectively; SEQ ID NOs: 201 and 208, respectively; SEQ ID NOs: 202 and 209, respectively; SEQ ID NOs: 203 and 210, respectively; SEQ ID NOs: 204 and 211, respectively; SEQ ID NOs: 205 and 212, respectively; SEQ ID NOs: 213 and 219, respectively; SEQ ID NOs: 214 and 220, respectively; SEQ ID NOs: 215 and 221, respectively; SEQ ID NOs: 216 and 222, respectively; SEQ ID NOs: 217 and 223, respectively; SEQ ID NOs: 218 and 224, respectively; SEQ ID NOs: 225 and 232, respectively; SEQ ID NOs: 226 and 233, respectively; SEQ ID NOs: 227 and 234, respectively; SEQ ID NOs: 228 and 235, respectively; SEQ ID NOs: 229 and 236, respectively; SEQ ID NOs: 230 and 237, respectively; or SEQ ID NOs: 231 and 238, respectively.

Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Fibroblast growth factor receptor 2b (FGFR2b) , wherein the antibody or antigen-binding fragment thereof comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of an antibody selected from the group consisting of FR2B-16, FR2B-19, FR2B-29, FR2B-31, FR2B-51, FR2B-53, FR2B-87, FR2B-89, FR2B-90, FR2B-97, FR2B-101, FR2B-110, FR2B-111, FR2B-112, FR2B-116, FR2B-117, FR2B-118 and FR2B-119. In some embodiments, the CDRs are the Kabat-defined CDRs, the Chothia-defined CDRs, or the IMGT-defined CDRs.

In some embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of human FGFR2b as determined by SPR.

In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a murine, humanized, or chimeric antibody or antigen-binding fragment thereof or a camelized single domain antibody.

In some embodiments, the antibody or antigen-binding fragment thereof is afucosylated.

In certain embodiments, the antibody or antigen-binding fragment thereof inhibits tumor cell proliferation. In some embodiments, the antibody or antigen-binding fragment thereof inhibits tumor cell proliferation by about 5%to about 35%as compared to treatment with a control antibody. In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor cell proliferation by at least 5%as compared to treatment with a control antibody. In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor cell proliferation by at least 10%as compared to treatment with a control antibody. In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor cell proliferation by at least 15%as compared to treatment with a control antibody. In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor cell proliferation by at least 20%as compared to treatment with a control antibody. In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor cell proliferation by at least 30%as compared to treatment with a control antibody.

In some embodiments, the antibody or antigen-binding fragment thereof is capable of inducing antibody dependent cell mediated cytotoxicity (ADCC) in FGFR2b-expressing cells. In one embodiment, the antibody or antigen-binding fragment thereof induces specific lysis in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%of FGFR2b-expressing cells.

In some embodiments, the antibody or antigen-binding fragment thereof inhibits tumor growth in a murine gastric cancer model, a murine breast cancer model, a murine ovarian cancer model, or a murine cholangiocarcinoma model. In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor growth in SNU-16 gastric cancer model. In one embodiment, the antibody or antigen-binding fragment thereof inhibits tumor growth by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%as compared to treatment with a control antibody.

In specific embodiment, the antibody or antigen binding fragment thereof is a full length antibody. In specific embodiment, the antibody or antigen binding fragment thereof is an antigen binding fragment. In some embodiments, the antigen binding fragment includes Fab, Fab', F(ab') 2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibody (e.g. scFv) , chimeric antibody, diabody and such polypeptides that comprise at least part of antibody sufficient to confer the specific antigen binding ability on the polypeptides.

Also provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of an antibody or antigen-biding fragment thereof provided herein and/or the light chain variable region or light chain of the antibody or antigen-binding fragment thereof provided herein. In specific embodiment, the nucleic acid molecule encodes the VH of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 145, 146, 147, 151, 152, 153, 154, 155, 156, 157, 165, 166, 167, 168, 169, 170, 177, 178, 179, 180, 181, 182 or 183. In specific embodiment, the nucleic acid molecule encodes the heavy chain of SEQ ID NO: 192, 193, 194, 199, 200, 201, 202, 203, 204, 205, 213, 214, 215, 216, 217, 218, 225, 226, 227, 228, 229 or 230. In specific embodiment, the nucleic acid molecule encodes the VL of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 148, 149, 150, 158, 159, 160, 161, 162, 163, 164, 171, 172, 173, 174, 175, 176, 184, 185, 186, 187, 188, 189 or 190. In specific embodiment, the nucleic acid molecule encodes the light chain of SEQ ID NO: 196, 197, 198, 206, 207, 208, 209, 210, 211, 212, 219, 220, 221, 222, 223, 224, 231, 232, 233, 234, 235, 236 or 237.

Also provided herein is an isolated vector comprising a polynucleotide provided herein. In some embodiments, the vector is an expression vector. In specific embodiment, vector including but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; phage such as λ phage or M13 phage and animal virus.

Also provided herein is a host cell comprising a polynucleotide provided herein, a vector provided herein. In one embodiment, the host cell is a cell selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB/20, NS0, PER-C6, HEK-293T, NIH-3T3, HeLa, BHK, Hep G2, SP2/0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, BMT10 cell, plant cell, insect cell, and human cell. In specific embodiment, the host cell is a CHO cell.

Also provided herein is a method (e.g., an in vitro method) of producing an antibody or antigen-binding fragment thereof that binds to FGFR2b, comprising expressing the antibody or antigen-binding fragment thereof provided herein in a host cell, and isolating the antibody or antigen-binding fragment thereof from the host cell.

Also provided herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR2b and is encoded by a polynucleotide provided herein.

Also provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, or a host cell provided herein; and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition comprising (i) antibodies or antigen-binding fragments thereof provided herein and (ii) a pharmaceutically acceptable carrier, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%of the antibodies or antigen-binding fragments thereof in the composition are afucosylated.

In some embodiments, the pharmaceutical composition comprising (i) antibodies or antigen-binding fragments thereof that specifically bind to FGFR2b and comprise the heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1-6, respectively; SEQ ID NOs: 9-14, respectively; SEQ ID NOs: 17-22, respectively; SEQ ID NOs: 25-30, respectively; SEQ ID NOs: 33-38, respectively; SEQ ID NOs: 41-46, respectively; SEQ ID NOs: 49-54, respectively; SEQ ID NOs: 57-62, respectively; SEQ ID NOs: 65-70, respectively; SEQ ID NOs: 73-78, respectively; SEQ ID NOs: 81-86, respectively; SEQ ID NOs: 89-94, respectively; SEQ ID NOs: 97-102, respectively; SEQ ID NOs: 105-110, respectively; SEQ ID NOs: 113-118, respectively; SEQ ID NOs: 121-126, respectively; SEQ ID NOs: 129-134, respectively; SEQ ID NOs: 137-142, respectively; SEQ ID NOs: 1, 242 and 3-6, respectively; SEQ ID NOs: 57-59, 243-244 and 62, respectively; or SEQ ID NOs: 81, 245, 83, 246-247 and 86, respectively and (ii) a pharmaceutically acceptable carrier, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%of the antibodies or antigen-binding fragments thereof in the composition are afucosylated. In one embodiment, fucosylation is undetectable in the composition.

In some embodiments, the pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein and another antibody. In some embodiments, the pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein and anti-PD1/PD-L1 antibody. The anti-PD1/PD-L1 antibody including but not limited to nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab.

Also provided herein is a method for inhibiting growth of tumor cells in a subject, comprising administering an effective amount of the antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In one embodiment, growth of tumor cell is inhibited by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% (e.g., as compared to treatment with a control antibody) .

Also provided herein is a method for reducing tumor cell metastasis in a subject, comprising administering an effective amount of the antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein.

Also provided herein is a method for treating FGFR2b expressing cancer in a subject, comprising administering an effective amount of the antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. Also provided herein is a method for treating diseases associated with overexpressing FGFR2b in a subject, comprising administering an effective amount of the antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In some embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, lung cancer, ovarian cancer, cholangiocarcinoma, colon cancer, prostate cancer, cervical cancer, pancreatic cancer, esophageal cancer, liver cancer, kidney cancer, head-and-neck tumors, mesothelioma, melanoma, sarcomas, brain tumors and endometrial cancer. In some embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, lung cancer, ovarian cancer, cholangiocarcinoma, pancreatic cancer, preferably triple negative breast cancer, non-small cell lung cancer, squamous lung carcinoma or intrahepatic cholangiocarcinoma.

In one embodiment, the breast cancer is triple negative breast cancer. In one embodiment, the lung cancer is non-small cell lung cancer or small cell lung cancer. In one embodiment, the non-small cell lung cancer is squamous cell carcinoma. In one embodiment, the non-small cell lung cancer is an adenocarcinoma. In one embodiment, the cholangiocarcinoma is an intrahepatic cholangiocarcinoma. In one embodiment, the brain tumor is glioma or glioblastoma. In one embodiment, the subject is human.

Also provided herein is a method for detecting FGFR2b in a sample comprising contacting said sample with an antibody or antigen-binding fragment thereof provided herein. In one embodiment, the sample is obtained from a cancer in a human subject.

Also provided herein is a kit comprising an antibody or antigen-binding fragment thereof provided herein, a polynucleotide provided herein, a vector provided herein, a host cell provided herein, or a pharmaceutical composition provided herein and a detection reagent.

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings set forth herein. The summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Further, the contents of all references, patents and published patent applications cited throughout this application are incorporated herein in entirety by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H show the binding potency of the selected antibodies.

FIGS. 2A-2G show the blocking potency of the selected antibodies.

FIGS. 3A-3F show the blocking activity to ERK phosphorylation of the selected antibodies.

FIGS. 4A-4B show binding of humanized anti-FGFR2b antibodies to human FGFR2b overexpressing on CHO-K1 cells in FACS binding assay.

FIGS. 5A-5B show blocking activity of humanized anti-FGFR2b antibodies on the interaction of FGF7 and human FGFR2b on CHO-K1 cell surface in FACS binding assay.

FIG. 6 shows blocking activity of humanized anti-FGFR2b antibodies on ERK phosphorylation induce by FGF7 treatment in SNU-16 cells.

FIG. 7 shows binding of anti-FGFR2IIIb antibody FR2B-101. h2 to both human and mouse FGFR2IIIb in ELISA binding assay.

FIGS. 8A-8H show different binding cross-reactivity of humanized anti-FGFR2b antibodies and reference antibodies to FGFR family members in ELISA binding assay.

FIG. 9 shows different binding activities of humanized anti-FGFR2b antibodies and reference antibodies to FGFR1b expressing on HEK293 cells in FACS binding assay.

FIG. 10 shows blocking activity of anti-FGFR2IIIb antibody FR2B-101. h2 in SNU-16 cell proliferation.

FIGS. 11A-11D show activity of anti-FGFR2IIIb antibodies in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in human PBMC or NK92-CD16-158V cells.

FIGS. 12A-12C show the serum concentration versus time curves of anti-FGFR2b antibodies after a single i.p. injection of 10 mg/kg antibodies in Nude mice.

FIGS. 13A-13B show anti-tumor activity of anti-FGFR2IIIb antibodies in SNU-16 gastric cancer cell xenograft model.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms “a” , “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a plurality of proteins; reference to “acell” includes mixtures of cells, and the like. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “comprising” , as well as other forms, such as “comprises" and “comprised” , is not limiting. In addition, ranges provided in the specification and appended claims include both end points and all points between the end points.

Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6^th ed., W.B. Saunders Company (2010) ; Sambrook J. &Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000) ; Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John &Sons, Inc. (2002) ; Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998) ; and Coligan et al., Short Protocols in Protein Science, Wiley, John &Sons, Inc. (2003) . The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definitions

In order to better understand the invention, the definitions and explanations of the relevant terms are provided as follows.

The term “antibody” or “Ab” , as used herein, generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) chains polypeptide held together by covalent disulfide bonds and non-covalent interactions. Light chains of an antibody may be classified into κ and λ light chain. Heavy chains may be classified into μ, δ, γ, α and ε, which define isotypes of an antibody as IgM, IgD, IgG, IgA and IgE, respectively. Antibodies may be of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype) , IgA1, IgA2, IgD, IgE or IgM antibody. The term “antibody” also encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity.

The term “antigen-binding fragment” or “antigen-binding fragment” of an antibody, which can be interchangeably used in the context of the application, refers to polypeptides comprising fragments of a full-length antibody, which retain the ability of specifically binding to an antigen that the full-length antibody specifically binds to, and/or compete with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, N.Y. (1989) ) , which is incorporated herein by reference for all purposes. Antigen binding fragments of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Under some conditions, antigen binding fragments include Fab, Fab', F (ab') ₂, Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibody (e.g. scFv) , chimeric antibody, diabody and such polypeptides that comprise at least part of antibody sufficient to confer the specific antigen binding ability on the polypeptides. Antigen-binding fragments of an antibody may be obtained from a given antibody (e.g., the monoclonal anti-human FGFR2b antibody provided in the instant application) by conventional techniques known by a person skilled in the art (e.g., recombinant DNA technique or enzymatic or chemical cleavage methods) , and may be screened for specificity in the same manner by which intact antibodies are screened.

The terms “variable region” or “variable domain” , as used herein, are used interchangeably and are common in the art. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR) . Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region is a mouse variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs) . In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs) .

The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In certain aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA &Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) . In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme. CDR boundaries for antibodies may also be defined or identified by the conventions of Chothia (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) or IMGT (the international ImMunoGeneTics database, http: //www. imgt. org) .

The term “monoclonal antibody” or “mAb” , as used herein, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

The term “human antibody” or “fully human antibody” , as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . However, the term “human antibody” , as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term “human monoclonal antibody” , as used herein, refers to antibodies displaying a single binding specificity, which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.

The term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

The term “chimeric antibody” , as used herein, refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The term “recombinant antibody” , as used herein, refers to an antibody that is prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.

The term “afucosylated” antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment thereof “lacking fucose” refers to an antibody or antigen-binding fragment thereof that lacks fucose in its constant region glycosylation. Methods of measuring fucose include any methods known in the art. In some embodiments, an antibody or antigen-binding fragment thereof described herein has reduced fucose content or lacks fucose (i.e., is “afucosylated” ) . In specific embodiments, an afucosylated antibody provided herein lacks fucose at Asn297. In some embodiments, fucose is undetectable in a composition comprising a plurality of afucosylated antibodies or antigen-binding fragments thereof.

In a specific example, cell lines with a knockout of both alleles of α1, 6-fucosyltransferase can be used to produce antibodies or antigen-binding fragments thereof with reduced fucose content. Alternatively, antibodies or antigen-binding fragments thereof with reduced fucose content or no fucose content can be produced by, e.g.: (i) culturing cells under conditions which prevent or reduce fucosylation; (ii) posttranslational removal of fucose (e.g., with a fucosidase enzyme) ; (iii) post-translational addition of the desired carbohydrate, e.g., after recombinant expression of a non-glycosylated glycoprotein; or (iv) purification of the glycoprotein so as to select for antibodies or antigen-binding fragments thereof which are not fucsoylated. See, e.g., Longmore GD &Schachter H (1982) Carbohydr Res 100: 365-92 and Imai-Nishiya H et al., (2007) BMC Biotechnol. 7: 84 for methods for producing antibodies thereof with no fucose content or reduced fucose content.

In some embodiments, an afucosylated antibody or antigen-binding fragment thereof provided herein has enhanced ADCC activity, which may be measured by the assay provided in Example 6 herein. In some embodiments, an afucosylated antibody or antigen-binding fragment thereof provided herein has enhanced ADCC activity compared to the fucosylated antibody or antigen-binding fragment thereof having the same amino acid sequence. In some embodiments, an afucosylated antibody or antigen-binding fragment thereof provided herein causes specific lysis greater than that with the fucosylated antibody or antigen-binding fragment thereof having the same amino acid sequence.

In some embodiments, compared to fucosylated FGFR2b antibodies or antigen-binding fragments thereof having the same amino acid sequence, the afucosylated FGFR2b antibody or antigen-binding fragment thereof enhances the ADCC activity by at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, or at least 75%. In some embodiments, the afucosylated FGFR2b antibodies or antigen-binding fragments thereof cause specific lysis that is at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, or at least 75%greater than specific lysis with fucosylated FGFR2b antibodies.

The term “anti-FGFR2b antibody” , “antibody that binds to FGFR2b” or “FGFR2b antibody” , as used herein, refers to an antibody that is capable of binding FGFR2b with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting FGFR2b.

The terms “FGFR2b” , “FGFR2IIIb” , “FGFR2b receptor” , “FGFR2b protein” , or “fibroblast growth factor receptor 2b” , which are used interchangeably herein, is a transmembrane receptor tyrosine kinase. The term “FGFR2b” may include human FGFR2b receptor, as well as variants, isoforms, and species homologs thereof. Accordingly, an antibody or antigen-binding fragment thereof, as defined and disclosed herein, may also bind FGFR2b from species other than human, for example mouse FGFR2b.

The term “human FGFR2b” , as used herein, refers to human sequence FGFR2b, such as the complete amino acid sequence of human FGFR2b. The human FGFR2b sequence may differ from human FGFR2b by having, e.g., conserved mutations or mutations in non-conserved regions and the FGFR2b has substantially the same biological function as the human FGFR2b.

The term “mouse FGFR2b” , as used herein, refers to mouse sequence FGFR2b, such as the complete amino acid sequence of mouse FGFR2b.

The term “Ka” , as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “Kd” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. Kd values for antibodies can be determined using methods well established in the art. The term “KD” as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M) . A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a system.

The term “high affinity” for an IgG antibody, as used herein, refers to an antibody having a KD of 1 x 10^-7 M or less, more preferably 5 x 10^-8 M or less, even more preferably 1x10^-9 M or less, even more preferably 5 x 10^-9 M or less and even more preferably 1 x 10^-10 M or less for a target antigen, for example, an FGFR2b receptor.

The term “isolated” , as used herein, refers to a state obtained from natural state by artificial means. If a certain “isolated” substance or component is present in nature, it is possible because its natural environment changes, or the substance is isolated from natural environment, or both. For example, a certain un-isolated polynucleotide or polypeptide naturally exists in a certain living animal body, and the same polynucleotide or polypeptide with a high purity isolated from such a natural state is called isolated polynucleotide or polypeptide. The term “isolated” excludes neither the mixed artificial or synthesized substance nor other impure substances that do not affect the activity of the isolated substance.

The term “isolated antibody” , as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds an FGFR2b protein is substantially free of antibodies that specifically bind antigens other than FGFR2b proteins) . An isolated antibody that specifically binds a human FGFR2b protein may, however, have cross-reactivity to other antigens, such as FGFR2b proteins from other species (e.g. mouse) . Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.

The term “vector” , as used herein, refers to a nucleic acid vehicle which can have a polynucleotide inserted therein. When the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can have the carried genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; phage such as λ phage or M13 phage and animal virus. The animal viruses that can be used as vectors, include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus) , pox virus, baculovirus, papillomavirus, papova virus (such as SV40) . A vector may comprise multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, a vector may comprise origin of replication.

The term “host cell” , as used herein, refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest. Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, and insect cells, and cells comprised within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell. ”

The term “identity” , as used herein, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm” ) . Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed. ) , 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.) , 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds. ) , 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds. ) , 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48: 1073.

The term “hybridoma” and the term “hybridoma cell line” , as used herein, may be used interchangeably. When the term “hybridoma” and the term “hybridoma cell line” are mentioned, they also include subclone and progeny cell of hybridoma.

The term “surface plasmon resonance” , as used herein, refers to and includes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system.

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” , as used herein, refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) . To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998) . In certain aspects, the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b comprises a constant region or portion thereof that is sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC) .

As used herein, the term “subject” includes any human or nonhuman animal, preferably humans.

The term “cancer” , as used herein, refers to any or a tumor or a malignant cell growth, proliferation or metastasis-mediated, solid tumors and non-solid tumors such as leukemia and initiate a medical condition. The cancer can be a “cancer that expresses FGFR2b” or a “FGFR2b expressing cancer” . Such terms refer to a cancer comprising cells that express FGFR2b. The cancer may be a primary tumor or may be advanced or metastatic cancer. Examples of cancer include, but are not limited to, gastric cancer, breast cancer, lung cancer, ovarian cancer, cholangiocarcinoma, colon cancer, prostate cancer, cervical cancer, pancreatic cancer, esophageal cancer, liver cancer, kidney cancer, head-and-neck tumors, mesothelioma, melanoma, sarcomas, brain tumors (e.g., gliomas, such as glioblastomas) and endometrial cancer.

The term “treatment” , “treating” or “treated” , as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, “treating” may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, “treatment” includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

The term “an effective amount” , as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. For instance, the “an effective amount” , when used in connection with treatment of FGFR2b-related diseases or conditions, refers to an antibody or antigen-binding fragment thereof in an amount or concentration effective to treat the said diseases or conditions.

The term “prevent” , “prevention” or “preventing” , as used herein, with reference to a certain disease condition in a mammal, refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.

The term “pharmaceutically acceptable” , as used herein, means that the vehicle, diluent, excipient and/or salts thereof, are chemically and/or physically is compatible with other ingredients in the formulation, and the physiologically compatible with the recipient.

As used herein, the term “apharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient pharmacologically and/or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) , and includes, but is not limited to pH adjuster, surfactant, adjuvant and ionic strength enhancer. For example, the pH adjuster includes, but is not limited to, phosphate buffer; the surfactant includes, but is not limited to, cationic, anionic, or non-ionic surfactant, e.g., Tween-80; the ionic strength enhancer includes, but is not limited to, sodium chloride.

As used herein, the term “adjuvant” refers to a non-specific immunopotentiator, which can enhance immune response to an antigen or change the type of immune response in an organism when it is delivered together with the antigen to the organism or is delivered to the organism in advance. There are a variety of adjuvants, including, but not limited to, aluminium adjuvants (for example, aluminum hydroxide) , Freund’s adjuvants (for example, Freund’s complete adjuvant and Freund’s incomplete adjuvant) , coryne bacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments now. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Anti-FGFR2b Antibodies

In some aspects, the invention comprises an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR2b.

In the context of the application, the “antibody” may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR grafted antibodies, camelized single domain antibody, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof, and derivatives thereof including Fc fusions and other modifications, and any other immunoreactive molecule as long as it exhibits preferential association or binding with a FGFR2b protein. Moreover, unless dictated otherwise by contextual constraints the term further comprises all classes of antibodies (i.e. IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) . In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a humanized monoclonal antibody.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including hybridoma techniques, recombinant techniques, phage display technologies, transgenic animals (e.g., a ) or some combination thereof. For example, monoclonal antibodies can be produced using hybridoma and art-recognized biochemical and genetic engineering techniques such as described in more detail in An, Zhigiang (ed. ) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1^st ed. 2009; Shire et al. (eds. ) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science + Business Media LLC, 1^st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) each of which is incorporated herein in its entirety by reference. It should be understood that a selected binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also an antibody of this invention. In a preferred embodiment, the anti-human FGFR2b monoclonal antibody is prepared by using hybridoma.

Generation of Hybridomas Producing Monoclonal Antibodies of the Invention

To generate hybridomas producing the antibodies of the invention, for instance, humanized monoclonal antibodies of the invention, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. Generation of hybridomas is well-known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

Generation of Transfectomas Producing Monoclonal Antibodies of the Invention

Antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202) . In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.

The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990) ) . Exemplary regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus (e.g., the adenovirus major late promoter (AdMLP) ) and polyoma. Alternatively, nonviral regulatory sequences can be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al. (1988) Mol. Cell. Biol. 8: 466-472) . The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In some embodiments, the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment (s) within the vector and the VL segment is operatively linked to the CL segment within the vector.

Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) . In some embodiments, the signal peptide used for antibody expression is selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 254-310. In some embodiments, the signal peptide used for antibody expression is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 254 or SEQ ID NO: 310. In specific embodiment, the signal peptide used for antibody expression is of the amino acid sequences set forth in SEQ ID NO: 254. In specific embodiment, the signal peptide used for antibody expression is of the amino acid sequences set forth in SEQ ID NO: 310.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017) . For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes may include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .

For expression of the light and heavy chains, the expression vector (s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. It is possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, for example, mammalian host cells, which can assemble and secrete a properly folded and immunologically active antibody.

Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO) cells (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159: 601-621) , NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338, 841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Anti-FGFR2b antibodies with certain properties

The antibodies of the invention are characterized by particular functional features or properties of the antibodies. In some embodiments, the isolated antibody or antigen-binding fragment thereof has one or more of the following properties:

(a) binding human FGFR2b with a KD of 1 x 10^-8 M or less;

(b) inhibiting tumor cell proliferation;

(c) inducing antibody dependent cell mediated cytotoxicity (ADCC) ;

(d) inhibiting tumor growth.

The antibody of the invention binds to human FGFR2b with high affinity. The binding of an antibody of the invention to FGFR2b can be assessed using one or more techniques well established in the art, for instance, ELISA. The binding specificity of an antibody of the invention can also be determined by monitoring binding of the antibody to cells expressing an FGFR2b protein, e.g., flow cytometry. For example, an antibody can be tested by a flow cytometry assay in which the antibody is reacted with a cell line that expresses human FGFR2b, such as CHO cells that have been transfected to express FGFR2b on their cell surface. Additionally or alternatively, the binding of the antibody, including the binding kinetics (e.g., KD value) can be tested in BIAcore binding assays. Still other suitable binding assays include ELISA assays, for example using a recombinant FGFR2b protein. For instance, an antibody of the invention binds to a human FGFR2b with a K_D of 1 x 10^-8 M or less, binds to a human FGFR2b with a K_D of 1 x 10^-9 M or less, binds to a human FGFR2b with a K_D of 5 x 10^-10 M or less, binds to a human FGFR2b with a K_D of 2 x 10^-10 M or less, binds to a human FGFR2b protein with a K_D of 1 x 10^-10 M or less, binds to a human FGFR2b protein with a K_D of 5 x 10^-11 M or less, binds to a human FGFR2b protein with a K_D of 3 x 10^-11 M or less, or binds to a human FGFR2b protein with a K_D of 2 x 10^-11 M or less.

Anti-FGFR2b antibodies comprising CDRs with sequence identity to specific sequences

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises:

A) one or more heavy chain CDRs (CDRHs) selected from at least one of the group consisting of:

(i) a CDRH1 with at least 90%sequence identity to a CDRH1 as set forth in SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129 or 137;

(ii) a CDRH2 with at least 90%sequence identity to a CDRH2 as set forth in SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 242 or 245; and

(iii) a CDRH3 with at least 90%, sequence identity to a CDRH3 as set forth in SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131 or 139;

B) one or more light chain CDRs (CDRLs) selected from at least one of the group consisting of:

(i) a CDRL1 with at least 90%sequence identity to a CDRL1 as set forth in SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140, 243 or 246;

(ii) a CDRL2 with at least 90%sequence identity to a CDRL2 as set forth in SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 244 or 247; and

(iii) a CDRL3 with at least 90%sequence identity to a CDRL3 as set forth in SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134 or 142; or

C) one or more CDRHs of A) and one or more CDRLs of B) .

The assignment of amino acids to each CDR may be in accordance with one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5^th Ed. ) , US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; IMGT (the international ImMunoGeneTics database, http: //www. imgt. org) ; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3^rd Ed., Wily-VCH Verlag GmbH and Co. unless otherwise noted.

Variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as set out above, such as, for example, the Kabat and IMGT numbering system) or by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in, and can be accessed through, the “Abysis" website at www. bioinf. org. uk/abs (maintained by A.C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and the VBASE2 website at www. vbase2. org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue) : D671 -D674 (2005) . Preferably sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB. See Dr. Andrew C.R. Martin's book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg. uk/abs) . The Abysis database website further includes general rules that have been developed for identifying CDRs which can be used in accordance with the teachings herein.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970) ) which has been incorporated into the GAP program in the GCG software package (available at http: //www. gcg. com) , using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17) : 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www. ncbi. nlm. nih. gov.

In other embodiments, the CDR amino acid sequences can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the respective sequences set forth above. As an illustrative example, the antibody may comprise a CDRH1 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to a CDRH1 as set forth in SEQ ID NO: 1.

Anti-FGFR2b antibodies comprising CDRs with amino acid addition, deletion and/or substitution

(i) a CDRH1 of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129 or 137, or a CDRH1 that differs in amino acid sequence from the CDRH1 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

(ii) a CDRH2 of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 242 or 245, or a CDRH2 that differs in amino acid sequence from the CDRH2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

(iii) a CDRH3 of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131 or 139, or a CDRH3 that differs in amino acid sequence from the CDRH3 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

(i) a CDRL1 of SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140, 243 or 246, or a CDRL1 that differs in amino acid sequence from the CDRL1 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

(ii) a CDRL2 of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 244 or 247, or a CDRL2 that differs in amino acid sequence from the CDRL2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

(iii) a CDRL3 of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134 or 142, or a CDRL3 that differs in amino acid sequence from the CDRL3 by an amino acid addition, deletion or substitution of not more than 2 amino acids; or

C) one or more CDRHs of A) and one or more CDRLs of B) .

In some embodiments, the CDRs of the isolated antibody or antigen-binding fragment thereof contain a conservative substitution of not more than 1 amino acid. The term “conservative substitution” , as used herein, refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc. ) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (for example, lysine, arginine and histidine) , amino acids having acidic side chains (for example, aspartic acid and glutamic acid) , amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine) . Therefore, a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993) ; Kobayashi et al., Protein Eng. 12 (10) : 879-884 (1999) ; and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997) , which are incorporated herein by reference) .

Anti-FGFR2b antibodies comprising CDRs

(i) a CDRH1 as set forth in SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129 or 137;

(ii) a CDRH2 as set forth in SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 242 or 245; and

(iii) a CDRH3 as set forth in SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131 or 139;

(i) a CDRL1 as set forth in SEQ ID NO: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132, 140, 243 or 246;

(ii) a CDRL2 as set forth in SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 244 or 247; and

(iii) a CDRL3 as set forth in SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134 or 142; or

C) one or more CDRHs of A) and one or more CDRLs of B) .

Anti-FGFR2b antibodies comprising a heavy chain variable region and a light chain variable region

(A) a heavy chain variable region:

(B) a light chain variable region:

In a specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of:

(a) SEQ ID NOs: 7 and 8, respectively;

(b) SEQ ID NOs: 15 and 16, respectively;

(c) SEQ ID NOs: 23 and 24, respectively;

(d) SEQ ID NOs: 31 and 32, respectively;

(e) SEQ ID NOs: 39 and 40, respectively;

(f) SEQ ID NOs: 47 and 48, respectively;

(g) SEQ ID NOs: 55 and 56, respectively;

(h) SEQ ID NOs: 63 and 64, respectively;

(i) SEQ ID NOs: 71 and 72, respectively;

(j) SEQ ID NOs: 79 and 80, respectively;

(k) SEQ ID NOs: 87 and 88, respectively;

(l) SEQ ID NOs: 95 and 96, respectively;

(m) SEQ ID NOs: 103 and 104, respectively;

(n) SEQ ID NOs: 111 and 112, respectively;

(o) SEQ ID NOs: 119 and 120, respectively;

(p) SEQ ID NOs: 127 and 128, respectively;

(q) SEQ ID NOs: 135 and 136, respectively; or

(r) SEQ ID NOs: 143 and 144, respectively.

(A) a heavy chain variable region:

(B) a light chain variable region:

(a) SEQ ID NOs: 145 and 148, respectively;

(b) SEQ ID NOs: 146 and 149, respectively;

(c) SEQ ID NOs: 147 and 150, respectively;

(d) SEQ ID NOs: 151 and 158, respectively;

(e) SEQ ID NOs: 152 and 159, respectively;

(f) SEQ ID NOs: 153 and 160, respectively;

(g) SEQ ID NOs: 154 and 161, respectively;

(h) SEQ ID NOs: 155 and 162, respectively;

(i) SEQ ID NOs: 156 and 163, respectively;

(j) SEQ ID NOs: 157 and 164, respectively;

(k) SEQ ID NOs: 165 and 171, respectively;

(l) SEQ ID NOs: 166 and 172, respectively;

(m) SEQ ID NOs: 167 and 173, respectively;

(n) SEQ ID NOs: 168 and 174, respectively;

(o) SEQ ID NOs: 169 and 175, respectively;

(p) SEQ ID NOs: 170 and 176, respectively;

(q) SEQ ID NOs: 177 and 184, respectively;

(r) SEQ ID NOs: 178 and 185, respectively;

(s) SEQ ID NOs: 179 and 186, respectively;

(t) SEQ ID NOs: 180 and 187, respectively;

(u) SEQ ID NOs: 181 and 188, respectively;

(v) SEQ ID NOs: 182 and 189, respectively; or

(w) SEQ ID NOs: 183 and 190, respectively.

In other embodiments, the amino acid sequences of the heavy chain variable region and/or the light chain variable region can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to the respective sequences set forth above.

In some further embodiments, the isolated antibody or antigen-binding fragment thereof may contain conservative substitution or modification of amino acids in the variable regions of the heavy chain and/or light chain. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10: 835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870; Hall et al. (1992) J. Immunol. 149: 1605-12; Kelley and O’ Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10: 341-6 and Beers et al. (2000) Clin. Can. Res. 6: 2835-43.

As described above, the term “conservative substitution” , as used herein, refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc. ) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (for example, lysine, arginine and histidine) , amino acids having acidic side chains (for example, aspartic acid and glutamic acid) , amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine) . Therefore, a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993) ; Kobayashi et al., Protein Eng. 12 (10) : 879-884 (1999) ; and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997) , which are incorporated herein by reference) .

Nucleic Acid Molecules Encoding Antibodies of the Invention

In some aspects, the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region and/or the light chain variable region of the isolated antibody as disclosed herein.

Nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below) , cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques) , a nucleic acid encoding such antibodies can be recovered from the gene library.

The isolated nucleic acid encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding nucleic acid to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3) . The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al. (1991) , supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably is an IgG1 or IgG4 constant region.

The isolated nucleic acid encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al., supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.

Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL-or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked” , as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

In some embodiments, the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region of the isolated antibody as disclosed herein. In some embodiments, the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the light chain variable region of the isolated antibody as disclosed herein.

Pharmaceutical Compositions

In some aspects, the invention is directed to a pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof as disclosed herein and a pharmaceutically acceptable carrier.

Components of the compositions

The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody and/or a drug. The pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another immune-stimulatory agent, anti-cancer agent, an antiviral agent, or a vaccine. A pharmaceutically acceptable carrier can include, for example, a pharmaceutically acceptable liquid, gel or solid carriers, an aqueous medium, a non-aqueous medium, an anti-microbial agent, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agent, a chelating agent, a diluent, adjuvant, excipient or a nontoxic auxiliary substance, other known in the art various combinations of components or more.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrating agents, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrin. Suitable anti-oxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, thioglycolic acid, Mercapto sorbitol, butyl methyl anisole, butylated hydroxy toluene and/or propylgalacte. As disclosed in the present invention, in a solvent containing an antibody or an antigen-binding fragment of the present invention discloses compositions include one or more anti-oxidants such as methionine, reducing antibody or antigen binding fragment thereof may be oxidized. The oxidation reduction may prevent or reduce a decrease in binding affinity, thereby enhancing antibody stability and extended shelf life. Thus, in some embodiments, the present invention provides a composition comprising one or more antibodies or antigen binding fragment thereof and one or more anti-oxidants such as methionine. The present invention further provides a variety of methods, wherein an antibody or antigen binding fragment thereof is mixed with one or more anti-oxidants, such as methionine, so that the antibody or antigen binding fragment thereof can be prevented from oxidation, to extend their shelf life and/or increased activity.

To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

Administration, Formulation and Dosage

The pharmaceutical composition of the invention may be administered in vivo, to a subject in need thereof, by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and therapeutic regimen.

Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

Formulations suitable for parenteral administration (e.g., by injection) , include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) , in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate) . Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Similarly, the particular dosage regimen, including dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc. ) .

Frequency of administration may be determined and adjusted over the course of therapy, and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dosage administered may be adjusted or attenuated to manage potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of a subject therapeutic composition may be appropriate.

It will be appreciated by one of skill in the art that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial harmful or deleterious side-effects.

In general, the antibody or the antigen binding fragment thereof of the invention may be administered in various ranges. These include about 5 μg/kg body weight to about 100 mg/kg body weight per dose; about 50 μg/kg body weight to about 5 mg/kg body weight per dose; about 100 μg/kg body weight to about 10 mg/kg body weight per dose. Other ranges include about 100 μg/kg body weight to about 20 mg/kg body weight per dose and about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg/kg body weight, at least about 250 μg/kg body weight, at least about 750 μg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, at least about 10 mg/kg body weight.

In any event, the antibody or the antigen binding fragment thereof of the invention is preferably administered as needed to subjects in need thereof. Determination of the frequency of administration may be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like.

In certain preferred embodiments, the course of treatment involving the antibody or antigen-binding fragment thereof of the instant invention will comprise multiple doses of the selected drug product over a period of weeks or months. More specifically, the antibody or antigen-binding fragment thereof of the instant invention may be administered once every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it will be appreciated that the dosages may be altered or the interval may be adjusted based on patient response and clinical practices.

Dosages and regimens may also be determined empirically for the disclosed therapeutic compositions in individuals who have been given one or more administration (s) . For example, individuals may be given incremental dosages of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or reduced or attenuated based respectively on empirically determined or observed side effects or toxicity. To assess efficacy of the selected composition, a marker of the specific disease, disorder or condition can be followed as described previously. For cancer, these include direct measurements of tumor size via palpation or visual observation, indirect measurement of tumor size by x-ray or other imaging techniques; an improvement as assessed by direct tumor biopsy and microscopic examination of the tumor sample; the measurement of an indirect tumor marker (e.g., PSA for prostate cancer) or a tumorigenic antigen identified according to the methods described herein, a decrease in pain or paralysis; improved speech, vision, breathing or other disability associated with the tumor; increased appetite; or an increase in quality of life as measured by accepted tests or prolongation of survival. It will be apparent to one of skill in the art that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of neoplastic condition, whether the neoplastic condition has begun to metastasize to other location in the individual, and the past and concurrent treatments being used.

Compatible formulations for parenteral administration (e.g., intravenous injection) will comprise the antibody or antigen-binding fragment thereof as disclosed herein in concentrations of from about 10 μg/ml to about 100 mg/ml. In certain selected embodiments, the concentrations of the antibody or the antigen binding fragment thereof will comprise 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, 100 μg/ml, 200 μg/ml, 300, μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml or 1 mg/ml.

Applications of the Invention

The antibodies, antibody compositions and methods of the present invention have numerous in vitro and in vivo utilities involving, for example, detection of FGFR2b.

The invention further provides methods for detecting the presence of human FGFR2b antigen in a sample, or measuring the amount of human FGFR2b antigen, comprising contacting the sample with a human monoclonal antibody, or an antigen binding fragment thereof, which specifically binds to human FGFR2b, under conditions that allow for formation of a complex between the antibody or portion thereof and human FGFR2b. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative of the presence of human FGFR2b antigen in the sample. Moreover, the anti-FGFR2b antibodies of the invention can be used to purify human FGFR2b via immunoaffinity purification.

Treatment of disorders including cancers

In some aspects, the present invention provides a method of treating a disorder in a mammal, which comprises administering to the subject (for example, a human) in need of treatment a therapeutically effective amount of the antibody or antigen-binding fragment thereof as disclosed herein. In some embodiments, the disorder is a cancer. In some embodiments, the disorder is a solid tumor. In some embodiments, the disorder is an epithelial tumor. In some embodiments, the disorder is FGFR2b expressing cancer.

FGFR2b is a transmembrane protein that is expressed on epithelial cells and is upregulated in many cancers, such as gastric cancer, cholangiocarcinoma (e.g. intrahepatic cholangiocarcinoma) , ovarian cancer, breast cancer (e.g. triple negative breast cancer) . In some aspects, the anti-FGFR2b antibodies or antigen-binding fragment thereof herein can be used to treat disorders characterized by the expression of FGFR2b, including, without limitation, gastric cancer, cholangiocarcinoma, ovarian cancer and breast cancer.

Effective doses of the compositions herein for the treatment of disease vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but nonhuman mammals may also be treated, e.g., companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy. Dosage levels can be readily determined by the ordinarily skilled clinician, and can be modified as required, e.g., as required to modify a subject's response to therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.

Combined use with chemotherapies

The antibody or antigen-binding fragments thereof may be used in combination with an anti-cancer agent, a cytotoxic agent or chemotherapeutic agent.

The term “anti-cancer agent” or “anti-proliferative agent” means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapies, radiation therapy and anti-metastatic agents and immunotherapeutic agents. It will be appreciated that, in selected embodiments as discussed above, such anti-cancer agents may comprise conjugates and may be associated with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments selected anti-cancer agents will be linked to the unpaired cysteines of the engineered antibodies to provide engineered conjugates as set forth herein. Accordingly, such engineered conjugates are expressly contemplated as being within the scope of the instant invention. In other embodiments, the disclosed anti-cancer agents will be given in combination with site-specific conjugates comprising a different therapeutic agent as set forth above.

As used herein the term “cytotoxic agent” means a substance that is toxic to the cells and decreases or inhibits the function of cells and/or causes destruction of cells. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins of bacteria (e.g., Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A) , fungal (e.g., α-sarcin, restrictocin) , plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S) , Momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and the tricothecenes) or animals, (e.g., cytotoxic RNases, such as extracellular pancreatic RNases; DNase I, including fragments and/or variants thereof) .

For the purposes of the instant invention a “chemotherapeutic agent” comprises a chemical compound that non-specifically decreases or inhibits the growth, proliferation, and/or survival of cancer cells (e.g., cytotoxic or cytostatic agents) . Such chemical agents are often directed to intracellular processes necessary for cell growth or division, and are thus particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, and thus inhibits cells from entering mitosis. In general, chemotherapeutic agents can include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell likely to become cancerous or generate tumorigenic progeny (e.g., TIC) . Such agents are often administered, and are often most effective, in combination, e.g., in regimens such as CHOP or FOLFIRI.

Examples of anti-cancer agents that may be used in combination with the site-specific constructs of the present invention (either as a component of a site specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, a camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, docetaxel, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogs, androgens, anti-adrenals, folic acid replenisher such as frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, an epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, polysaccharide complex (JHS Natural Products, Eugene, OR) , razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; cyclophosphamide; thiotepa; taxoids, chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) , topoisomerase inhibitor RFS 2000; difluorometlhylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as troxacitabine (a1, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines, rIL-2; topoisomerase 1 inhibitor; rmRH; Vinorelbine and Esperamicins and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Combined use with radiotherapies

The present invention also provides for the combination of the antibody or antigen-binding fragments thereof thereof with radiotherapy (i.e., any mechanism for inducing DNA damage locally within tumor cells such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like) . Combination therapy using the directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed conjugates may be used in connection with a targeted anti-cancer agent or other targeting means. Typically, radiation therapy is administered in pulses over a period of time from about 1 to about 2 weeks. The radiation therapy may be administered to subjects having head and neck cancer for about 6 to 7 weeks. Optionally, the radiation therapy may be administered as a single dose or as multiple, sequential doses.

Kits

The present invention also provides kits comprising one or more antibodies or antigen-binding fragments thereof described herein or conjugates thereof. In a specific embodiment, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof provided herein. Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The present invention also provides kits that can be used in diagnostic methods. In one embodiment, a kit comprises an antibody or antigen-binding fragment thereof described herein, preferably a purified antibody or antigen-binding fragment thereof, in one or more containers. In a specific embodiment, kits described herein contain a substantially isolated FGFR2b antigen (e.g., human FGFR2b) that can be used as a control. In another specific embodiment, the kits described herein further comprise a control antibody or antigen-binding fragment thereof which does not react with a FGFR2b antigen. In another specific embodiment, kits described herein contain one or more detection reagents for detecting the binding of an antibody or antigen-binding fragment thereof to a FGFR2b antigen (e.g., the antibody or antigen-binding fragment thereof can be conjugated to a detection reagent such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody or antigen-binding fragment thereof which recognizes the first antibody or antigen-binding fragment thereof can be conjugated to a detection reagent) . Examples of detection reagent may include a fluorescent label (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate label (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase) , radioisotope, luminescent label, chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection. In specific embodiments, a kit provided herein can include a recombinantly produced or chemically synthesized FGFR2b antigen. The FGFR2b antigen provided in the kit can also be attached to a solid support. In a more specific embodiment, the detecting means of the above described kit includes a solid support to which a FGFR2b antigen is attached. Such a kit can also include a non-attached reporter-labeled anti-human antibody or antigen-binding fragment thereof or anti-mouse/rat antibody or antigen-binding fragment thereof. In this embodiment, binding of the antibody or antigen-binding fragment thereof to the FGFR2b antigen can be detected by binding of the said reporter-labeled antibody or antigen-binding fragment thereof.

EXAMPLES

The present invention, thus generally described, will be understood more readily by reference to the following Examples, which are provided by way of illustration and are not intended to be limiting of the instant invention. The Examples are not intended to represent that the experiments below are all or the only experiments performed.

EXAMPLE 1

I. Screening of anti-FGFR2b monoclonal antibodies

Antibodies against human FGFR2IIIb (FGFR2b) were isolated by screening hybridoma clones from immunized mice. Briefly, for each group, 5 SJL and/or Balb/c mice were immunized with human FGFR2 IIIb/FGFR2 IIIc protein antigen (25-50 ug by intraperitoneal injection or 5-10 ug by HOCK injection) and/or human FGFR2 IIIb/FGFR2 IIIc overexpressing HEK293F cell line (0.5-1x10⁷ cells/injection) . The primary immunization was followed by several boosts until animals developed satisfactory antiserum titers (1: 50,000; 3-fold over pre-bleed serum) suitable for hybridoma development.

In group 1 and 2, SJL and Balb/c mice were immunized with human FGFR2 beta IIIb-hFc protein (Kactus Biosystems, LLC; Accession: P21802-3 Arg152-Glu378; Cat. No. FGR-HM2BB) by intraperitoneal (i.p. ) injection. Immune tolerance break was performed by intraperitoneal injection of 100 μg anti-CD25 Ab two days before primary immunization (Day -2) and 100 μg anti-CD40 Ab on Day 7.

In group 3, SJL mice were immunized with human FGFR2 beta IIIb-hFc protein by HOCK Injection. Immune tolerance break was performed as group 1 and 2.

In group 4 and 5, SJL and Balb/c mice were immunized with HEK293F cell line overexpressing human FGFR2 alpha IIIb (Sino Biological, LLC; Accession: NM_000141.4; Cat. No. HG10824-M, replacing the IIIc domain with IIIb domain by molecular cloning method) by intraperitoneal injection.

In group 6, 7, and 8, SJL mice were immunized with both human FGFR2 beta IIIb-hFc protein and human FGFR2 beta IIIc-hFc protein (Sino Biological, LLC; Accession: NP_001138387.1; Cat. No. 16483-H08H) by intraperitoneal injection or HOCK injection in both legs.

After the immunization, the mice were sacrificed to perform hybridoma fusion and screening according to the standard protocol. Hybridoma screening was performed to test: 1) the binding of the supernatant to human FGFR2 beta IIIb-hFc or human FGFR2 beta IIIc-hFc protein in ELISA assay; 2) the binding of the supernatant to CHOK1-FGFR2 IIIb or CHOK1-FGFR2 IIIc cells (Sino Biological, LLC; Accession: NM_000141.4; Cat. No. HG10824-M) in FACS assay; 3) the blocking of the supernatant in human FGFR2 IIIb/FGF7 binding assays in ELISA. For ELISA binding screen, the hybridoma supernatant or the reference antibodies (100 ul/well) was added to the coated plate (1 ug/ml of coating antigen) at 37℃ for 1 hour. After secondary antibody incubation and TMB development, OD450 was measured using Molecular device spectra max plus384. For FACS binding screen, the hybridoma supernatants or the reference antibodies were diluted with FACS buffer (0.1ml/well) and added to the 96-well plate containing 2×10⁵ cells/well. The plates were incubated at 4℃ for 1 hour. After staining with fluorescence conjugated secondary antibody (1: 1000) at 4℃ for 1 hour, the plates were analyzed by BD FACS CantoTM II. For ELISA blocking screen, the hybridoma supernatant or the reference antibodies (50 ul/well) was added to the coated plate (1ug/ml of coating antigen) . Then 50 ul of 1.58 ug/ml biotinylated FGF7-his (Sino Biological, LLC; Uniprot accession: P21781, Cat. No. 10210-H07E) was added and incubated at 37℃ for 1 hour. After secondary antibody incubation and TMB development, OD450 was measured.

119 FGFR2 IIIb-reactive, FGFR2c-non-reactive hybridoma clones with blocking activity were identified. Briefly, total RNA was reverse transcribed into cDNA using anti-sense primers following the technical manual of PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Cat#6110A) . Then the antibody fragments of VH and VL were amplified according to the SOP of Biointron Biologic Inc. and the PCR fragments were cloned into pUC19-T vector. Colony PCR was performed to screen for clones and no less than five positive clones were sequenced. Chimeric antibodies with human IgG1 Fc carrying S239D/I332E mutation were produced in 10 ml CHO cell expression system for further characterization.

Reference antibodies FR-21 (mouse Bemarituzumab, US 10, 689, 448) , Bemarituzumab (US 10, 689, 448) , and Hu36-2 (WO 2021129672) were chosen as the positive controls for FGFR2 IIIb screening and characterization assays. Anti-HEL-mouse IgG1 isotype control antibody (Biointron LLC; Cat. No. B118301) was chosen as the negative control. The heavy chain and light chain amino acid sequences of the reference antibodies are listed in Table 1 below.

Exemplary signal peptides used for antibody expression include without limitation to: MHSSALLCCLVLLTGVRA (SEQ ID NO: 310) .

Table 1

Heavy chain (HC) and Light chain (LC) amino acid sequences of the reference antibodies

II. Anti-FGFR2 IIIb antibody characterization

The antibodies were evaluated for binding activity to CHOK1-FGFR2 IIIb cells and blocking activity of CHOK1-FGFR2 IIIb/FGF7 interaction in FACS. For FACS binding screen, the antibodies (100 ul/well) were added to 96-well plate containing CHOK1-FGFR2 IIIb cells or SNU-16 cells (2×10⁵ cells/well) . The plates were incubated at 4℃ for 1 hour. After stained with fluorescence dye conjugated secondary antibody (1: 1000) , the cells were analyzed by flow cytometry. For FACS blocking screen, the antibodies (50 ul/well) were added to 96-well plate containing CHOK1-FGFR2 IIIb cells (2×10⁵ cells/well) pre-incubated with 400 ng/ml biotinylated FGF7-his and 10 ug/ml heparin at 4℃ for 30 mins. The plates were incubated at 4℃for 1 hour. After stained with fluorescence dye conjugated secondary antibody (1: 1000) , the cells were analyzed by flow cytometry.

18 anti-FGFR2 IIIb antibodies with high binding affinity and/or blocking activity were selected. The sequences of the anti-FGFR2 IIIb antibodies are shown in Tables 2-3 below. The CDRs were determined by the Kabat system. The binding and blocking potency of the selected antibodies are summarized in Table 4.

Exemplary signal peptides used for antibody expression include without limitation to:

MGWSCIILFLVATATGVHS (SEQ ID NO: 254) ,

MEWPLIFLLLLSGTAGVQS (SEQ ID NO: 255) ,

MKVLSLLYLLTAIPGILS (SEQ ID NO: 256) ,

MECNWILPFILSVTSGVYS (SEQ ID NO: 257) ,

MGRLTSSFLLLIVPAYVLS (SEQ ID NO: 258) ,

MGFSRIFLFLLSVTTGVHS (SEQ ID NO: 259) ,

MAVLALLLCLVTFPSCVLS (SEQ ID NO: 260) ,

MAVLGLLFCLVTFPSCVLS (SEQ ID NO: 261) ,

MNFGLSLIFLVLVLKGVQC (SEQ ID NO: 262) ,

MDRLTSSFLLLIVPAYVLS (SEQ ID NO: 263) ,

MGWSCIILILVAAATGVHS (SEQ ID NO: 264) ,

MECNWILPFILSVTSGVHS (SEQ ID NO: 265) ,

MECNWILPLILSVTSGVYS (SEQ ID NO: 266) ,

MNFGLSLIFLALILKGVQC (SEQ ID NO: 267) ,

MGWSWIFLFLLSETAGVLS (SEQ ID NO: 268) ,

MEWTWVFLFLLSVTAGVHS (SEQ ID NO: 269) ,

MDRLTSSFLLLTVPAYVLS (SEQ ID NO: 270) ,

MGWSYIILFLVATATGVHS (SEQ ID NO: 271) ,

MEWPCIFLFLLSVTEGVHS (SEQ ID NO: 272) ,

MAVLVLLFCLVTFPSCVLS (SEQ ID NO: 273) ,

MGWSWIFFFLLSGTAGVHC (SEQ ID NO: 274) ,

MEWSGVFIFLLSVTAGVHS (SEQ ID NO: 275) ,

MGWSWIFLFLLSEIAGVLS (SEQ ID NO: 276) ,

MGWSWIFLFLLSGTAGVLS (SEQ ID NO: 277) ,

MGRSWIFLFLLSGTAGVLS (SEQ ID NO: 278) ,

MESQTQVFVYMLLWLSGVDG (SEQ ID NO: 279) ,

MESQTQVFLSLLLWVSGTCG (SEQ ID NO: 280) ,

METDTLLLWVLLLWVPGSTG (SEQ ID NO: 281) ,

MHFQVQIFSFLLISASVIMSRG (SEQ ID NO: 282) ,

MSSAQFLGLLLLCFQGTRC (SEQ ID NO: 283) ,

MESQIQVFVFVFLWLSGVDG (SEQ ID NO: 284) ,

MESQTLVFISILLWLYDADG (SEQ ID NO: 285) ,

MKFPSQLLLLLLFGIPGMIC (SEQ ID NO: 286) ,

MVSTPQFLVFLLFWIPASRG (SEQ ID NO: 287) ,

MEFHTQVFVFVFLWLSGVDG (SEQ ID NO: 288) ,

MESQTLVFISILLWLYGADG (SEQ ID NO: 289) ,

MRAPAQIFGFLLLLFPGTRC (SEQ ID NO: 290) ,

MSVPTQVLGLLLLCLTGARC (SEQ ID NO: 291) ,

MSVPTQVLGLLLLWLTGARC (SEQ ID NO: 292) ,

MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 293) ,

MDFQVQIFSFLLISASVILSRG (SEQ ID NO: 294) ,

MMVLAQFLAFLLLWFPGARC (SEQ ID NO: 295) ,

MDFQVQIFSFLLMSASVIMSRG (SEQ ID NO: 296) ,

MRAPAQFLGILLLWFPGARC (SEQ ID NO: 297) ,

MRPSIQFLGLLLFWLHGAQC (SEQ ID NO: 298) ,

MRPSIQFLGLLLFWLHGTQC (SEQ ID NO: 299) ,

MDFQVQIFSFLLISASVIISRG (SEQ ID NO: 300) ,

MDSQAQVLMLLLLWVSGTCG (SEQ ID NO: 301) ,

MRPSIQFLGLLLFGLHGAQC (SEQ ID NO: 302) ,

MDLQVQIISFLLISVTVLMSRG (SEQ ID NO: 303) ,

MKLPVRLLVLMFWIPASSS (SEQ ID NO: 304) ,

MESQIQAFVFVFLWLSGVDG (SEQ ID NO: 305) ,

MESQTQVLMFLLLWVSGACA (SEQ ID NO: 306) ,

MVFTPQILGLMLFWISASRG (SEQ ID NO: 307) ,

MESQTQVLMSLLFWVSGTCG (SEQ ID NO: 308) ,

MRAPAQIFGFLLLLFPGSRC (SEQ ID NO: 309) .

Table 2

Table 3

Variable domain amino acid sequences of anti-FGFR2b antibodies

Table 4

Binding and blocking potency of anti-FGFR2b antibodies

Anti-FGFR2 IIIb antibodies were also screened for their ability to block ERK phosphorylation induced by the downstream signaling of FGFR2 IIIb/FGF7 interaction. For ERK phosphorylation assay, SNU-16 cells were starved in RPMI1640 medium (Invitrogen, Cat. No. 11875-093) supplemented with 0.1%BSA medium at 37℃ for 24 hours. Anti-FGFR2 IIIb antibodies were added to the starved SNU-16 cells for 30 minutes. FGF7 at a final concentration of 400 ng/ml was added to the cells for 10 minutes. The cell pellets were harvested carefully by centrifugation and then processed for phosphor-ERK determination with Advanced ERK phospho-T202/Y204 kit (Cisbio, Cat. No. 64AERPEG) according to manufacturer’s manual. EC50, IC50, Top and Bottom values were derived from best-fit binding curves (non-linear fit, 4-parameters) with GraphPad Prism 8.02 software. As shown in Table 5, different clones of anti-FGFR2 IIIb antibody had different blocking activities to ERK phosphorylation.

Table 5

Blocking activity to ERK phosphorylation of anti-FGFR2b antibodies

EXAMPLE 2

I. Humanization of anti-FGFR2b antibodies

Humanization design

FR2B-16, FR2B-89, FR2B-97, and FR2B-101 were humanized by CDR grafting plus back mutation. The structure of parental antibody was modelled by MOE homology modelling program. Humanized antibodies were designed using CDR grafting. Briefly, the CDRs of parental antibody were grafted into the human acceptors to obtain humanized light chains and humanized heavy chains for each parental antibody. Three to four heavy chains (VH1, VH2, VH3 and VH4) and two to four light chains (VL1, VL2, VL3 and VL4) were paired with each other for affinity ranking experiment. The PTM risk of all sequences was analyzed, and appropriate PTM removal mutations were designed of heavy chain and light chain. The sequences of humanized antibodies are shown in Tables 6-8.

MGWSCIILFLVATATGVHS (SEQ ID NO: 254) , and MHSSALLCCLVLLTGVRA (SEQ ID NO: 310) .

Table 6

CDR amino acid sequences of humanized antibodies

Table 7

Variable domain amino acid sequences of humanized antibodies

Table 8

Heavy chain (HC) and Light chain (LC) amino acid sequences of humanized antibodies

II. Affinity ranking of chimeric, humanized antibodies and PTM removal antibodies

For affinity ranking, antibodies were captured on the sensor chip through Fc capture method. FGFR2IIIb-His (Kactus, Cat. No. FGR-HM1BB) was used as the analyte. The surface was regenerated before the injection of another antibody. The process was repeated until all antibodies were analyzed. The off-rates were obtained from fitting the experimental data locally to 1: 1 interaction model using the Biacore 8K evaluation software. The data of dissociation (kd) and association (ka) rate constants were obtained using Biacore 8K evaluation software. The equilibrium dissociation constants (KD) were calculated from the ratio of kd over ka. The antibodies were ranked by their dissociation rate constant kd. As shown in Table 9, based on the ranking result, the top 3 clones and suitable PTM removal mutation were selected.

Table 9

The binding kinetics of humanized antibodies to human FGFR2b

III. Humanized antibodies characterization

Binding and blocking activity of humanized antibodies were evaluated. As shown in FIGs. 4-5, humanized antibodies FR2B-89. h1-6, FR2B-101. h1-6, FR2B-97. h5-6 all bind to human FGFR2b and blocked the interaction between human FGFR2b with FGF7 with similar potency comparing to the parental antibodies. The binding and blocking potency of the selected humanized antibodies are summarized in Table 10.

Table 10

Binding and blocking potency of humanized antibodies

The chimeric and selected humanized antibodies were also evaluated for their ability to block ERK phosphorylation induced by the downstream signaling of FGFR2 IIIb/FGF7 interaction, as shown in Table 11 and Figure 6.

Table 11

Blocking activity to ERK phosphorylation of selected humanized antibodies

EXAMPLE 3

Binding of anti-FGFR2IIIb antibodies to mouse FGFR2b

Anti-FGFR2IIIb antibody FR2B-101. h2 was evaluated for its ability to bind mouse FGFR2IIIb (Kactusbio, LLC: FGF-MM1BB) in ELISA binding assay. As shown in Figure 7, anti-FGFR2IIIb antibody FR2B-101. h2 binds to mouse FGFR2IIIb in a dose response manner.

EXAMPLE 4

Binding selectivity of anti-FGFR2b antibodies to FGFRs

Anti-FGFR2IIIb humanized antibodies were also assayed for binding specificity against closely related FGFR family members: human FGFR1 IIIb/IIIc, FGFR3 IIIb/IIIc, and FGFR4 (Sino Biological, LLC) by either ELISA binding (Figure 8) or FACS binding analysis (Figure 9) . As shown in Figure 8 and Figure 9, different clones of anti-FGFR2IIIb antibody had different binding selectivity against other FGFR family members.

The binding kinetics of humanized antibodies to human FGFR2b and human FGFR1b were determined by Biacore. The result is shown as Table 12.

Table 12

The binding kinetics of humanized antibodies to human FGFR2b and human FGFR1b

EXAMPLE 5

Anti-FGFR2 IIIb antibodies inhibited SNU-16 cell proliferation in vitro

The effect of anti-FGFR2 IIIb antibodies on tumor cell proliferation in vitro was measured in SNU-16 cells. Approximately 5,000 SNU-16 cells were plated onto a 96-well plate in RPMI1640 media (Invitrogen; Cat. No. 11875-093) with no fetal bovine serum (FBS) to serum starve the cells, which were incubated at 37℃ with 5%CO₂ for 16 hours/overnight. Next, SNU-16 cells were treated with varying concentrations of anti-FGFR2 IIIb antibodies diluted in RPMI media without FBS for 30 minutes. SNU-16 cells were then treated with 100 ng/mL FGF7 with 1 μg/mL heparin (final concentrations) diluted in RPMI media and incubated at 37℃ with 5%CO₂ for 7 days. To measure the cell viability, Alamar Blue was added to the culture medium and incubated at 37℃ with 5%CO₂, following the manufacturer’s protocol. Fluorescent signal was read on a Perkin Elmer Victor Microplate Reader. The experiment was performed in duplicate. The result is shown in Figure 10.

EXAMPLE 6

Anti-FGFR2 IIIb antibodies induced ADCC in various target cells

A panel of anti-FGFR2b antibodies was evaluated for the ability to enhance the killing of Ba/F3 murine interleukin-3 dependent pro-B cell line transfected with FGFR2b (Ba/F3-FGFR2b) by PBMC or NK cell line NK-92 overexpressing CD16-158V (NK92-CD16-158V) . In vitro assays to determine the ADCC activity of the anti-FGFR2 IIIb antibodies were performed.

Ba/F3-FGFRIIIb WT, Ba/F3-FGFRIIIb AHCYL1, and KATO-III cells were used as target cells. ADCC assay testing was performed using PBMC or NK92-CD16-158V cells at an effector to target (E/T) ratio of 30: 1 and 5: 1, respectively. The target cells were incubated for 5 hours in the presence of effectors and increasing concentrations of antibody. The ADCC assay was validated using the positive control antibody Bemarituzumab. Cytotoxicity was determined by quantifying LDH release as per the manufacturer's instructions (Cyto Tox Non Radioactive Cytotoxicity Assay, Promega) . Maximal lysis was determined in the presence of 5 %Triton X-100, and spontaneous release was determined in the absence of antibody. Percentage of specific lysis was calculated as follows: Specific cytotoxicity %= (experimental-spontaneous release) / (maximal-spontaneous release ) x 100 The ADCC results are shown in Figure 11. Afucosylated anti-FGFR2 IIIb antibodies induced greater specific lysis than fucosylated anti-FGFR2 IIIb antibodies with S239D/I332E mutation in FGFR2IIIb-expressing Ba/F3 cells. Further, anti-FGFR2 IIIb antibodies showed little to no ADCC activity in wildtype Ba/F3 cells (see Figure 11C) .

EXAMPLE 7

Pharmacokinetic study of anti-FGFR2 IIIb antibodies in nude mice after single i.p. injection

The pharmacokinetic properties of anti-FGFR2 IIIb antibodies following a single intraperitoneal (i.p. ) administration (10 mg/kg) were determined in female nude mice (N = 6/group, n = 3/data point) . The concentrations of anti-FGFR2 IIIb antibodies over time in serum quantified by ELISA are depicted in Figure 12. The mean PK parameters are summarized in Table 13.

The serum concentration of anti-FGFR2b antibodies in study animals was subjected to a non compartmental pharmacokinetic analysis by using the PK solver add-in of Windows Office Excel software. The linear/log trapezoidal rule was applied in obtaining the PK parameters.

Table 13a

Pharmacokinetic Parameters of anti-FGFR2b antibodies (FR2B-16, FR2B-19, FR2B-31, FR2B-30) after a single i.p. injection of 10 mg/kg antibodies in Nude mice

Table 13b

Pharmacokinetic Parameters of anti-FGFR2b antibodies (Bemarituzumab-SI and FR2B-101. h2-SI) after a single i.p. injection of 10 mg/kg antibodies in Nude mice

Table 13c

Pharmacokinetic Parameters of anti-FGFR2b antibodies (Bemarituzumab-Afu and FR2B-101. h2-Afu) after a single i.p. injection of 10 mg/kg antibodies in Nude mice

EXAMPLE 8

Anti-FGFR2 IIIb antibodies inhibited tumor growth in SNU-16 CDX model

This example describes in vivo functional evaluation of the anti-FGFR2b antibody FR2B-101. h2 with the purpose of demonstrating anti-tumor activity. The antibodies were evaluated for their ability to inhibit the tumor growth in SNU-16 gastric cancer CDX model. A Bemarituzumab analogue (IgG1 with S239D/I332E mutation) was included for comparison.

Female Balb/c nude mice were injected subcutaneously with 10 million SNU-16 tumor cells with Matrigel in the dorsal areas. When the tumor size reaches about 150 mm3, the mice are grouped randomly and 0.2 mg/kg of mAbs are administered i.p. twice per week in a volume of 10 ml/kg. Tumor sizes are determined twice a week by measuring in two dimensions [length (a) and width (b) ] . Tumor volume is calculated according to V =ab²/2 and expressed as mean tumor volume+ SEM. The number of mice in each treatment group is 6 mice. Statistical analysis can be performed, e.g., using Student's t test.

Figure 13 A showed that both FR2B-101. h2 and Bemarituzumab administered at a low dose level of 0.2 mg/kg twice per week strongly inhibited the growth of SNU-16 gastric tumor xenografts, with FR2B-101. h2 being more potent inhibiting growth of the xenograft. Figure 13 B showed that in another independent efficacy study of SNU-16 gastric tumor xenografts, both afucosylated and S239D/I332E mutated FR2B-101. h2 are more potent than afucosylated Bemarituzumab in tumor growth inhibition.

Those skilled in the art will further appreciate that the present disclosure may be embodied in other specific forms without departing from the spirit or central attributes thereof. In that the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it is to be understood that other variations are contemplated as being within the scope of the present disclosure. Accordingly, the present disclosure is not limited to the particular embodiments that have been described in detail herein. Rather, reference should be made to the appended claims as indicative of the scope and content of the disclosure.

Claims

An antibody or antigen-binding fragment thereof that specifically binds to Fibroblast growth factor receptor 2b (FGFR2b) , comprising heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences selected from the group consisting of:

(a) SEQ ID NOs: 1-6, respectively;

(b) SEQ ID NOs: 9-14, respectively;

(c) SEQ ID NOs: 17-22, respectively;

(d) SEQ ID NOs: 25-30, respectively;

(e) SEQ ID NOs: 33-38, respectively;

(f) SEQ ID NOs: 41-46, respectively;

(g) SEQ ID NOs: 49-54, respectively;

(h) SEQ ID NOs: 57-62, respectively;

(i) SEQ ID NOs: 65-70, respectively;

(j) SEQ ID NOs: 73-78, respectively;

(k) SEQ ID NOs: 81-86, respectively;

(l) SEQ ID NOs: 89-94, respectively;

(m) SEQ ID NOs: 97-102, respectively;

(n) SEQ ID NOs: 105-110, respectively;

(o) SEQ ID NOs: 113-118, respectively;

(p) SEQ ID NOs: 121-126, respectively;

(q) SEQ ID NOs: 129-134, respectively;

(r) SEQ ID NOs: 137-142, respectively;

(s) SEQ ID NOs: 1, 242 and 3-6, respectively;

(t) SEQ ID NOs: 57-59, 243-244 and 62, respectively; and

(u) SEQ ID NOs: 81, 245, 83, 246-247 and 86, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(A) a heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, or 143;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, or 143; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, or 143; and

(B) a light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136 or 144;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136 or 144; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136 or 144.
The antibody or antigen-binding fragment thereof of claim 2, comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences of:

(a) SEQ ID NOs: 7 and 8, respectively;

(b) SEQ ID NOs: 15 and 16, respectively;

(c) SEQ ID NOs: 23 and 24, respectively;

(d) SEQ ID NOs: 31 and 32, respectively;

(e) SEQ ID NOs: 39 and 40, respectively;

(f) SEQ ID NOs: 47 and 48, respectively;

(g) SEQ ID NOs: 55 and 56, respectively;

(h) SEQ ID NOs: 63 and 64, respectively;

(i) SEQ ID NOs: 71 and 72, respectively;

(j) SEQ ID NOs: 79 and 80, respectively;

(k) SEQ ID NOs: 87 and 88, respectively;

(l) SEQ ID NOs: 95 and 96, respectively;

(m) SEQ ID NOs: 103 and 104, respectively;

(n) SEQ ID NOs: 111 and 112, respectively;

(o) SEQ ID NOs: 119 and 120, respectively;

(p) SEQ ID NOs: 127 and 128, respectively;

(q) SEQ ID NOs: 135 and 136, respectively; or

(r) SEQ ID NOs: 143 and 144, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(A) a heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 145, 146, 147, 151, 152, 153, 154, 155, 156, 157, 165, 166, 167, 168, 169, 170, 177, 178, 179, 180, 181, 182 or 183;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 145, 146, 147, 151, 152, 153, 154, 155, 156, 157, 165, 166, 167, 168, 169, 170, 177, 178, 179, 180, 181, 182 or 183; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 145, 146, 147, 151, 152, 153, 154, 155, 156, 157, 165, 166, 167, 168, 169, 170, 177, 178, 179, 180, 181, 182 or 183; and

(B) a light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO: 148, 149, 150, 158, 159, 160, 161, 162, 163, 164, 171, 172, 173, 174, 175, 176, 184, 185, 186, 187, 188, 189 or 190;

(ii) comprising an amino acid sequence at least 85%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 148, 149, 150, 158, 159, 160, 161, 162, 163, 164, 171, 172, 173, 174, 175, 176, 184, 185, 186, 187, 188, 189 or 190; or

(iii) comprising an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with the amino acid sequence of SEQ ID NO: 148, 149, 150, 158, 159, 160, 161, 162, 163, 164, 171, 172, 173, 174, 175, 176, 184, 185, 186, 187, 188, 189 or 190.
The antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences of:

(a) SEQ ID NOs: 145 and 148, respectively;

(b) SEQ ID NOs: 146 and 149, respectively;

(c) SEQ ID NOs: 147 and 150, respectively;

(d) SEQ ID NOs: 151 and 158, respectively;

(e) SEQ ID NOs: 152 and 159, respectively;

(f) SEQ ID NOs: 153 and 160, respectively;

(g) SEQ ID NOs: 154 and 161, respectively;

(h) SEQ ID NOs: 155 and 162, respectively;

(i) SEQ ID NOs: 156 and 163, respectively;

(j) SEQ ID NOs: 157 and 164, respectively;

(k) SEQ ID NOs: 165 and 171, respectively;

(l) SEQ ID NOs: 166 and 172, respectively;

(m) SEQ ID NOs: 167 and 173, respectively;

(n) SEQ ID NOs: 168 and 174, respectively;

(o) SEQ ID NOs: 169 and 175, respectively;

(p) SEQ ID NOs: 170 and 176, respectively;

(q) SEQ ID NOs: 177 and 184, respectively;

(r) SEQ ID NOs: 178 and 185, respectively;

(s) SEQ ID NOs: 179 and 186, respectively;

(t) SEQ ID NOs: 180 and 187, respectively;

(u) SEQ ID NOs: 181 and 188, respectively;

(v) SEQ ID NOs: 182 and 189, respectively; or

(w) SEQ ID NOs: 183 and 190, respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, wherein the heavy chain constant region is selected from the group consisting of human immunoglobulins IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions, preferably IgG1 heavy chain constant region, more preferably the IgG1 heavy chain constant region is afucosylated or comprises one or more mutations, the mutation comprising S239D/I332E, S239D/A330L/I332E.
The antibody or antigen-binding fragment thereof of claim 6, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 239 or 240, preferably, the heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 239 or 240.
The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof further comprises a light chain constant region, wherein the light chain constant region is selected from the group consisting of human immunoglobulins IgGκ and IgGλ light chain constant regions, preferably IgGκ light chain constant region.
The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 241, preferably, the light chain constant region having the amino acid sequence set forth in SEQ ID NO: 241.
The antibody or antigen-binding fragment thereof of any one of claims 1 and 4-5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 192, 193, 194, 199, 200, 201, 202, 203, 204, 205, 213, 214, 215, 216, 217, 218, 225, 226, 227, 228, 229 or 230, and the light chain having at least 85%, at least 90%, at least 95%, or at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 196, 197, 198, 206, 207, 208, 209, 210, 211, 212, 219, 220, 221, 222, 223, 224, 231, 232, 233, 234, 235, 236 or 237.
The antibody or antigen-binding fragment thereof of claim 10, comprising a heavy chain and a light chain comprising the amino acid sequences of:

(a) SEQ ID NOs: 192 and 196, respectively;

(b) SEQ ID NOs: 193 and 197, respectively;

(c) SEQ ID NOs: 194 and 198, respectively;

(d) SEQ ID NOs: 199 and 206, respectively;

(e) SEQ ID NOs: 200 and 207, respectively;

(f) SEQ ID NOs: 201 and 208, respectively;

(g) SEQ ID NOs: 202 and 209, respectively;

(h) SEQ ID NOs: 203 and 210, respectively;

(i) SEQ ID NOs: 204 and 211, respectively;

(j) SEQ ID NOs: 205 and 212, respectively;

(k) SEQ ID NOs: 213 and 219, respectively;

(l) SEQ ID NOs: 214 and 220, respectively;

(m) SEQ ID NOs: 215 and 221, respectively;

(n) SEQ ID NOs: 216 and 222, respectively;

(o) SEQ ID NOs: 217 and 223, respectively;

(p) SEQ ID NOs: 218 and 224, respectively;

(q) SEQ ID NOs: 225 and 232, respectively;

(r) SEQ ID NOs: 226 and 233, respectively;

(s) SEQ ID NOs: 227 and 234, respectively;

(t) SEQ ID NOs: 228 and 235, respectively;

(u) SEQ ID NOs: 229 and 236, respectively;

(v) SEQ ID NOs: 230 and 237, respectively; or

(w) SEQ ID NOs: 231 and 238, respectively.
An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding fragment thereof of any one of claims 1-11 and/or the light chain variable region or light chain of the antibody or antigen-binding fragment thereof of any one of claims 1-11.
A vector comprising the polynucleotide of claim 12.
A host cell comprising the polynucleotide of claim 12 or the vector of claim 13.
A pharmaceutical composition comprising at least one antibody or antigen-binding fragment thereof of any one of claims 1-11 and a pharmaceutically acceptable carrier.
A method for preparing antibody or antigen-binding fragment thereof as defined in any of claims 1-11 comprising the steps of:

- expressing the antibody or antigen-binding fragment thereof of any one of claims 1-11 in the host cell of claim 14; and

- isolating the antibody or antigen-binding fragment thereof from the host cell.
A method for inhibiting growth of tumor cells in a subject, comprising administering an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-11 or the pharmaceutical composition of claim 15 to the subject.
A method for reducing tumor cell metastasis in a subject, comprising administering an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-11 or the pharmaceutical composition of claim 15 to the subject.
A method for treating FGFR2b expressing cancer in a subject, comprising administering an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-11 or the pharmaceutical composition of claim 15 to the subject.
The method of claim 19, wherein the cancer is selected from the group consisting of gastric cancer, breast cancer, lung cancer, ovarian cancer, cholangiocarcinoma, pancreatic cancer, preferably triple negative breast cancer, non-small cell lung cancer, squamous lung carcinoma or intrahepatic cholangiocarcinoma.
A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11 and a detection reagent.