Anti-FGFR2B antibodies and their uses

Abstract

The present disclosure provides anti-FGFR2B antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions comprising same, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to antibodies, particularly anti-FGFR2B antibodies and antigen-binding fragments thereof, and methods for preparing said antibodies and their use for treating or preventing FGFR2B-related diseases or conditions. [Background technology]

[0002] Targeted therapies that inhibit oncogenic driver mutations have achieved significant success in chronic myeloid leukemia (CML) with BCR-ABL fusion, melanoma with BRAF V600E mutation, lung cancer with EGFR mutation, and breast cancer with HER2 amplification. However, the types of cancer with specific driver gene mutations are limited (Francavilla C, O'Brien CS. Fibroblast growth factor receptor signaling dysregulation and targeting in breast cancer. Open Biol. 2022 Feb;12(2):210373). To improve patient outcomes, the development of novel therapeutics targeting other cancer driver mutations is urgently needed.

[0003] Receptor tyrosine kinases (RTKs) are single-pass membrane proteins whose overexpression is associated with breast cancer and other cancers and with shorter disease-free survival (Templeton AJ, Diez-Gonzalez L, Ace O, Vera-Badillo F, Seruga B, Jordan J, Amir E, Pandiella A, Ocana A. Prognostic relevance of receptor tyrosine kinase expression in breast cancer: a meta-analysis. Cancer Treat Rev. 2014 Oct;40(9):1048-55, and Butti R, Das S, Gunasekaran VP, Yadav AS, Kumar D, Kundu GC. Receptor tyrosine kinases (RTKs) in breast cancer: signaling, therapeutic implications and challenges. Mol. Cancer. 2018 Feb 19;17(1):34). Upon ligand stimulation, RTKs activate several pathways, including mitogen-activated protein kinases (MAPKs), Janus kinases (JAKs) / signal transducers and activators of transcription (STATs), phospholipase C gamma (PLCγ), and phosphoinositide 3-kinase (PI3-K) (Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2010 Jun 25;141(7):1117-34.). RTK signaling regulates the response of cancer cells to perturbations in the extracellular environment, which is composed of proteins from fibroblasts, adipocytes, immune cells, the extracellular matrix, and the extended vasculature.

[0004] Fibroblast growth factor receptors (FGFRs) and their isoforms are known as RTKs. Upon binding to FGFs and specific cofactors, dimerization of the FGFR kinase domain induces phosphorylation of tyrosine (Y) residues, resulting in full receptor activation and the phosphorylation and recruitment of adaptor proteins. FGFRs are characterized by multiple alternative splicing sequences of their mRNA, resulting in the generation of various isoforms (Ornitz et al., J. Biol. Chem. 271: 15292, 1996; see also Swiss-Prot P21802 and isoforms P21802-1 to -20 for sequences of FGFR2 and its isoforms). Among these, FGFR2 type IIIb (also known as K-sam-II) is a receptor with high affinity for both FGF1 and the FGF family (FGF7, FGF10, and FGF22).

[0005] The expression patterns of FGFR2 isoforms and their ligands suggest that FGFR2 is involved in epithelial-stromal interactions (Finch et al., Dev. Dyn. 203:223, 1995), potentially mediating effects from the tumor microenvironment and promoting developmental abnormalities and cancer progression. It has been reported that KGF (FGF7) and KGFR (FGFR2IIIb) are overexpressed in many pancreatic and breast cancers (Ishiwata et al., Am. J. Pathol. 153:213, 1998, and Francavilla C, O'Brien CS. Fibroblast growth factor receptor signaling dysregulation and targeting in breast cancer. Open Biol. 2022 Feb;12(2):210373), and their coexpression correlates with poor prognosis (Cho et al., Am. J. Pathol. 170:1964, 2007). Somatic mutations in the FGFR2 gene were found in 12% of a large panel of endometrial (uterine) cancers. In some cases examined, these mutations were essential for tumor cell survival (Dutt et al., Proc. Natl. Acad. Sci. USA 105:8713, 2008). In two tumors, FGFR2 mutations were found to be consistent with the S252W substitution associated with Aper syndrome. Amplification and overexpression of FGFR2 are associated with undifferentiated, diffuse gastric cancer, which has a particularly poor prognosis (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al., Gastroenterol. 131: 1530, 2006). Summary of the Invention

[0006] There is a need in the art for antibodies that bind to FGFR2b with high affinity and inhibit FGFR2b signaling.

[0007] The present invention provides anti-FGFR2B antibodies or antigen-binding fragments thereof, and methods of making and using same, eg, for treating FGFR2B-related diseases or conditions.

[0008] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises one to three selected from HCDR1, HCDR2, and HCDR3 of the heavy chain variable region (VH), and the amino acid sequence of VH is an sequence set forth in any one of SEQ ID NOs: 43 to 49.

[0009] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises one to three selected from LCDR1, LCDR2, and LCDR3 of the light chain variable region (VL), and the amino acid sequence of the VL is a sequence set forth in any one of SEQ ID NOs: 50 to 56.

[0010] According to one aspect, the present invention provides an isolated FGFR2B antibody or antigen-binding fragment thereof, which comprises three CDRs of the heavy chain variable region (VH), i.e., HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region (VL), i.e., LCDR1, LCDR2, and LCDR3, and wherein the amino acid sequence of the VH is any one of SEQ ID NOs: 43 to 49, and the amino acid sequence of the VL is any one of SEQ ID NOs: 50 to 56.

[0011] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising three CDRs of a heavy chain variable region (VH), i.e., HCDR1, HCDR2, and HCDR3, and three CDRs of a light chain variable region (VL), i.e., LCDR1, LCDR2, and LCDR3, wherein the VH and VL are: (1) a VH comprising the amino acid sequence set forth in SEQ ID NO: 43 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 50; (2) a VH comprising the amino acid sequence set forth in SEQ ID NO: 44 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 51; (3) a VH comprising the amino acid sequence set forth in SEQ ID NO: 45 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 52; (4) VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and VL comprising the amino acid sequence set forth in SEQ ID NO: 53; (5) VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and VL comprising the amino acid sequence set forth in SEQ ID NO: 54; (6) VH comprising the amino acid sequence set forth in SEQ ID NO: 48 and VL comprising the amino acid sequence set forth in SEQ ID NO: 55; or (7) VH comprising the amino acid sequence set forth in SEQ ID NO: 49 and VL comprising the amino acid sequence set forth in SEQ ID NO: 56 The present invention provides an FGFR2B antibody or antigen-binding fragment thereof selected from the group consisting of:

[0012] According to one aspect, the invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining regions (HCDRs), one to three of HCDR1, HCDR2, and HCDR3, and (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3; (2) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or 7, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or 8, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; (3) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11; (4) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 13, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14; (5) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; or (6) Provided is an FGFR2B antibody or an antigen-binding fragment thereof, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0013] According to one aspect, the invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of light chain complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, and: (1) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; (2) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; (3) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33; (5) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36; (6) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 38, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 39; or (7) An anti-FGFR2B antibody or an antigen-binding fragment thereof is provided, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 41, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0014] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises heavy chain complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, and: (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; (2) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; (3) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; (4) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33; (5) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 13, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36; (6) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 38, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 39; or (7) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 41, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 42.

[0015] In one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth in Table 1 below. [Table C1]

[0016] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH), and the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 43 to 49.

[0017] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a light chain variable region (VL), and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 50 to 56.

[0018] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 43, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50.

[0019] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51.

[0020] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52.

[0021] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53.

[0022] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54.

[0023] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55.

[0024] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56.

[0025] In one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises any combination of heavy chain variable region (VH) and light chain variable region (VL) listed in Table 2 below. [Table C2]

[0026] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 64.

[0027] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 65.

[0028] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66.

[0029] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 67.

[0030] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 68.

[0031] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 62, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 69.

[0032] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 63, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 70.

[0033] According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises an Fc region. In a specific embodiment, the Fc region is modified by mutating one or more amino acids (e.g., introducing amino acid substitutions) to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. According to a preferred embodiment, the one or more amino acids have mutations at the following positions according to the EU numbering system: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, and 295. , 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439. In one preferred embodiment, the one or more amino acids have mutations at the following positions, numbered according to the EU numbering system: L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339 and P396. In a preferred embodiment, the one or more amino acid mutations are substitutions selected from the following, numbered according to the EU numbering system: L235V, G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L. In a preferred embodiment, the amino acid mutations comprise the following substitutions, numbered according to the EU numbering system: L235V, F243L, R292P, Y300L, and P396L (VLPYLL).In one preferred embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises an Fc region having the following substitutions according to the EU numbering system: L235V, F243L, R292P, Y300L, and P396L (VLPYLL).

[0034] According to one aspect, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided by the present invention comprise an Fc region having one or more of the following sets of substitutions: (1) L235V, F243L, R292P, Y300L, and P396L; (2) S239D and I332E; (3)S239D, A330L and I332E.

[0035] According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC) in any combination set forth in Table 3 below: [Table C3]

[0036] In one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a hypofucosylated or defucosylated constant region.

[0037] In one aspect, the isolated antibody or antigen-binding fragment thereof provided by the present invention binds to FGFR2b but not to FGFR2c.

[0038] According to one embodiment, the anti-FGFR2B antibody provided by the present invention is a monoclonal antibody.

[0039] According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention is a full-length antibody, a single domain antibody (e.g., VHH), Fab, Fab', Fab'-SH, (Fab')2, a single-chain antibody (e.g., scFv), Fv, or dAb (domain antibody).

[0040] According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises an Fc region, wherein the amino acid sequence of the Fc region is identical to or a variant of the sequence of the Fc region of human IgG1, IgG2, or IgG4.

[0041] In another aspect, the present invention provides an antagonistic anti-FGFR2B antibody comprising the CDRs of an antibody provided by the present invention. In one embodiment, such an antagonistic anti-FGFR2B antibody comprises an Fc region variant that enhances the effector function of the antibody. In one embodiment, the effector function is ADCC. In one embodiment, the antagonistic anti-FGFR2B antibody comprises an Fc region variant that is human IgG1 VLPYLL. In one embodiment, the antagonistic anti-FGFR2B antibody comprises an Fc region variant that is hypofucosylated or defucosylated human IgG1 or human IgG4.

[0042] In certain embodiments, the antibodies of the invention have one or more of the following properties: (1) Cross-reactive with human, cynomolgus monkey, mouse, and rat FGFR2B orthologues; (2) binds to human FGFR2B, particularly the extracellular domain of human FGFR2B, with high affinity, e.g., a KD value of less than 100 nM, e.g., less than 50 nM, e.g., less than 30 nM, preferably less than 10 nM or 5 nM, where the KD value is preferably measured using a surface plasmon resonance assay; (3) binds to human FGFR2B expressed on the surface of cells (e.g., T cells) with high affinity, e.g., an EC50 value of less than 100 nM, e.g., less than 50 nM, e.g., less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM or 5 nM, where the EC50 value is preferably measured using a FACS assay; (4) Inhibits the binding of human FGFR2B to its ligands FGF7, FGF10, and / or FGF22. The inhibition rate is at least 50%, for example, at least 60%, 70%, 80%, 85%, or 90%. Here, the inhibition rate is determined, for example, by ELISA. The IC50 value is preferably less than 10 nM, more preferably less than 1 nM. (5) exhibits binding affinity and / or specificity equivalent to or similar to any antibody listed in any of Tables 1 to 3; (6) inhibits (e.g., competitively inhibits) the FGFR2B binding of any antibody listed in Tables 1 to 3; (7) Binds to the same or overlapping epitope as any of the antibodies listed in Tables 1 to 3; (8) binds to an epitope different from the epitope of any antibody shown in Tables 1 to 3; (9) It has biological activity equivalent to or similar to any of the antibodies shown in Tables 1 to 3.

[0043] In another aspect, the present invention provides an isolated nucleic acid encoding any of the antibodies or fragments thereof provided by the present invention. Preferably, the nucleic acid encodes the heavy or light chain, or the heavy or light chain variable region, of an antibody of the present invention. In a preferred embodiment, the nucleic acid further comprises a sequence encoding a signal peptide.

[0044] In another aspect, the present invention provides a recombinant vector or expression vector comprising one or more nucleic acids provided by the invention, the vector being suitable for recombinant production of an antibody or antigen-binding fragment thereof provided by the invention. In one embodiment, the vector is an expression vector.

[0045] In another aspect, the present invention provides a host cell comprising one or more nucleic acids or recombinant or expression vectors provided by the present invention.

[0046] In another aspect, the present invention provides a method for producing an anti-FGFR2B antibody or antigen-binding fragment thereof, the method comprising culturing a host cell containing an expression vector encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to cause the host cell to express an antibody or fragment capable of binding to FGFR2B, and optionally recovering the expressed antibody or fragment from the host cell.

[0047] In another aspect, the present invention provides an immunoconjugate targeting FGFR2b or a pharmaceutically acceptable salt or solvate thereof, comprising an anti-FGFR2B antibody of the present invention or an antigen-binding fragment thereof conjugated to a payload.

[0048] In some embodiments, the payload is a drug, such as a cytotoxic agent.

[0049] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector or host cell provided by the present invention, and optionally at least one pharmaceutically acceptable auxiliary substance, such as a pharmaceutical carrier or pharmaceutical excipient.

[0050] In another aspect, the present invention provides a pharmaceutical combination comprising an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, or host cell provided by the present invention and one or more additional therapeutic agents.

[0051] In another aspect, the present invention also provides use of an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, or host cell provided by the present invention in the preparation of a medicament for treating an FGFR2B-related disease or condition.

[0052] In another aspect, the present invention also provides a method for killing FGFR2B-positive cancer cells in vitro or in vivo, the method comprising contacting a cell population containing FGFR2B-positive cancer cells with an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, pharmaceutical composition or drug combination provided by the present invention, or administering to a subject in need thereof an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, pharmaceutical composition or drug combination provided by the present invention.

[0053] In another aspect, the present invention provides use of an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, pharmaceutical composition, or pharmaceutical combination of the present invention in the preparation of a medicament for treating cancer, preferably selected from breast cancer (e.g., triple-negative breast cancer), gastric cancer, GEJ cancer, esophageal cancer, lung cancer (e.g., squamous cell NSCLC), ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, bile duct cancer, and pancreatic cancer.

[0054] In another aspect, the present invention provides the use of an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, pharmaceutical composition, or pharmaceutical combination in the treatment or prevention of cancer, preferably selected from breast cancer (e.g., triple-negative breast cancer), gastric cancer, GEJ cancer, esophageal cancer, lung cancer (e.g., squamous cell NSCLC), ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, bile duct cancer, and pancreatic cancer.

[0055] According to another aspect, the present invention also provides a method for treating or preventing an FGFR2B-related disease or condition, the method comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof provided by the present invention, or a nucleic acid, vector, host cell, or pharmaceutical composition or drug combination comprising the same. According to one embodiment, the FGFR2B-related disease or condition is cancer, such as breast cancer or gastric cancer.

[0056] The anti-FGFR2B antibodies or antigen-binding fragments thereof of the present invention may be combined with other therapeutic agents or treatments for treating or preventing FGFR2B-related diseases or conditions.

[0057] In another aspect, the present invention also provides a method for detecting FGFR2B in a sample using the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention, which can be used for the diagnosis / detection of an FGFR2B-related disease or condition.

[0058] The present invention also encompasses any combination of the aspects described in this disclosure. Any aspect or any combination thereof described in this disclosure is applicable to all of the antibodies against FGFR2B or fragments thereof, methods, and uses of the present invention described in this disclosure. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 shows epitope binning of the humanized anti-FGFR2B monoclonal antibody of the present invention. [Figure 2] FIG. 2 shows the ADCC of the chimeric anti-FGFR2B mAb of the present invention against KATO-III cells. [Figure 3] FIG. 3 shows the binding specificity of the humanized anti-FGFR2B monoclonal antibodies of the present invention tested for binding to other FGFR family members. [Figure 4] FIG. 4 shows the species cross-reactivity of the humanized anti-FGFR2B monoclonal antibody of the present invention. [Figure 5] FIG. 5 shows the results of an ELISA test for the inhibition of humanized mAbs on the interaction between FGFR2b and FGF7. [Figure 6] FIG. 6 shows the flow cytometry results for binding of FGFR2b antibodies to 293T_hFGFR2b (A) and 293T_hFGFR2c (B) cells. [Figure 7]FIG. 7 shows the ADCC reporter biological activity of humanized FGFR2b antibodies targeting KATO-III cells and KYSE-180 cells. [Figure 8] Figure 8 shows the inhibition of FGF7-induced MCF7 cell proliferation by several FGFR2b antibodies. MCF7 cells were incubated in serum-free medium and treated with either untreated (medium) or hIgG isotype or several FGFR2b antibodies (30 μg / mL) for 72 hours in the presence or absence of FGF7 (25 ng / mL). Cell proliferation was assessed by the Cell Titer-Glo® luminescent cell viability assay. [Figure 9] Figure 9 shows the results of an HTRF assay to detect FGF7- or FGF10-induced phosphorylation of FGFR2 and ERK1 / 2 proteins in SNU-16 cells. (A-B) FGF7-induced phosphorylation of FGFR2 (A) and ERK1 / 2 (B) was inhibited by several FGFR2b antibodies. (C-D) FGF10-induced phosphorylation of FGFR2 (C) and ERK1 / 2 (D) was inhibited by several FGFR2b antibodies. [Figure 10] Figure 10 shows the ADCC activity of multiple FGFR2b antibodies targeting KATO-III cells by human PBMCs. (A) The primary ADCC activity in donors with the FcγRIIIA genotype was 158V / V. (B) The primary ADCC activity in donors with the FcγRIIIA genotype was 158V / F. [Figure 11] Figure 11 shows the plasma concentration-time curves. [Figure 12] FIG. 12 shows the efficacy of humanized mAbs against the SNU16 tumor model in Balb / c nude mice. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention provides anti-FGFR2B antibodies or antigen-binding fragments thereof that have unique CDR sequences that bind to human FGFR2B with high affinity and specificity and preferably mediate ADCC more effectively. The anti-FGFR2B antibodies or antigen-binding fragments thereof provided by the present invention can be used alone or in combination with other therapies to treat diseases or conditions such as cancer, inflammation, and autoimmune diseases.

[0061] definition Unless otherwise indicated, the present invention will be practiced using conventional techniques within the level of skill of one in the art of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology.

[0062] In order to make the present invention more readily understandable, some scientific and technical terms are defined below. Unless otherwise specified in this disclosure, all scientific and technical terms used in this disclosure have the meaning commonly understood by those skilled in the art to which this invention pertains. For definitions and terms in this field, reference may be made in particular to the specialist publication "Current Protocols in Molecular Biology (Ausubel)." Abbreviations for amino acid residues are indicated by the standard three-letter and / or one-letter codes used for the 20 L-amino acids commonly used in this field.

[0063] As used in this application and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0064] The term "about" means a value or integer within an acceptable error range for a particular value or integer, as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, as practiced by one of ordinary skill in the art. Alternatively, "about" can refer to a range of up to 5%, 10%, or 20% (i.e., ±5%, ±10%, or ±20%).

[0065] When connecting two or more alternatives, the term "and / or" shall be construed to mean any one alternative or any two or more alternatives in combination.

[0066] As used herein, the term "comprise" or "include" means including the stated elements, integers, or steps, but does not exclude other elements, integers, or steps. In this disclosure, the term "comprise" or "include" includes "consisting of" the stated elements, integers, or steps, unless otherwise specified. For example, reference to an antibody variable region "containing" a particular sequence is intended to encompass an antibody variable region consisting of that particular sequence.

[0067] The term "FGFR2B" refers to the four-member FGFR family (FGFR1-4), whose cognate ligands, fibroblast growth factors (FGFs), comprise a family of 22 members (FGF1-14 and FGF16-23). ​​In the present disclosure, unless otherwise specified, this term refers to native FGFR2B from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length," unprocessed FGFR2B, as well as any form of FGFR2B or fragment thereof generated by intracellular processing. This term also encompasses naturally occurring variants of FGFR2B, such as splice variants and allelic variants. In one embodiment, FGFR2B refers to full-length FGFR2B from humans, or a fragment thereof (e.g., a mature fragment lacking the signal peptide). According to one aspect, human FGFR2B refers to mature FGFR2B identical to the amino acid sequence set forth in Accession No. Uniprot#P21802 (amino acid residues 1-21 are a leader peptide), or a fragment thereof (such as the extracellular domain comprising AA22-377 thereof). According to one aspect, this term also encompasses fusion proteins comprising FGFR2B or a fragment thereof (such as the extracellular domain thereof), such as a fusion protein comprising the human FGFR2B extracellular domain and an Fc region.

[0068] In the present disclosure, the term "FGFR2B ligand" or "FGF7 / 10" refers to the natural ligand of FGFR2B or a functional variant thereof.

[0069] The term "antibody" broadly refers to an immunoglobulin (Ig) molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, or an antigen-binding fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding properties of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, and non-limiting examples are described below. In this disclosure, the term "antibody" can refer to any form of antibody that possesses the desired biological activity. Thus, the term is used in the broadest sense. Examples include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, CrossMab antibodies, or camelized single-domain antibodies.

[0070] In this disclosure, the term "affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, in this disclosure, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Methods for measuring binding affinity are known in the art, including surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., ForteBio).

[0071] The terms "specific binding" or "specifically binding," with respect to the interaction of an antibody, binding protein, or peptide with a second chemical entity, mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical entity. For example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. Generally, if an antibody is specific for epitope "A," then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody. According to the present disclosure, a specific binding protein has a K of 1 nm, e.g., 10 nm or less. D and binds to the corresponding antigen.

[0072] In this disclosure, "k on The term "k" (also written as "k on"), as known in the art, refers to the on-rate constant of binding of a binding protein (e.g., an antibody) to an antigen to form a complex, e.g., an antibody / antigen complex. on " is also synonymous with the term "association rate constant" or "ka," and these terms are used interchangeably in this disclosure. This value indicates the rate at which an antibody binds to its target antigen, or the rate of complex formation between an antibody and an antigen, as shown in the following formula: Antibody (“Ab”) + Antigen (“Ag”) → Ab-Ag

[0073] In this disclosure, "k off The term "Koff" (also "koff") refers to the dissociation rate constant, or "dissociation rate constant," for a binding protein (e.g., an antibody) to dissociate from a binding complex (e.g., an antibody / antigen complex), as known to those of skill in the art. This value indicates the rate at which an antibody dissociates from its target antigen, or the rate at which an Ab-Ag complex dissociates into free antibody and antigen over time, as shown in the following equation: Ab + Ag ← Ab-Ag

[0074] In this disclosure, the term "KD" (also written as "Kd") refers to the "equilibrium dissociation constant," which may be obtained by titration measurements at equilibrium or by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD) are used to describe the binding affinity of an antibody to an antigen. Methods for determining the association and dissociation rate constants are well known in the art. Fluorescence-based techniques offer high sensitivity and the ability to test samples in physiological buffer at equilibrium. BIAcore (登録商標) Other experimental techniques and instruments such as (Biomolecular Interaction Analysis) assays can also be used (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). For example, Octet (登録商標) Biolayer interferometry (BLI) using the RED96 system (Pall Forte Bio LLC) is another affinity assay technique. Additionally, KinExA, available from Sapidyne Instruments (Boise, ID), (登録商標) A Kinetic Exclusion Assay (Kinetic Exclusion Assay) can also be used.

[0075] The terms "antagonistic anti-FGFR2B antibody," "FGFR2B inhibitor," "FGFR2B antagonist antibody," "antagonist FGFR2B antibody," and "FGFR2B antibody antagonist" are used interchangeably in the present disclosure. These terms include antibodies capable of inhibiting and / or blocking FGFR2B-mediated biological signaling activity. According to certain aspects, the FGFR2B antagonist antibody inhibits or suppresses FGFR2B-induced signaling pathways and / or suppresses or reduces FGFR2B-mediated cellular responses, such as cancer cell proliferation and cancer cell survival, by, for example, inhibiting or substantially reducing binding of FGFR2B to an FGFR2B ligand.

[0076] The term "FGFR2B-related disease or condition," as used herein, refers to a non-physiological condition associated with the expression, function, or activity of FGFR2B, or associated with the activity of FGFR2B-mediated signaling. Such diseases or conditions include, but are not limited to, cancer, inflammation, and autoimmune diseases. In a preferred embodiment, blocking FGFR2B signaling is beneficial to the disease.

[0077] The terms "immune response" and "immune reaction" are used interchangeably in this disclosure and refer to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, and granulocytes, as well as soluble macromolecules (e.g., antibodies, cytokines, and complement) produced by these cells or the liver, which result in selective damage, destruction, or elimination of pathogens invading the human body, cells or tissues infected by pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. According to one aspect, the FGFR2B antibody antagonists of the invention inhibit or reduce immune reactions, for example, reducing immune rejection in graft-versus-host disease. According to one aspect, the FGFR2B antibody agonists of the invention enhance anti-tumor immune responses.

[0078] The term "signal transduction," as used herein, refers to a biochemical causal process that typically is initiated by a protein-protein interaction, such as the binding of FGF7 / 10 (ligand) to FGFR2B (receptor), resulting in the transmission of a signal from one part of a cell to another. Generally, transduction involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that result in the signal transduction. The penultimate process typically involves nuclear events, resulting in changes in gene expression.

[0079] As used herein, the terms "activity" and "biological activity," or "biological property" and "biological feature," are used interchangeably in the present disclosure and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize FGFR2B activity in vivo or in vitro, the ability to enhance or activate FGFR2B in vivo or in vitro, the IC50 for FGFR2B inhibition of FGF7 / 10, the IC50 for inhibition of FGFR2B-FGF7 / 10-mediated cell proliferation, the in vivo stability of the antibody, and the immunogenicity of the antibody. Other distinguishing antibody biological properties or characteristics known in the art include, for example, (species) cross-reactivity (i.e., general cross-reactivity with non-human homologs of the targeted peptide or with other proteins or tissues) and the ability to maintain high levels of antibody expression in mammalian cells. The above properties or characteristics can be observed, determined, or assessed using techniques well known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasmon resonance assays of tissue sections of different origins (including human, primate, and other origins), in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry.

[0080] The terms "whole antibody," "full-length antibody," and "intact antibody" are used interchangeably in this disclosure and refer to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this disclosure) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this disclosure) and a light chain constant region. The light chain constant region consists of one domain, namely, CL. The VH and VL regions can be further divided into conserved regions (commonly called "framework regions" (FR)) and intervening hypervariable regions (commonly called "complementarity-determining regions" (CDRs)). A "complementary determining region" or "CDR region" or "CDR" is a region in the variable domain of an antibody that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains multiple residues that contact antigens ("antigen contact sites"). CDRs are primarily involved in binding to an epitope. The CDRs of each heavy and light chain are usually referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. The CDRs present in the heavy chain variable domain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, respectively, and the CDRs present in the light chain variable domain of an antibody are referred to as LCDR1, LCDR2, and LCDR3, respectively. Each VH or VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The constant region is not involved directly in binding the antibody to an antigen, but has multiple effector functions.

[0081] For a given VH or VL amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined by any one or a combination of several well-known schemes, such as the Chothia scheme (Chothia et al., "Canonical Structures for the Hypervariable Regions of Immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987)), the Kabat scheme (Kabat et al., "Sequences of Proteins of Immunological Interest," 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London), and the North scheme (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)). The boundaries of the CDRs of the anti-FGFR2B antibodies of the present invention can be determined by any method or a combination of methods known to those skilled in the art, as well as by manual evaluation.

[0082] Antibody light chains are classified into two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant region. Antibody heavy chains are classified into five major classes, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of their constant region. Some of these classes can be further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0083] An "IgG antibody" means that the heavy chain constant region of the antibody is in the form of IgG. For example, an IgG2 antibody means that the heavy chain constant region is of the IgG2 isotype.

[0084] The term "antigen-binding fragment" of an antibody, as used herein, includes antibody fragments or derivatives. Typically, an antigen-binding fragment contains at least a fragment of the antigen-binding or variable region of an antibody (e.g., one or more CDRs) and retains at least some of the binding properties of the antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., sc-Fv); nanobodies; and multispecific antibodies composed of multiple antibody fragments. When antigen-binding activity is expressed in molar concentrations, a binding fragment or derivative typically retains at least 10% of the antigen-binding activity of the antibody from which it is derived. Preferably, a binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding activity of the antibody from which it is derived.

[0085] An antibody or antigen-binding fragment thereof may include conservative or non-conservative amino acid substitutions (referred to as "conserved variants" or "functionally conserved variants" of an antibody) that do not significantly alter its biological activity. In a preferred aspect, the conservative substitutions are exemplary conservative substitution residues shown in Table A, preferably the preferred conservative amino acid substitution residues shown in Table A. [Table A]

[0086] An epitope is a region of an antigen to which an antibody binds. Epitopes are formed from consecutive amino acids or non-contiguous amino acids juxtaposed on the tertiary structure of a protein.

[0087] As used herein, the term "isolated anti-FGFR2B antibody or antigen-binding fragment thereof" refers to an anti-FGFR2B antibody or antigen-binding fragment thereof in an isolated state. For example, "isolated" means that a molecule is substantially free of nucleic acids, proteins, lipids, sugars, or other substances, such as cellular debris or growth medium. However, as will be appreciated by those of skill in the art, the term "isolated" does not contemplate the complete absence of such substances, nor does it imply the absence of water, buffers, or salts, unless present in amounts that would substantially interfere with the experimental or therapeutic use of the antibody described in this disclosure. According to certain embodiments, the purity of an isolated antibody or antigen-binding fragment is greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0088] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; that is, the individual antibodies comprising the population are identical except for minor amounts of naturally occurring mutations. Monoclonal antibodies are highly specific and bind to a single epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies that target (i.e., have specificity for) multiple different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, but should not be construed as requiring a particular method for producing the antibody.

[0089] The term "chimeric antibody," as used herein, refers to an antibody that has the variable region of a first antibody and the constant region of a second antibody derived from different species. Typically, the variable region sequences are obtained from antibodies of laboratory animals such as rodents, and the constant region sequences are obtained from human antibodies. This selection makes the resulting chimeric antibody less likely to provoke an adverse immune response in humans than antibodies derived from laboratory animals.

[0090] The term "humanized antibody," as used herein, refers to antibody forms containing sequences derived from both human and non-human (e.g., mouse, rat) antibodies. Humanized antibodies typically contain at least one, and typically two, variable regions, with all or substantially all of the hypervariable loops of the variable regions corresponding to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions corresponding to those of a human immunoglobulin. Humanized antibodies may also contain at least a portion of a constant region (e.g., an Fc region) derived from a human immunoglobulin, as appropriate. As is well known to those skilled in the art, amino acid mutations may be introduced into a humanized antibody (e.g., in the variable region, framework region, and / or constant region (if present)) to, for example, improve certain antibody properties. Such antibody forms are also included within the scope of the "humanized antibody" of the present invention.

[0091] As is well known to those skilled in the art, an antibody may have a sugar chain present in the cell that produces the antibody. For example, an antibody produced by a mouse, a mouse cell, or a hybridoma derived from a mouse cell may contain a mouse sugar chain. Alternatively, an antibody produced by a rat, a rat cell, or a hybridoma derived from a rat cell may contain a rat sugar chain.

[0092] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native Fc regions and variant Fc regions. Native Fc regions encompass various naturally occurring immunoglobulin Fc sequences, including various Ig subtypes and their cognate Fc regions (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520). In one embodiment, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In this disclosure, unless otherwise specified, amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0093] As used herein, the terms "Fc region variant" and "variant Fc region" are used interchangeably in this disclosure and refer to an Fc region polypeptide comprising amino acid mutations relative to a native sequence Fc region. Fc region variants of the present invention are defined according to the amino acid mutations that comprise them. Thus, for example, L235V refers to an Fc region variant in which leucine is substituted with valine at position 235 of the EU index relative to the parent polypeptide. Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring and non-naturally occurring amino acids. Variants may also include non-naturally occurring amino acids.

[0094] The term "Fc receptor" or "FcR," as used herein, refers to a receptor that binds to the Fc region of an antibody. In certain embodiments, the FcR is a native-sequence human FcR. In certain embodiments, the FcR is an FcγR (gamma receptor), which includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses. It also includes allelic variants and alternatively spliced ​​forms of these receptors. FcγRII includes FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic region (e.g., Annu. Rev. Immunol. 15: 203-234 (1997)). For a review of FcR, see, e.g., Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995)). Other FcRs, including those identified in the future, are encompassed by the term "FcR" in this disclosure. The terms "Fc receptor" or "FcR" also include the neonatal receptor FcRn. It plays a role in transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and regulating immunoglobulin homeostasis.Methods for measuring FcRn binding are known (see, e.g., Ghetie and Ward, Immunol. Today 18 (12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279 (8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)). WO 2000 / 42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).

[0095] The term "pharmaceutically acceptable auxiliary substance" refers to diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), pharmaceutical excipients, pharmaceutical carriers, stabilizers, and the like, administered with an active substance.

[0096] The term "pharmaceutical composition" means a composition that contains an active ingredient, present in a form such that the biological activity of such active ingredient is effective, and that does not contain additional ingredients that would cause unacceptable toxicity to a subject to which the composition is administered.

[0097] The term "therapeutic agent" as used herein includes substances that are effective in the prevention or treatment of cancer and related diseases.

[0098] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents cell function or causes cell death or destruction.

[0099] "Chemotherapeutic agents" include small chemical molecules useful in the treatment of cancer or immune system disorders.

[0100] A "small molecule drug" refers to a low-molecular-weight compound capable of modulating biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules have better cell membrane permeability, are less susceptible to degradation, and are less likely to provoke an immune response than large molecules.

[0101] The term "immunomodulator," as used herein, refers to a natural or synthetic active agent or drug that modulates (e.g., suppresses or enhances) the immune response. The immune response can be a humoral or cellular response. In one example, an immunomodulator includes an immunosuppressant that inhibits an immune response, e.g., an immunosuppressant that beneficially inhibits the immune response in inflammatory and autoimmune diseases. In another example, a modulator includes an active agent or drug that enhances the immune response, e.g., an active agent or drug that beneficially enhances an anti-cancer immune response in cancer treatment.

[0102] The terms "cancerous" and "cancer" refer to or describe physiological disorders in mammals that are generally characterized by uncontrolled cell growth. This definition includes benign tumors, malignant tumors, dormant tumors, and micrometastases. "Cancer" includes, but is not limited to, solid tumors and hematologic cancers. Examples of various cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, and the like.

[0103] The term "isolated nucleic acid" refers to a polynucleotide (e.g., genomic, cDNA, synthetic, or a combination thereof) that is not associated by human intervention with all or part of a naturally occurring polynucleotide, or is operably associated with a non-naturally occurring polynucleotide, or is not found in nature as part of a larger sequence.

[0104] The term "vector," as used herein, refers to any recombinant polynucleotide construct that can be used for transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Furthermore, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to in the present disclosure as "expression vectors." An expression vector refers to a nucleic acid that is capable of replicating and expressing a target gene when the vector is transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, amplification within the host.

[0105] In this disclosure, "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell to be transformed. Examples include, but are not limited to, transfection, viral infection, electroporation, lipofection, particle bombardment, etc. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replicating either as an autonomously replicating plasmid or as part of the host chromosome. Also included are cells that transiently express the inserted DNA or RNA for a period of time.

[0106] The term "recombinant host cell" (or simply "host cell") refers to a cell into which exogenous DNA has been introduced. In one embodiment, the host cell contains two or more (e.g., a plurality) nucleic acids encoding an antibody, such as those described in U.S. Pat. No. 7,262,028. Such terms are intended to refer not only to the particular subject cell but also to the progeny of that cell. Because certain variations may arise in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used in this disclosure. According to certain aspects, host cells include prokaryotic and eukaryotic cells selected from all kingdoms of life. In other embodiments, eukaryotic cells include protist, fungal, plant, and animal cells. In another embodiment, exemplary host cells include, but are not limited to, the prokaryotic cell line Escherichia coli, the mammalian cell lines CHO, HEK293, Jurkat, COS, NS0, SP2, and PER.C6, the insect cell line Sf9, and the fungal cell Saccharomycetes cerevisiae.

[0107] As used herein, the terms "subject" or "patient" or "individual" include human or non-human animals. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0108] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, effectively prevents or ameliorates one or more symptoms of a disease or condition, or the onset of a disease or condition. A therapeutically effective dose refers to the amount of an antibody or antigen-binding fragment thereof sufficient to result in improvement of symptoms, e.g., the amount necessary to treat, cure, prevent, or ameliorate the associated pathology, or to accelerate the rate of treatment, cure, prevention, or amelioration. When only an active ingredient is administered to an individual, the therapeutically effective dose refers to that ingredient alone. When multiple ingredients are administered in combination, the therapeutically effective dose refers to the total amount of active ingredients that contribute to the therapeutic effect, regardless of the combination, order of administration, or whether they are administered simultaneously. An effective amount of a therapeutic agent is an amount that improves a diagnostic criterion or parameter by at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0109] As used herein, the terms "to treat" or "treat" or "treatment" include 1) therapeutic measures that cure, alleviate, or relieve the symptoms of a diagnosed condition or disease and / or arrest the progression of a diagnosed condition or disease, and 2) prophylactic measures that prevent or delay the onset of a condition or disease. Subjects receiving treatment therefore include individuals who are afflicted with a disease, individuals who are susceptible to a disease, and individuals in whom it is desired to prevent a disease. In one aspect, the invention relates to the treatment of a disease or condition. In another aspect, the invention relates to the prevention of a disease or condition.

[0110] According to certain aspects of the present invention, "treatment" of a disease or condition refers to ameliorating the disease or condition (i.e., alleviating, preventing, or inhibiting the progression of the disease or at least one of its clinical symptoms). According to other aspects, "treatment" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be discernible to the patient. According to other aspects, "treatment" refers to physical modulation of the disease or condition (e.g., stabilization of discernible symptoms), physiological modulation (e.g., stabilization of physical parameters), or both. Methods for assessing the treatment and / or prevention of disease are generally known in the art unless expressly stated otherwise in this disclosure.

[0111] According to yet another aspect of the invention, "prevention" of a disease or condition includes suppressing the onset or development of the disease or condition, or symptoms of a particular disease or condition. In some aspects, subjects with a family history of cancer are candidates for preventative treatment. Generally, in the context of cancer, "prevention" refers to administering an agent before the symptoms or conditions of cancer appear, particularly to subjects at risk of cancer.

[0112] According to certain embodiments, after "treating" cancer with the methods of the present invention, an individual patient is considered to have been successfully treated if the patient exhibits one or more of the following: a reduction in the number or complete elimination of cancer cells; a reduction in tumor size; a reduction in or elimination of cancer cell invasion into surrounding organs (e.g., metastasis of cancer cells to soft tissue or bone); a reduction in or elimination of tumor metastasis; a reduction in or elimination of tumor growth; a palliative treatment for one or more symptoms associated with a particular cancer; a reduction in incidence and mortality; an improvement in quality of life; a reduction in the incidence, frequency, or tumorigenicity of tumors; a reduction in the number or frequency of cancer stem cells within the tumor; differentiation of tumor cells to a non-neoplastic state; or some combination of these effects.

[0113] "Inhibition of tumor growth" refers to any mechanism that inhibits tumor cell growth. In some embodiments, tumor cell growth is inhibited by slowing tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by halting tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by killing tumor cells. In some embodiments, tumor cell growth is inhibited by inducing apoptosis in tumor cells. In some embodiments, tumor cell growth is inhibited by inducing differentiation of tumor cells. In some embodiments, tumor cell growth is inhibited by depriving tumor cells of nutrients. In some embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In some embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

[0114] As used herein, "sequence identity" refers to the degree of sequence identity based on a one-to-one comparison of nucleotides or amino acids within a comparison window. "(Percentage) sequence identity" can be calculated as follows: Compare two optimally aligned sequences within the comparison window. Determine the number of positions in the two sequences that have the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Trh, Gly, Val, Leu, Ile, Phe, Tr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and met) to obtain the number of matching positions. Divide the number of matching positions by the total number of positions within the comparison window (i.e., the window size). Multiply the result by 100 to obtain the percentage sequence identity. Optimal alignment for determining percent sequence identity can be achieved using various methods known in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including the algorithms necessary to achieve maximal alignment over the entire length of the sequence or over the target sequence region being compared. In the present invention, for antibody sequences, amino acid sequence identity is determined by optimally aligning a candidate antibody sequence with a reference antibody sequence, and in a preferred embodiment, is determined according to the Kabat numbering system.

[0115] Anti-FGFR2B antibodies and their production The antibodies of the present invention can be produced by any method suitable for producing antibodies. Any suitable form of FGFR2B can be used as the immunogen (antigen) for producing the antibodies. For example, any mutant of FGFR2B or a fragment thereof can be used as the immunogen. In one embodiment, hybridoma cells producing mouse monoclonal anti-human FGFR2B antibodies can be produced by methods well known in the art. Examples of these methods include, but are not limited to, the hybridoma technology first developed by Kohler et al. (1975) (Nature 256: 495-497). Preferably, mouse spleen cells are isolated and fused with a mouse myeloma cell line by PEG or electrofusion according to standard protocols. Hybridoma cells secreting antibodies with FGFR2B-binding activity are then screened. The DNA sequence of the immunoglobulin variable region can be detected from the hybridoma cells of the present invention by a degenerate primer PCR-based method.

[0116] Antibodies derived from rodents (e.g., mice) can cause undesirable antibody immunogenicity when used as therapeutic agents in vivo. Repeated use can trigger immune responses against therapeutic antibodies in humans. Such immune responses can result in, at a minimum, a loss of therapeutic efficacy and, in severe cases, potentially fatal allergic reactions. One approach to reducing the immunogenicity of rodent antibodies is to create chimeric antibodies by fusing mouse variable regions with human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the presence of intact rodent variable regions in chimeric antibodies can potentially cause harmful immunogenicity in patients.

[0117] Grafting CDRs from rodent variable regions onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences. For humanized antibodies of the present invention, murine CDR regions can be inserted into human germline frameworks using methods known in the art. See Winter et al., U.S. Pat. No. 5,225,539, and Queen et al., U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370.

[0118] The precise amino acid sequence boundaries of the variable region CDRs of antibodies of the present invention can be determined using any well-known method, such as Kabat, Chothia, AbM, Contact, or North. It should be noted that the boundaries of the variable region CDRs of the same antibody may differ when defined using different definition systems. That is, the CDR sequences of the variable regions of the same antibody defined using different alignment systems will be different. Therefore, when defining an antibody having a specific CDR sequence as defined in the present invention, the scope of such an antibody also includes antibodies that contain the specific CDR sequence within their variable region sequence but have CDR boundaries that differ from those specified in the present invention due to the application of a different scheme (e.g., a different definition system, a combination thereof, etc.).

[0119] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs differ from antibody to antibody, only a small portion of the amino acid positions in the CDRs are directly involved in antigen binding. To provide a "smallest binding unit" for antigen binding, the minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM, and North schemes. The smallest binding unit can be a subset of CDR residues. As will be understood by those skilled in the art, the remaining residues of the CDR sequence can be determined according to the antibody structure and protein folding. Therefore, the present invention also encompasses any modifications of the CDRs presented in this disclosure. According to one embodiment, in a variant of the CDR of the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention, the amino acid residues of the smallest binding unit remain unchanged, while other CDR residues defined according to Kabat or IMGT can be replaced with conservative amino acid residues.

[0120] In one embodiment, the present invention also provides antibodies with altered effector function(s). The term "effector function" refers to a biological activity attributable to the Fc region of an antibody. Such functions may vary depending on the class of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these classes are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; recruitment of immune cells; and antibody cross-linking mediated by binding of the Fc region to FcR receptors on the cell surface. As will be appreciated by those skilled in the art, an appropriate antibody Fc region sequence can be selected depending on desired requirements, such as whether or not to recruit the immune system to kill target cells or whether or not to cross-link antibodies through interaction with FcR. For example, if immune system recruitment and target cell killing are desirable properties of an antibody of interest, the Fc region of the antibody can be selected or further modified to enhance binding to activating FcγR receptors and / or complement, e.g., to promote ADCC or CDC effector function. Furthermore, by selecting or mutating the Fc region so that the antibody selectively binds to one or more Fc receptors and reduces or eliminates binding to another Fc receptor or receptors, antibody effector function can be tailored, such as increasing the antibody's cross-linking ability with varying degrees of ADCC activity.See, for example, Xinhua Wang et al., IgG Fc engineering to modulate antibody effector functions, Protein Cell 2018, 9 (1): 63-73, DOI 10.1007 / s13238-017-0473-8; Shields RL, High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR, 2001, J Biol Chem. 2001 Mar 2; 276 (9): 6591-604. Epub 2000 Nov 28, etc.

[0121] According to one aspect, Fc region mutants can be produced by introducing one or more amino acid modifications into the Fc region of an antibody provided by the present invention. Fc region mutants can have a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions. For example, various modifications that can be introduced into human IgG1 to enhance or reduce its FcγR binding and enhance or reduce the corresponding functions are summarized in Bruhns and Joensson, published in Immunol Rev. 2015 Nov; 268(1):25-51, page 44.

[0122] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises one to three selected from HCDR1, HCDR2, and HCDR3 of the heavy chain variable region (VH), and the amino acid sequence of VH is any one of the sequences set forth in SEQ ID NOs: 43 to 49.

[0123] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises one to three selected from LCDR1, LCDR2, and LCDR3 of the light chain variable region (VL), and the amino acid sequence of the VL is a sequence set forth in any one of SEQ ID NOs: 50 to 56.

[0124] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising: three CDRs of the heavy chain variable region (VH), i.e., HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region (VL), i.e., LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of the VH is a sequence set forth in any one of SEQ ID NOs: 43 to 49, and the amino acid sequence of the VL is a sequence set forth in any one of SEQ ID NOs: 50 to 56.

[0125] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising three CDRs of a heavy chain variable region (VH), i.e., HCDR1, HCDR2, and HCDR3, and three CDRs of a light chain variable region (VL), i.e., LCDR1, LCDR2, and LCDR3; wherein the VH and VL are: (1) a VH comprising the amino acid sequence set forth in SEQ ID NO: 43 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 50; (2) a VH comprising the amino acid sequence set forth in SEQ ID NO: 44 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 51; (3) a VH comprising the amino acid sequence set forth in SEQ ID NO: 45 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 52; (4) VH comprising the amino acid sequence set forth in SEQ ID NO: 46 and VL comprising the amino acid sequence set forth in SEQ ID NO: 53; (5) VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and VL comprising the amino acid sequence set forth in SEQ ID NO: 54; (6) VH comprising the amino acid sequence set forth in SEQ ID NO: 48 and VL comprising the amino acid sequence set forth in SEQ ID NO: 55; or (7) VH comprising the amino acid sequence set forth in SEQ ID NO: 49 and VL comprising the amino acid sequence set forth in SEQ ID NO: 56 The present invention provides an anti-FGFR2B antibody or an antigen-binding fragment thereof selected from the group consisting of:

[0126] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising heavy chain complementarity-determining regions (HCDRs), 1 to 3 of HCDR1, HCDR2, and HCDR3, and satisfying any of the following:

[0127] (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0128] (2) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or 7, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or 8, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0129] (3) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0130] (4) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 13, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0131] (5) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0132] (6) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0133] According to one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising light chain complementarity-determining regions (LCDRs), 1 to 3 of LCDR1, LCDR2, and LCDR3, and satisfying any of the following:

[0134] (1) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0135] (2) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0136] (3) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0137] (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0138] (5) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0139] (6) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 38, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 39, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0140] (7) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 41, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 42, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0141] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises heavy chain complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, and satisfies any of the following:

[0142] (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0143] (2) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0144] (3) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0145] (4) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0146] (5) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 13, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto. LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, or a sequence optionally having at least one and three, two or one or less further amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, or a sequence optionally having at least one and three, two or one or less further amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36, or a sequence optionally having at least one and three, two or one or less further amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0147] (6) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto. and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, or a sequence optionally having at least one and three, two or one or less further amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 38, or a sequence optionally having at least one and three, two or one or less further amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 39, or a sequence optionally having at least one and three, two or one or less further amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0148] (7) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21, or a sequence optionally having at least one and three, two, or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto. LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 41, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 42, or a sequence optionally having at least one and three, two or one or less amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0149] In one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 set forth in Table 1 below. [Table 1]

[0150] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH), and the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 43 to 49.

[0151] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a light chain variable region (VL), and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 50 to 56.

[0152] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 43, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50.

[0153] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51.

[0154] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52.

[0155] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53.

[0156] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54.

[0157] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55.

[0158] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56.

[0159] According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) in any combination listed in Table 2 below: [Table 2]

[0160] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 64.

[0161] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 65.

[0162] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66.

[0163] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 67.

[0164] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 68.

[0165] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 62, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 69.

[0166] According to one aspect, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 63, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 70.

[0167] According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises an Fc region. In a specific embodiment, the Fc region is modified by mutating one or more amino acids (e.g., introducing amino acid substitutions) to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. According to a preferred embodiment, the one or more amino acids have mutations at the following positions according to the EU numbering system: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, and 295. , 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439. In one preferred embodiment, the one or more amino acid mutations are at the following positions according to the EU numbering system: L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396. ... The substitutions are selected from the following: L235V, G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L. In a preferred embodiment, the amino acid mutations are numbered according to the EU numbering system. The substitutions include the following: L235V, F243L, R292P, Y300L, and P396L (VLPYLL). In a preferred embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention is numbered according to the EU numbering system.It contains an Fc region with the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL).

[0168] According to one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain (HC) and a light chain (LC) in any combination listed in Table 3 below: [Table 3]

[0169] In one embodiment, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a hypofucosylated or defucosylated constant region.

[0170] According to one aspect of the present invention, the amino acid mutations described herein include amino acid substitutions, insertions, or deletions. Preferably, the amino acid mutations described herein are amino acid substitutions, preferably conservative substitutions.

[0171] According to a preferred embodiment, the amino acid mutations of the present invention occur in regions outside the CDRs (e.g., within the FRs). More preferably, the amino acid mutations of the present invention occur outside the heavy chain variable region and / or outside the light chain variable region. According to one embodiment, the amino acid mutations occur outside the heavy chain constant region and / or outside the light chain constant region.

[0172] In certain aspects, antibodies of the invention containing amino acid mutations have properties that are the same or similar to the specific antibodies described in this disclosure.

[0173] In certain embodiments, the anti-FGFR2B antibodies of the present invention comprise post-translational modifications to the CDRs, the light chain variable region, the heavy chain variable region, the light chain, or the heavy chain.

[0174] In one embodiment, the anti-FGFR2B antibody provided by the present invention is a full-length antibody, a single domain antibody such as VHH, Fab, Fab', Fab'-SH, (Fab')2, a single-chain antibody such as scFv, Fv, dAb (domain antibody), or a bi(multi)specific antibody.

[0175] In one embodiment, the anti-FGFR2B antibodies provided by the present invention are antibodies in the form of any IgG isotype, for example, antibodies in the form of IgG1, IgG2, IgG3, or IgG4.

[0176] In one aspect, the antibodies provided herein have been modified to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites in an antibody can be readily achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. For example, glycosylation can be altered to increase the affinity of the antibody for an "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be introduced to eliminate one or more variable region framework glycosylation sites, thereby removing glycosylation at these sites. Such aglycosylation can increase the affinity of the antibody for the antigen. Such methods are described, for example, in U.S. Patent No. 5,426,300. If the antibody contains an Fc region, the carbohydrates attached thereto can be altered. In some applications, modifications to remove undesired glycosylation sites, such as removing fucose modules, are useful for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be made to modify complement dependent cytotoxicity (CDC).

[0177] In certain aspects, it may be desirable to produce cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody have been substituted with cysteine ​​residues.

[0178] In certain aspects, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dialkanes, poly-1,3,6-trialkanes, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0179] In certain embodiments, the antibodies of the invention have one or more of the following properties: (1) cross-reacts with human, cynomolgus monkey, mouse, and rat FGFR2B orthologues; (2) binds to human FGFR2B, particularly the extracellular domain of human FGFR2B, with high affinity, for example, with a K value of less than 100 nM, for example, less than 50 nM, for example, less than 30 nM, preferably less than 10 nM or 5 nM, where the K value is preferably measured using a surface plasmon resonance assay; (3) binds to human FGFR2B expressed on the surface of cells (e.g., T cells) with high affinity, e.g., an EC50 value of less than 100 nM, e.g., less than 50 nM, e.g., less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM or 5 nM, where the EC50 value is preferably measured using a FACS assay; (4) inhibiting the binding of human FGFR2B to the ligand FGF7 / 10 by at least 50%, e.g., at least 60%, 70%, 80%, 85%, or 90%, as determined, for example, by ELISA, and preferably having an IC50 value of less than 10 nM, more preferably less than 1 nM; (5) exhibits binding affinity and / or specificity equivalent to or similar to any of the antibodies shown in Table 2; (6) inhibits (e.g., competitively inhibits) the FGFR2B binding of any antibody shown in Table 2; (7) binds to an epitope identical to or overlapping with the epitope of any antibody shown in Table 2; (8) binds to an epitope different from the epitope of any of the antibodies shown in Table 2; (9) Has biological activity equivalent to or similar to any of the antibodies shown in Table 2.

[0180] According to one embodiment, the FGFR2B antibodies of the present invention are antagonistic antibodies that bind to an FcR (e.g., FcγR)-binding Fc region, such as a human IgG1, IgG2, or IgG4 Fc region or a variant thereof (e.g., IgG4 with S228P), preferably a human IgG1 Fc region or a variant thereof. The binding affinity of such a variant to FcγR is preferably equal to or stronger than that of the parent Fc region (e.g., a native-sequence Fc region) to FcγR. The antibody preferably comprises a human IgG1 or IgG4 Fc region sequence identical to the Fc region sequence of the constant region sequence set forth in SEQ ID NO: 74 or 75, or a human IgG1 or IgG4 Fc region variant that has at least 95%, 96%, 97%, or 99% identity to the Fc region sequence of the constant region sequence set forth in SEQ ID NO: 74 or 75, or a human IgG1 or IgG4 Fc region variant that includes 10 or less, 5 or less, or 1 to 3 amino acid mutations relative to the Fc region sequence of the constant region sequence set forth in SEQ ID NO: 74 or 75.

[0181] According to one embodiment, the antibody comprises an Fc region variant, and the binding affinity of the Fc region variant to FcγR is increased by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to, for example, the parent Fc region (e.g., a native-sequence Fc region). According to one embodiment, the antibody of the present invention comprising an Fc region variant has enhanced FcγR-mediated effector function compared to an antibody comprising the corresponding parent Fc region (e.g., a native-sequence Fc region). Preferably, the Fc region of the antibody comprises the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL). According to one aspect, the antibody comprises a human IgG1 Fc region having the same sequence as the Fc region sequence of the constant region sequence set forth in SEQ ID NO: 21; or a human IgG1 Fc region variant having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the Fc region sequence of the constant region sequence set forth in SEQ ID NO: 21; or the antibody comprises 10 or fewer, 5 or fewer, or 1 to 3 amino acid mutations compared to the Fc region sequence of the constant region sequence set forth in SEQ ID NO: 21, as well as mutations that reduce the binding affinity of the Fc region to FcγR, preferably L235V, F243L, R292P, Y300L, and P396L substitutions.

[0182] According to certain embodiments, the FGFR2B antagonist antibodies of the invention have one or more of the following properties: (1) binds to human FGFR2B with high affinity, for example, with a KD value of less than 10 nM, more preferably less than 5 nM, preferably as measured using a surface plasmon resonance assay; (2) binds to human FGFR2B expressed on the surface of cells (e.g., activated CD4+ T cells) with high affinity, e.g., an EC50 value of less than 10 nM, more preferably less than 5 nM, where the EC50 value is preferably measured using a FACS assay; (3) inhibiting the binding of FGFR2B to the ligand FGF7 / 10 by at least 70%, preferably at least 80%, 85%, or 90%, as determined, for example, by ELISA, and preferably having an IC50 value of less than 10 nM, more preferably less than 1 nM; (4) blocking FGFR2B-mediated signaling activity; (5) Inhibits the growth of cancer cells.

[0183] Antibody expression The present invention relates to host cells comprising one or more expression vectors, and to methods for producing antibodies or antigen-binding fragments thereof of the invention, comprising culturing the host cells and purifying and recovering the antibodies or antigen-binding fragments.

[0184] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.). Enzymatic reactions and purification techniques may be performed according to manufacturer's instructions, or may be accomplished by common techniques in the art or as described in this disclosure. The techniques and procedures described above can generally be performed according to conventional methods well known in the art or as described in the various general and specific references cited and discussed herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0185] In one aspect, the present invention provides nucleic acids encoding any of the above-described anti-FGFR2B antibodies or antigen-binding fragments thereof. For example, the present invention provides nucleic acids encoding the heavy chain, light chain, variable region, or portion comprising a complementarity-determining region described in the present disclosure. In one aspect, the nucleic acid encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 17 or 18. In one aspect, the nucleic acid encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 19 or 20.

[0186] In one aspect, one or more vectors containing the nucleic acid are provided. In one embodiment, the vector is an expression vector. The expression vector is selected depending on the host cell in which the vector is intended to be expressed. Typically, the expression vector comprises a promoter and other regulatory sequences (e.g., enhancers) operably linked to the nucleic acid encoding the anti-FGFR2B antibody or antigen-binding fragment thereof. In one embodiment, the expression vector further comprises a sequence encoding an antibody constant region.

[0187] In one aspect, the present invention provides host cells, e.g., prokaryotic or eukaryotic cells, for expressing the recombinant antibodies of the invention. In one embodiment, Escherichia coli can be used as a prokaryotic host for cloning and expressing nucleic acids of the invention. Other suitable microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, e.g., Salmonella, Serratia, and various Pseudomonas species. Expression vectors containing expression control sequences (e.g., origins of replication) compatible with the host cell can also be prepared in these prokaryotic hosts. In one embodiment, mammalian host cells are used to express and produce the anti-FGFR2B antibody polypeptides of the invention. For example, hybridoma cell lines expressing endogenous immunoglobulin genes, mammalian cell lines harboring exogenous expression vectors, including normal human cells, or immortalized animal or human cells. Many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including, for example, CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, hybridomas, and the like.

[0188] In one aspect, the present invention provides a method for preparing an anti-FGFR2B antibody, comprising introducing an expression vector into mammalian host cells and culturing the host cells for a sufficient period of time to express the antibody within the host cells, or more preferably, to secrete the antibody into the culture medium in which the host cells are cultured for antibody production. Recovery of the antibody from the culture medium can be carried out using standard protein purification techniques. Antibody molecules prepared by the methods described herein can be purified using known and available techniques, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The specific conditions available for producing a particular protein will vary depending on factors such as net charge, hydrophobicity, and hydrophilicity, which will be readily apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by various well-known analytical methods, such as size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0189] Antibodies expressed by different cell lines or in transgenic animals will likely have different glycosylation from each other, however, all antibodies encoded by the nucleic acids provided in this disclosure or comprising the amino acid sequences provided in this disclosure are part of the present invention, regardless of their type of glycosylation.

[0190] immune complex The present invention relates to immunoconjugates comprising any of the anti-FGFR2B antibodies or antigen-binding fragments thereof of the present invention conjugated with a payload. In a preferred embodiment, such immunoconjugates comprise one or more drugs (e.g., cytotoxic agents, small molecule compounds, immunostimulants, etc.) or markers as payloads.

[0191] Assay The physical / chemical properties and / or biological activity of the anti-FGFR2B antibodies provided in the present disclosure can be identified, screened, or characterized by various assays known in the art. In one aspect, the antigen-binding activity of the antibodies of the present invention can be tested by known methods, such as ELISA and Western blot. Binding to FGFR2B can be determined by methods known in the art. Examples of such methods are described in the present disclosure.

[0192] The present invention also provides assay methods for identifying anti-FGFR2B antibodies having desired biological activities. Such biological activities include, for example, binding to FGFR2B (e.g., binding to human FGFR2B), enhancing FGFR2B-mediated signal transduction (e.g., enhancing NFκB-mediated transcription), and enhancing T effector cell function (e.g., enhancing effector T cell proliferation and / or enhancing cytokine production (e.g., gamma interferon) by effector T cells). Antibodies having such biological activities in vivo and / or in vitro are also provided.

[0193] According to a particular embodiment, the antibodies of the invention are tested for such biological activity.

[0194] Cells used in the above in vitro assay methods include cell lines that naturally express FGFR2B and cell lines modified to express FGFR2B, such as tumor cell lines, etc. Such cells also include cell lines that do not normally express FGFR2B but have been modified to express FGFR2B by transformation with DNA encoding FGFR2B.

[0195] Of course, the immunoconjugates or immunofusions of the invention may be used instead of or in addition to anti-FGFR2B antibodies to perform any of the above assays.

[0196] Of course, combinations of anti-FGFR2B antibodies and other active agents can also be used to perform any of the above assays.

[0197] Pharmaceutical Composition The pharmaceutical composition of the present invention can comprise an antibody of the present invention and pharmaceutically acceptable auxiliary substances. According to another aspect, the pharmaceutical composition of the present invention can be included in a pharmaceutical kit. According to another aspect, the pharmaceutical composition of the present invention can be included in a kit, such as a diagnostic kit.

[0198] As used herein, the term "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, and the like. Pharmaceutical carriers suitable for the present invention include sterile liquids, such as water and oils, including fats and oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Physiological saline, aqueous dextrose, and aqueous glycerin can also be used as liquid carriers, particularly for injectable solutions.

[0199] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, milk powder, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, fat-soluble milk powder, glycerol, propylene, diol, water, ethanol, etc. For information on the use of excipients and their methods of use, see also "Handbook of Pharmaceutical Excipients," fifth edition, R.C. Rowe, P.J. Seskey, and S.C. Owen, Pharmaceutical Press, London, Chicago. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin, etc.

[0200] The present invention provides pharmaceutical compositions comprising one or more of a monoclonal antibody that binds to FGFR2B, or an antigen-binding fragment thereof, or a nucleic acid, a vector, or a host cell, or an immunoconjugate or an immunofusion. Of course, the anti-FGFR2B antibody or an antigen-binding fragment thereof, or a nucleic acid, a vector, or a host cell, or an immunoconjugate or an immunofusion thereof provided by the present invention may be formulated in the pharmaceutical composition together with appropriate pharmaceutical carriers, excipients, and other co-administered agents suitable for the preparation of pharmaceuticals in order to provide improved transportability, deliverability, tolerance, etc.

[0201] Pharmaceutical formulations containing the anti-FGFR2B antibodies described in this disclosure can be prepared by mixing the anti-FGFR2B antibodies or antigen-binding fragments thereof of the present invention having the desired purity with one or more pharmaceutically acceptable excipients, preferably in the form of an aqueous solution or a lyophilized formulation. Examples of lyophilized antibody preparations are described in U.S. Patent No. 6,267,958. Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter of which discloses a preparation containing a histidine acetate buffer.

[0202] The pharmaceutical compositions or preparations of the present invention may contain one or more other active ingredients necessary for the treatment of a specific disease, preferably active ingredients with complementary activities that do not adversely affect each other. For example, it may be desirable to also contain another therapeutic agent. In some embodiments, the other therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, another antibody, an immunomodulatory agent, or another biopolymer drug.

[0203] According to one embodiment, the pharmaceutical composition of the present invention may comprise a nucleic acid encoding an anti-FGFR2B antibody or an antigen-binding fragment thereof.

[0204] Methods and Uses The present invention provides methods for preventing, diagnosing, or treating an FGFR2B-related disease or condition, comprising administering to a patient in need thereof an effective amount of an anti-FGFR2B antibody, or antigen-binding fragment thereof, or an immunoconjugate or immunofusion comprising same, or a pharmaceutical composition comprising same, or a nucleic acid, vector, or host cell, as described herein.

[0205] In one aspect, the present invention provides the use of an anti-FGFR2B antibody or an antigen-binding fragment thereof, or an immunoconjugate or immunofusion or pharmaceutical composition comprising same, in the manufacture or preparation of a medicament for treating or preventing an FGFR2B-related disease or condition in a subject.

[0206] In one aspect, the anti-FGFR2B antibodies and antigen-binding fragments thereof provided by the present invention, and pharmaceutical compositions comprising them, can be used as therapeutic agents for treating or preventing FGFR2B-related diseases or conditions in a subject. For an FGFR2B-related disease in a subject identified using standard methods, the anti-FGFR2B antibodies and antigen-binding fragments thereof disclosed in the present invention, pharmaceutical compositions, immunoconjugates, or immunofusions comprising them, or the nucleic acids, vectors, or host cells described in the present disclosure can be administered.

[0207] In some embodiments, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent or treatment. In some embodiments, such therapeutic agents include, for example, chemotherapeutic agents, radiotherapeutic agents, cytokines, vaccines, other antibodies, immunomodulators, or other biopolymer drugs. In some embodiments, therapeutic treatments include surgery; and radiation therapy, local or concentrated radiation therapy, etc.

[0208] The combination therapy includes combined administration (i.e., two or more therapeutic agents may be contained in the same preparation or in two or more separate preparations) and separate administration, in which case the administration of the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention may occur before, simultaneously with, or after the administration of the additional therapeutic agent and / or adjuvant and / or treatment.

[0209] According to one aspect, the FGFR2B-related disease or condition of the present invention refers to a disease or condition related to abnormal FGFR2B expression, activity, and / or signal transduction in a subject. Examples include, but are not limited to, cancer, inflammation, autoimmune disease, etc. According to one aspect, in an FGFR2B-related disease or condition, the nucleic acid (level or content) encoding FGFR2B is increased, or the expression of FGFR2B is increased, or the protein level or activity of FGFR2B is increased, or the signal transduction mediated by FGFR2B is increased. According to another aspect, in an FGFR2B-related disease or condition, the nucleic acid (level or content) encoding FGFR2B is decreased, or the expression of FGFR2B is decreased, or the protein level or activity of FGFR2B is decreased, or the signal transduction mediated by FGFR2B is decreased.

[0210] According to certain embodiments, treatment of such diseases or conditions may be benefited by inhibiting FGFR2B at the nucleic acid or protein level, or by inhibiting binding of FGFR2B to a ligand, or by inhibiting FGFR2B-mediated signaling.

[0211] According to another aspect, treatment of such diseases or conditions may benefit from increasing FGFR2B at the nucleic acid or protein level, or by enhancing FGFR2B-mediated signaling.

[0212] In one embodiment, the FGFR2B-related disease or condition is cancer. Particular cancers include, but are not limited to, solid tumors, breast cancer, urothelial cancer, melanoma, kidney cancer, ovarian cancer, head and neck cancer, gastric cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, prostate cancer, lymphocytic leukemia, and sarcoma. Preferably, the antibody for preventing, diagnosing, or treating FGFR2B-related cancer is an FGFR2B agonist.

[0213] In one embodiment, the FGFR2B-related disease or condition is an inflammatory and / or autoimmune disease. In one embodiment, the FGFR2B-related inflammatory and / or autoimmune disease is selected from idiopathic dermatitis, rheumatoid arthritis, asthma (e.g., allergic asthma), COPD, autoimmune uveitis, multiple sclerosis, lupus (e.g., systemic lupus erythematosus), ulcerative colitis, scleroderma, and graft-versus-host disease (GVHD). Preferably, the antibody for treating or preventing an FGFR2B-related inflammatory and / or autoimmune disease is an FGFR2B antagonist.

[0214] In certain embodiments, the subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease described herein (e.g., cancer). In particular embodiments, the subject may be undergoing or have previously undergone other treatments, e.g., chemotherapy and / or radiation therapy.

[0215] The antibodies or antigen-binding fragments of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, preparations, pharmaceutical combinations, and kits comprising the same) can be administered by any suitable technique. Examples include oral, parenteral, intrapulmonary, or intranasal administration. They can also be administered intralesionally if localized treatment is required. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, e.g., injection, e.g., intravenous or subcutaneous, depending on the site of administration and whether administration is brief or chronic. Various modes of administration are contemplated by the present disclosure. Examples include, but are not limited to, a single dose, multiple doses at various time points, a bolus dose, and pulse infusion.

[0216] The antibodies or antigen-binding fragments of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, preparations, pharmaceutical combinations, and kits comprising the same) should be formulated and administered in accordance with good medical practice, taking into consideration factors such as the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of pharmaceutical delivery, the method of administration, the administration schedule, and other factors known to medical professionals. Optionally, the antibodies can be combined with one or more agents currently used to prevent or treat the disease. The effective amounts of these other agents will vary depending on the amount of antibody present in the formulation, the condition being treated, the method of treatment, and other factors discussed above.

[0217] To prevent or treat disease, the antibodies or antigen-binding fragments of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, preparations, pharmaceutical combinations, and kits comprising the same) will be administered (alone or in combination with one or more additional therapeutic agents) at an appropriate dose depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody, and the judgment of the treating physician. The antibody is suitably administered to the patient once or in a series of multiple treatments.

[0218] According to certain embodiments, any of the anti-FGFR2B antibodies or antigen-binding fragments thereof provided in the present disclosure can be used to detect the presence of FGFR2B in a biological sample. The term "detection," as used in the present disclosure, includes quantitative detection or qualitative detection. According to certain embodiments, the biological sample is blood, serum, or other liquid sample derived from a living body. According to certain embodiments, the biological sample includes cells or tissues. According to some embodiments, the biological sample is derived from a lesion associated with a hyperproliferative or cancerous lesion.

[0219] According to one aspect, the antibodies or antigen-binding fragments thereof of the present invention can be used to diagnose an FGFR2B-related disease or condition, such as cancer, for example, to evaluate (e.g., monitor) the treatment or progression of a disease described herein and / or for their diagnosis and / or staging in an individual. According to a particular aspect, a labeled anti-FGFR2B antibody or antigen-binding fragment thereof is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and indirectly detectable moieties, such as enzymes or ligands, via enzymatic reactions or molecular interactions. According to one aspect, the present disclosure provides a kit for diagnosing an FGFR2B-related disease, the kit comprising an antibody or antigen-binding fragment thereof of the present invention.

[0220] According to certain embodiments provided herein, the sample is obtained prior to treatment with an anti-FGFR2B antibody or antigen-binding fragment thereof. According to certain embodiments, the sample is obtained prior to treatment with another therapeutic agent. According to certain embodiments, the sample is obtained during or after treatment with another therapeutic agent.

[0221] The present invention includes any combination of the specific embodiments described in this disclosure. While specific details and examples are described to explain preferred embodiments of the present invention, it should be understood that these are merely illustrative and are used as examples. Furthermore, the present invention also encompasses modifications based on the preferred embodiments of the present invention that would be obvious to one skilled in the art. For all purposes, all publications, patents, and patent applications cited in this disclosure, including citations, are incorporated herein by reference in their entirety. [Example]

[0222] Example 1: Generation of anti-FGFR2b monoclonal antibodies Anti-FGFR2b monoclonal antibodies (mAbs) were generated by conventional hybridoma fusion technology. mAbs that showed binding specificity to FGFR2b in enzyme-linked immunosorbent assay (ELISA) were selected for further characterization.

[0223] Construction of expression vector pcDNA3.1-human FGFR2b DNA encoding the full-length human FGFR2b isoform (Uniprot accession number P21802-2) was inserted into the pcDNA3.1(+) vector (Synbio Technologies) by seamless cloning, and the construct was confirmed by DNA sequencing. Large-scale DNA preparations were performed using Qiagen's Plasmid Maxiprep System and used for immunization.

[0224] Immunization, hybridoma fusion, and cloning Mice were immunized intramuscularly with 100 μg of the pcDNA3.1-human FGFR2b vector prepared above, followed by three intramuscular boosts of 10 μg of recombinant human FGFR2b extracellular domain (ECD) fused to a human Fc moiety at the C-terminus (Sino Biological, human FGFR2b-Fc, product number 16485-H02H). Antibody immune responses were monitored by FGFR2b-specific ELISA. Ten days after serum screening, mice with the highest anti-FGFR2b antibody serum titers were boosted intravenously with 10 μg of FGFR2b-Fc. Three days after the boost, spleen cells were harvested and fused with mouse myeloma cells to maintain their viability, generating hybridoma cell lines. Primary screening of FGFR2b-specific antibodies was performed using supernatants from mouse hybridoma clones to select hybridoma cell lines producing FGFR2b-specific antibodies that bound only to FGFR2b but not to FGFR2c. ELISA and flow cytometry assays demonstrated that several antibodies, including 55D6, 38D4, 39C2, 35B11, 52E2, 61B7, and 30C7, exhibited strong affinity for human FGFR2-IIIb but showed no detectable binding to human FGFR2c. These antibodies did not bind to human FGFR1, FGFR3c, FGFR3b, or FGFR4. Interestingly, all of these FGFR2b-specific antibodies inhibited the interaction of FGF7 with FGFR2b as determined by ELISA.

[0225] Example 2: V gene cloning and generation of chimeric antibodies 1. Cloning and sequencing of hybridoma antibody V genes A lead antibody with a desirable profile was selected for V gene cloning. The sequences of the mouse anti-human FGFR2b light and heavy chain variable regions were obtained by polymerase chain reaction (PCR) amplification. PCR was performed according to Wang, Z. et al., 2000 (Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity. J. Immunol. Methods 233, 167-177). Total RNA was extracted from positive hybridoma cells using the MiniBest Universal RNA Extraction Kit (TaKaRa), and cDNA was synthesized using an Oligo(dT) primer with a First Strand cDNA Synthesis Kit (TaKaRa). The variable regions of the mouse IgG gene were amplified by PCR using a different isotype primer for the heavy chain variable region and a kappa chain primer for the light chain variable region. The PCR product was subcloned into a TA cloning vector. For each variable gene construct, 10 or more single colonies were subjected to DNA sequencing by Synbio Technologies (Suzhou, China). The amino acid sequences of Vh and Vk were derived from the DNA sequencing results.

[0226] 2. Construction of Chimeric Antibodies Seven antibodies, including 55D6, 38D4, 39C2, 35B11, 52E2, 61B7, and 30C7, were selected as lead antibodies for generating chimeric antibodies with human IgG1 constant regions. Their sequences are shown in Table 4. After sequence analysis and confirmation, cDNAs for the heavy and light chain variable regions were synthesized and fused to the constant region sequence of human kappa human IgG1. To enhance the ADCC of the antibody, L235V, F243L, R292P, Y300L, and P396L (VLPYLL) were introduced into the Fc domain of this chimeric mAb. To promote antibody secretion, signal peptide sequences (MEFGLSWVFLVALFRGVQC and MDMRVPAQLLGLLLLWLRGARC) were added to the N-terminus of the heavy and light chains. The resulting chimeric antibody genes were cloned into expression vectors. Large-scale DNA preparation was performed using Qiagen's Plasmid Maxi-Prep System.

[0227] 3. Expression and Purification of Chimeric Antibodies Co-transfection of heavy and light chains was performed using Invitrogen's ExpiFectamine according to the manufacturer's protocol. (登録商標) ExpiCHO-S cells (5-6x106 cells / ml) in ExpiCHO expression medium were incubated with ExpiFectamine (登録商標) Equal amounts of heavy chain vector and light chain vector DNA were transfected at a final concentration of 0.8 μg / ml using CHO Reagent. Plasmid DNA or ExpiFectamine (登録商標) CHO reagents were placed in the cold OptiPRO (登録商標) Dilute with medium and mix by swirling or inverting the tube. (登録商標) The CHO / plasmid DNA mixture was incubated at room temperature for 1-5 minutes and then gently transferred to the shake flask containing the cells. The transfected cells were incubated at 37°C in a humidified 5% CO2 atmosphere on an Orbilt shaker (shaking speed 125 rpm). 18-22 hours after transfection, ExpiCHO was transfected. (登録商標)Feed was added, and the conditioned medium was collected on day 10. The supernatant was centrifuged at 4,000 rpm for 20 minutes and filtered through a 0.22 μm filter to remove cell debris. The filtered supernatant was loaded onto a pre-equilibrated Protein A affinity column. The Protein A resin was washed with equilibration buffer (PBS), and the antibody was eluted using 25 mM citric acid (pH 3.5). The purified antibody solution was adjusted to pH 6.0-7.0 using 1 M Tris base (pH 9.0). Endotoxin was controlled at less than 1 EU / mg. Finally, the purified antibody was characterized by SDS-PAGE.

[0228] The anti-human FGFR2b specific antibody Bema_VLPYLL (VH and VL sequences are patent WO2015 / 017600A1, FIVEPRIMETHERAPEUTICS) was also expressed as a positive control. Fc is human IgG1_VLPYLL.

[0229] [Table 4-1] [Table 4-2]

[0230] Example 3: Epitope characterization of chimeric anti-FGFR2b mAbs Epitope characterization of the chimeric mAb and Bema_VLPYLL was performed using BLI. 100 nM human FGFR2b-biotin protein (Katcus, product code: FGF-HM4ABB) was loaded onto an SA biosensor. After a washing step, the biosensor was immersed in a solution of the first antibody, and the first antibody was allowed to bind for 90 seconds. A clear binding signal was observed. The biosensor was washed with KD buffer and then incubated with the second antibody. This signal indicated whether the epitopes of the two antibodies competed with each other.

[0231] Using this epitope competition assay (Figure 1), we found that 55D6, 38D4, 39C2, and Bema_VLPYLL recognize the same epitope, and that any of these antibodies can fully compete with the other antibodies for binding to human FGFR2b. Unlike the above antibodies, 52E2 binds to a completely different epitope on FGFR2b. Interestingly, 35B11, 61B7, and 30C7 not only compete with 52E2 for binding to FGFR2b, but also compete with Bema or other antigens for binding. Two other benchmark antibodies, 2-10 (Daiichi Sankyo) and GP369 (Aveo Therapeutic), were also expressed and their epitopes tested. 2-10 and GP369 can compete with Bema_VLPYLL for binding, but cannot compete with 52E2 for binding to FGFR2b. The results are not shown here.

[0232] Example 4: ADCC activity of anti-FGFR2b chimeric mAb The ADCC activity of the FGFR2b antibody was evaluated using an ADCC reporter bioassay, a bioluminescent reporter gene assay that quantifies antibody biological activity via FcγRIIIa-mediated pathway activation. Jurkat-NFATLuc-FcγRIIIa-V176 cells (Jurkat cells (Shanghai Institute of Biological Sciences, Cat. No. SCSP-513)) were transfected with the PGL4.30-Luc / NFAT-RE / Hygro plasmid (Promega) and screened with hygromycin. The Jurkat-nfat-Luc cell line stably expressed the FcγRIIIA-V176 (SEQ ID NO: 37) sequence. The sequence was inserted into the vector pVitro-neo (InvivoGen) and transformed into the plasmid pVitro-neo-pcDNa3.1-FcγRII. IA-V176 was obtained. The resulting cell line was transfected with the pVitro-neo-FcγriiA-V176 plasmid, and the stable-expressing cell line Jurkat-nFAT-Luc-FcγRIIIA-V176 was screened using the antibiotic G418. Jurkat cells (Shanghai Institute for Biological Sciences, product code: SCSP-513) were transfected with the PGL4.30-Luc / NFAT-RE / Hygro plasmid (Promega) and screened with hygromycin. The cell line Jurkat-nfat-Luc was stably expressed.

[0233] The FcγRIIIA-V176 (SEQ ID NO: 37) sequence was inserted into the vector pVitro-neo (InvivoGen) to generate the plasmid pVitro-neo-pcDNa3.1-FcγRIIIA-V176. The resulting cell line was transfected with the pVitro-neo-FcγRIIIA-V176 plasmid, and the stable expression cell line Jurkat-nFAT-Luc-FcγRIIIA-V176 was screened using the antibiotic G418. Effector cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 μg / mL hygromycin, 250 μg / mL G418, 1 mM sodium pyruvate, and 0.1 mM MEM non-essential amino acids. Human FGFR2 gene-amplified cancer cell lines (KATO-III cells and KYSE-180 cells) were used as target cells for the ADCC reporter bioassay. The gastric cancer cell line, KATO-III, was maintained in IMDM medium containing 20% ​​FBS and 1x penicillin-streptomycin, while the esophageal squamous cell carcinoma cell line, KYSE-180, was maintained in RPMI-1640 medium supplemented with 10% FBS and 1x penicillin-streptomycin. Target cells were seeded at 20,000 cells / well in a 96-well white-bottom assay plate and incubated with serial dilutions of antibody. After 30 minutes of incubation at 37°C, 1.2x105 Jurkat-NFATLuc-FcγRIIIa-V176 reporter cells were added per well to the assay plate and incubated for 5 hours at 37°C. One-Glo (登録商標) Luciferase Assay Reagent (Promega, Part Number: E6120) was added, and luminescence was measured using a SpectraMax M5 microplate reader. Samples and controls were run in duplicate, and the mean reporter signal values ​​of the sample dilutions were plotted as relative luminescence units (RLU) versus antibody concentration. Dose-response curves were fitted with a four-parameter model using PrismGraphpad statistical software.

[0234] To confirm the ADCC activity of the chimeric monoclonal antibodies, we used an ADCC reporter bioassay. As shown in Figure 2, 55D6, 38D4, 39C2, 35B11, 52E2, 61B7, and 30C7 exhibited effective ADCC activity against KATO-III cells (FGFR2b amplified and highly overexpressed).

[0235] Example 5: Humanization of a mouse antibody 55D6, 38D4, 39C2, 35B11, and 52E2 were selected for humanization. The humanization process to generate highly optimized mAbs consists of five steps. The first step is the selection of a receptor human framework (FR) suitable for antigen binding activity, immunogenicity, expression, stability, and pharmacokinetics. The selected germline variants were IGHV1-46*01 (for VH of 55D6, 39C2, and 38D4), IGHV1-69-2*02 (for VH of 35B11), IGHV1-2*02 (for VH of 52E2), IGKV3-11*01 (for VL of 55D6), IGKV1-39*01 (for VL of 39C2, 38D4, and 52E2), and IGKV2-30*02 (for VL of 35B11). In the second step, the CDRs of a mouse antibody are grafted onto the FRs of the human antibody described in the first step to generate a CDR-grafted mAb. Next, three-dimensional structural models of the parent mouse antibody and the CDR-grafted mAb are simulated. Finally, following the guidance of the (3D) Fv structural model, amino acids in the framework regions near the CDR regions at the VH / VL interface and within the structure are backmutated to the corresponding amino acids in the FRs of the mouse antibody. The third step is to prepare expression vectors for multiple versions of the humanized antibody. The fourth step is to express and purify the humanized antibody. The final step is to evaluate the humanized antibody in multiple dimensions.

[0236] Using this humanization procedure, we obtained humanized antibodies 55D6, 38D4, 39C2, 35B11, and 52E2, designated Hu55D6, Hu38D4, Hu39C2, Hu35B11, and Hu52E2, respectively. The constant regions of the humanized mAbs are human IgG1 and kappa. To enhance ADCC, these humanized antibodies were expressed in low-fucose forms by adding the fucose analog 2-deoxy-2-fluoro-L-fucose (Biosynth, W-203582) to the expression medium. As a positive control, Bema (constant regions are human IgG1 and kappa) was also expressed in low-fucose forms to compare the biological activity of the five humanized mAbs. The VH and VL sequences of the humanized mAbs are shown in the table below.

[0237] [Table 5-1] [Table 5-2]

[0238] Example 6: Kinetic binding of humanized monoclonal antibodies to human FGFR2b The kinetic binding of a humanized monoclonal antibody (mAb) to human FGFR2b (Sino Biological, product code 16485-H08H) was measured using Bio-Layer Interferometry (BLI). 100 nM of the humanized mAb was dissolved in 1x kinetics buffer (1x PBS, pH 7.4, 0.02% Tween 20, 0.1% BSA) and loaded onto four pre-wetted Protein A biosensors. The biosensors were then incubated with various concentrations of human FGFR2b. All binding data were collected at 30°C. The experiment consisted of five steps: 1. baseline acquisition (60 s); 2. antibody loading onto the Protein A biosensor (60 s); 3. second baseline acquisition (60 s); 4. antigen binding for k measurement (120 s); and 5. antigen dissociation for k measurement (180 s). Four concentrations of antigen (100 nM, 33.3 nM, 11.1 nM, and 0 nM) diluted in 1x kinetics buffer were used. Baseline and dissociation steps were performed in 1x kinetics buffer. KD was determined from the ratio of koff to kon. The biosensor was regenerated for 5 seconds in regeneration buffer (10 mM glycine-HCl, pH 1.7) and then neutralized for 5 seconds in neutralization buffer (1x PBS, pH 7.4, 0.02% Tween 20, 0.1% BSA). This process was repeated three times.

[0239] As shown in Table 6 below, Hu55D6, Hu38D4, Hu39C2, Hu35B11 and Hu52E2 bind to human FGFR2b with high affinity, and the KD of these humanized mAbs is superior to Bema.

[0240] [Table 6]

[0241] Example 7: Binding specificity of humanized monoclonal antibodies to human FGFR2b To avoid side effects due to off-target binding, we used ELISA to measure the binding specificity of humanized antibodies to FGFR family members. Briefly, human FGFR2b (Sino Biological, Cat. No. 16485-H08H), FGFR2c (Sino Biological, Cat. No. 10824-H08H), FGFR1 (Sino Biological, Cat. No. 10616-H08H), FGFR3b (Sino Biological, Cat. No. 10648-H08H), FGFR3c (Sino Biological, Cat. No. 10644-H08H), or FGFR4 (Sino Biological, Cat. No. 10538-H08H) was immobilized on a plate, and the humanized mAb was serially diluted in PBS and incubated for 1 hour. Next, goat pAb against human IgG-HRP was added, followed by TMB, and binding was detected at OD450nm. Finally, the data were analyzed using GraphPad Prism.

[0242] ELISA analysis revealed that Hu55D6, Hu38D4, Hu39C2, Hu35B11, and Hu52E2 specifically bound to FGFR2b, but not to other FGFR family members (see Figure 3).

[0243] Example 8: Species cross-reactivity of humanized mAbs To further aid in the selection of animal models, we used an ELISA assay to detect whether our candidate antibodies bind to mouse, rat, or monkey FGFR2b. Briefly, human FGFR2b-His (Sino Biological, Cat. No. 16485-H08H), mouse FGFR2b-His (Sino Biological, Cat. No. 51128-M08H), cynomolgus monkey FGFR2b-His (Sino Biological, Cat. No. FGF-CM1BB), or rat FGFR2b-mFc (in-house expression, Uniprot, sequence F1LSG7) was immobilized on a plate. The humanized mAb was serially diluted in PBS and incubated for 1 hour. Next, goat pAb against human IgG-HRP was added, followed by TMB, and binding was detected at OD450nm. Finally, the data were analyzed using GraphPad Prism.

[0244] ELISA analysis showed that Hu55D6, Hu38D4, Hu39C2, Hu35B11, and Hu52E2 not only bound to human FGFR2b but also cross-linked to mouse, rat, and cynomolgus monkey FGFR2b. These results support the feasibility of evaluating the efficacy of these antibodies in mouse models and the potential of evaluating the preclinical toxicity of these humanized antibodies in rats and monkeys (see Figure 4).

[0245] Example 9: ELISA assay for inhibiting binding of FGFR2b to FGF7 0.5 μg / ml hFGFR2b-Fc (SinoBiological, Catalog No. 16485-H02H, Lot No. LC13JL2910) was coated onto an ELISA plate and incubated overnight at 4°C. The plate was washed three times and blocked at 37°C for 1 hour. The plate was washed and then incubated with 50 μl of diluted humanized mAb and 50 μl of biotin-labeled FGF7-Fc (in-house produced; FGF7-Fc was expressed and purified using EZ-Link). (登録商標) Sulfo-NHS-LC-Biotin Kit (ThermoFisher, Catalog No. A39257) was added to each well and incubated at 37°C for 1 hour. After washing the plate six times, 100 μl of 1:5000 diluted HRP-conjugated streptavidin (Abcam, Catalog No. ab7403, Lot No. GR3259274-11) was added per well. After incubation at room temperature for 1 hour, mixed TMB substrate reagent was added, incubated at room temperature for 5 minutes, and stopped by adding 0.1 M H2SO4. OD450nm was measured using a microplate reader, and the IC50 value for inhibition of binding between the humanized anti-FGFR2b monoclonal antibody and the ligand FGF7 was calculated.

[0246] Blocking ELISA results showed that all five humanized mAbs potently inhibited the interaction between FGFR2b and FGF7, and the IC50 of these mAbs was comparable to that of Bema (see Figure 5).

[0247] The blocking activity of all five humanized mAbs against the interaction between FGFR2b and FGF10 was measured by BLI. Briefly, FGFR2b-biotion was loaded onto an SA biosensor and incubated with the test antibody or isotype control, after which the sensor was immersed in an FGF10 solution. The five humanized mAbs also inhibited the interaction between FGFR2b and FGF10, but the data are not shown here.

[0248] Example 10: Flow cytometry binding analysis of FGFR2b antibodies in HEK293T cells stably expressing human FGFR2b and FGFR2c HEK293T cells (Chinese Academy of Sciences Cell Bank, product code: GNHu44) were stably transfected with expression vectors expressing human FGFR2-IIIb (293T-hFGFR2b) (Uniprot accession number P21802-3) and human FGFR2-IIIc (293T-hFGFR2c) (Uniprot accession number P21802), respectively. 293T-hFGFR2b / 2c cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1× penicillin-streptomycin, and 1 μg / mL puromycin. Cells were washed and cultured at 5 × 10 4 Cells were seeded at 1000 kJ / well into a 96-well plate containing cold wash buffer (1x PBS containing 2% FBS) and incubated with multiple antibody dilutions for 60 minutes at 4°C. After two washes with cold wash buffer, 0.5 μg / mL of FITC-conjugated goat anti-human IgG1 antibody (Abcam, catalog no. ab98623, lot no. GR3319406) was immediately added to the cells and incubated for 30 minutes at 4°C. After two washes with cold wash buffer, cells were resuspended in 120 μL of cold PBS and analyzed by flow cytometry. FITC median fluorescence intensity (MFI) values ​​were fitted to antibody concentrations, and the binding EC50 values ​​of several antibodies were calculated using Prism 6.02 statistical software. The results are shown in Figure 6. It can be seen that all five humanized FGFR2b antibodies bound to 293T-hFGFR2b cells but not to 293T-hFGFR2c cells.

[0249] Example 11: ADCC reporter bioassay of humanized mAbs The ADCC reporter bioassay described in Example 3 was again used to measure the ADCC activity of the humanized FGFR2b antibodies. All humanized FGFR2b antibodies exhibited strong ADCC activity against KATO-III cells (ATCC, Cat#HTB-103) and KYSE-180 cells (low FGFR2b expression) (Cobioer, Cat#CBP60456), with Hu52E2 exhibiting a higher maximum RLU value than Bema and other FGFR2b antibodies (see Figure 7).

[0250] Example 12: Inhibition of FGF7-induced MCF7 cell proliferation by FGFR2b antibody MCF7 cells (Cobioer, Cat. No. CBP60380) derived from a patient with triple-negative breast cancer (TNBC) were maintained in EMEM medium containing 10% FBS and 1x penicillin-streptomycin. Cells were seeded at 10,000 cells per well in a 96-well plate in complete growth medium and cultured overnight for adhesion. Cells were then incubated in serum-free medium for 24 hours and treated with 30 μg / mL of human IgG isotype or multiple FGFR2b antibodies in the presence or absence of FGF7 (25 ng / mL) for 72 hours. Cell proliferation was monitored using CellTiter-Glo (Promega, Cat. No. G7571). (登録商標) Viability was assessed by a luminescent cell viability assay. Samples and controls were tested in duplicate, and the average reporter signal of the sample dilutions was plotted against antibody concentration in relative luminescence units using Prism 6.02 statistical software. As shown in Figure 8, MCF7 cells were stimulated with FGF7 protein, and Bema and several FGFR2b antibodies effectively inhibited FGF7-induced MCF7 cell proliferation.

[0251] Example 13: HTRF assay to detect phosphorylation of FGFR2 and ERK1 / 2 proteins induced by FGF7 or FGF10 in SNU-16 cells FGFR2b is a receptor tyrosine kinase involved in cell survival, proliferation, migration, and angiogenesis, primarily via the RAS-MAPK signaling pathway. To confirm whether FGFR2b antibodies inhibit downstream signaling induced by FGF7 and FGF10, we used a cell-based fluorescence resonance energy transfer (HTRF) assay to measure endogenous phosphorylated ERK1 / 2 (Thr202 / Tyr204) and phosphorylated FGFR2 (Tyr653 / 654) in SNU-16 cells (Cobioer, Cat. No. CBP60502), a gastric cancer cell line harboring FGFR2b amplification. Phosphorylated ERK1 / 2 and phosphorylated FGFR2 (Tyr653 / 654) were induced by the ligands FGF7 and FGF10. Briefly, SNU-16 cells were cultured in RPMI-1640 medium containing 10% FBS and 1x penicillin-streptomycin until they reached 90% confluence. Cells were seeded into 96-well plates in FBS-free RPMI-1640 medium and incubated overnight at 37°C. Then, cells were treated with 15 μg / mL or 0.15 μg / mL of human IgG isotype or multiple FGFR2b antibodies for 1.5 hours. Subsequently, cells were treated with a ligand-inducing complex containing 30 ng / mL FGF7 / 10 and 20 μg / mL heparin for 5 minutes at 37°C. After activation, cells were incubated with lysis buffer at room temperature for at least 30 minutes with shaking. After the lysis step was complete, 16 μL of cell lysate was transferred to a 96-half-well white plate and 4 μL of the mixed antibody solution prepared in detection buffer was added. The incubation complex was incubated for 4 hours at room temperature or overnight at 4°C, and then analyzed using a compatible HTRF (High Temperature Radioisotope) assay. (登録商標) Phosphorylated ERK1 / 2 (Thr202 / Tyr204) and phosphorylated FGFR2 (Tyr653 / 654) were measured by fluorescence emission at two different wavelengths (665 nm and 620 nm) using a reader. The HTRF ratio was calculated as (665 nm signal / 620 nm signal) * 104, reflecting the cellular phosphorylation level induced by each ligand.

[0252] As shown in FIG. 9, several FGFR2b antibodies and Bema significantly inhibited the phosphorylation of FGFR2 and ERK1 / 2 induced by FGF7 or FGF10.

[0253] Example 14: ADCC activity of FGFR2b antibodies targeting KATO-III cells by PBMCs In vitro testing to measure the ADCC activity of FGFR2b antibodies was performed using flow cytometry analysis. Briefly, KATO-III cells (ATCC, Cat# HTB-103) were maintained in IMDM medium containing 20% ​​FBS and 1x penicillin-streptomycin, and freshly isolated PBMCs from healthy donors were obtained from SailyBio (Shanghai, China). Primary ADCC assays were performed using effector cells collected from two independent donors over two days. The ADCC assays were performed using freshly isolated human PBMCs as effector cells at an effector / target cell ratio (E / T) of 40:1. Target cells were first transfected with CellTrace (登録商標) Effector cells were labeled with FarRed stain and incubated with increasing concentrations of antibodies for 16 hours. Target cell lysis was indicated by double-positive (propidium iodide and far-red staining) results by flow cytometry analysis. Maximum lysis was measured in the presence of 5% TrionX-100, and spontaneous release was measured in the absence of antibody. Specific lysis was calculated as a percentage of maximum lysis minus spontaneous lysis using the following formula: (Specific cell lysis rate)% = (experimental value - spontaneous lysis) / (maximum lysis rate - spontaneous lysis) × 100

[0254] As a result, as shown in Figure 10, several anti-FGFR2b humanized antibodies induced comparable specific KATO-III cell lysis by PBMCs from two donors with FcγRIIIA genotypes 158V / V and 158V / F.

[0255] Example 15: Pharmacokinetic evaluation of humanized mAbs The pharmacokinetic profiles of anti-FGFR2b lead molecules and the benchmark drug Bema were directly compared in Sprague Dawley (SD) rats after a single intravenous dose of 30 mg / kg. Eighteen female rats were randomly divided into six groups (three rats per group) and administered 30 mg / kg Bema or Hu39C2, Hu55D6, Hu52E2, Hu35B11, or Hu38D4 via a single slow bolus injection at a volume of 10 mL / kg. Plasma samples from each group were collected pre-dose (0 min), 30 min, 2 h, 8 h, 24 h, 48 h, and at D4, D7, D10, D14, D21, and D28. PK analysis was performed using a partially validated ELISA assay with a detection range of 0.156–20 ng / mL. Microplate wells were pre-coated with human FGFR2b protein, His tag (Acrobiosystems, FGB-H5223). After blocking, standards (STD), quality control (QC) samples, matrix blank samples, and test samples were added to the wells. After washing, biotin anti-human IgG4 (BD Pharmingen) was added. (登録商標) , 555879) is added to the microplate wells, followed by the addition of streptavidin labeled with horseradish peroxidase (HRP). Tetramethylbenzidine (TMB) is added to the microplate wells to develop a colorimetric signal (blue) in the presence of HRP. Once color develops, the reaction is stopped by adding stop solution to each well. Optical density (OD) is measured using a microplate reader set at 450 nm and 620 nm. The optical density (OD) values ​​of QC and test samples are converted to concentrations by comparison with a standard curve regressed with a four-parameter logistic model analyzed simultaneously. The mean, average, and mean plasma concentration-time curves are shown in Figure 11. Relevant PK parameters (Table 7) were calculated and evaluated by non-compartmental analysis (NCA) using Phoenix software.

[0256] [Table 7]

[0257] Example 16: Antitumor effect in SNU16 xenograft model using nude mice In vitro studies demonstrated that a humanized FGFR2b monoclonal antibody induced ADCC effects against SNU16 (Cobioer, Cat:CBP60502). Therefore, an in vivo model was established and used to evaluate antitumor activity. Briefly, SNU16 tumors were dissected into 3-5 mm3 fragments, and SNU16 tumor fragments soaked in Matrigel (Nova) were subcutaneously injected into the right flank of each female Balb / c nude mouse using a trocar. 16 days after inoculation, 35 mice with tumors measuring approximately 80 mm3 were selected and randomly divided into seven groups (n = 5). Mice were then intraperitoneally injected with either an isotype control or a humanized FGFR2b antibody at a dose of 10 mg / kg twice weekly for 4 weeks. At the end of the study, animals were sacrificed by CO2 inhalation. Tumor size and volume were measured twice weekly. Results were analyzed using PrismGraphPad and expressed as mean ± SEM.

[0258] Hu55D6, Hu38D4, Hu39C2, Hu35B11, and Hu52E2 significantly inhibited tumor growth, as shown in Figure 12. The tumor size and TGI of the treatment groups are summarized in Table 8.

[0259] [Table 8] [Sequence List Free Text]

[0260] [Table SL1]

[0261] [Table SL2]

[0262] [Table SL3]

[0263]

Table SL4

[0264]

Table SL5

[0265]

Table SL6

[0266]

Table SL7

[0267]

Table SL8

[0268]

Table SL9

Claims

1. 1. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising three CDRs of a heavy chain variable region (VH), i.e., HCDR1, HCDR2, and HCDR3, and three CDRs of a light chain variable region (VL), i.e., LCDR1, LCDR2, and LCDR3, wherein the VH and VL are: (1) VH comprising the amino acid sequence set forth in SEQ ID NO: 43, and VL comprising the amino acid sequence set forth in SEQ ID NO: 50; (2) VH comprising the amino acid sequence set forth in SEQ ID NO: 44, and VL comprising the amino acid sequence set forth in SEQ ID NO: 51; (3) VH comprising the amino acid sequence set forth in SEQ ID NO: 45, and VL comprising the amino acid sequence set forth in SEQ ID NO: 52; (4) VH comprising the amino acid sequence set forth in SEQ ID NO: 46, and VL comprising the amino acid sequence set forth in SEQ ID NO: 53; (5) VH comprising the amino acid sequence set forth in SEQ ID NO: 47, and VL comprising the amino acid sequence set forth in SEQ ID NO: 54; (6) VH comprising the amino acid sequence set forth in SEQ ID NO: 48 and VL comprising the amino acid sequence set forth in SEQ ID NO: 55; or (7) VH comprising the amino acid sequence set forth in SEQ ID NO: 49, and VL comprising the amino acid sequence set forth in SEQ ID NO: 56 An anti-FGFR2B antibody or antigen-binding fragment thereof selected from:

2. 1. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising: Heavy chain complementarity determining regions (HCDRs), including 1-3 of HCDR1, HCDR2, and HCDR3; and (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3; (2) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or 7, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or 8, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6; (3) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11; (4) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 13, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14; (5) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18; or (6) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; and / or a light chain complementarity determining region (LCDR), comprising 1-3 of LCDR1, LCDR2, and LCDR3; and (1) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; (2) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; (3) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; (4) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33; (5) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36; (6) LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 38, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 39; or (7) An anti-FGFR2B antibody or an antigen-binding fragment thereof, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 41, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

42.

3. 1. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising a heavy chain complementarity determining region (HCDR), HCDR1, HCDR2, and HCDR3, and a light chain complementarity determining region (LCDR), LCDR1, LCDR2, and LCDR3; and (1) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; (2) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 25, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 27; (3) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 30; (4) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33; (5) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 12, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15 or 13, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36; (6) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 17, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 37, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 38, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 39; or (7) An anti-FGFR2B antibody or an antigen-binding fragment thereof, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 40, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 41, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

42.

4. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in the table below: Table 1 An anti-FGFR2B antibody or an antigen-binding fragment thereof comprising:

5. 1. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region (VH) comprising an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 43 to 49; and / or An anti-FGFR2B antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL), wherein the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 50 to 56.

6. 1. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), and (1) VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 43, and VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50; (2) VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44, and VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51; (3) VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45, and VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52; (4) VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46, and VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53; (5) VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47, and VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54; (6) VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48, and VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55; or (7) An anti-FGFR2B antibody or an antigen-binding fragment thereof, wherein the VH comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49, and the VL comprises an amino acid sequence identical to or having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

56.

7. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising any combination of a heavy chain variable region (VH) and a light chain variable region (VL) as set forth in the table below: Table 2 An anti-FGFR2B antibody or an antigen-binding fragment thereof comprising:

8. 8. The isolated antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the antibody is a chimeric or humanized antibody, optionally wherein the antibody is a humanized antibody.

9. comprising an Fc region variant, wherein the Fc region variant has enhanced binding to FcγR and / or enhanced ability to mediate ADCC; Preferably, the Fc region variants comprise one or more of the following sets of substitutions: (1) L235V, F243L, R292P, Y300L, and P396L; (2) S239D and I332E; (3) S239D, A330L, and I332E 9. The isolated antibody or fragment of any one of claims 1 to 8, comprising:

10. 10. The isolated antibody or antigen-binding fragment of any one of claims 1 to 9, wherein the antibody comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO: 71 or 72, preferably SEQ ID NO:

72.

11. 1. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) and a light chain (LC), and (1) the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 64; (2) the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 65; (3) the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 66; (4) The HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 67; (5) The HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 68; (6) The HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 62, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 69; or (7) An anti-FGFR2B antibody or antigen-binding fragment thereof, wherein the HC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 63, and the LC comprises an amino acid sequence identical to or having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

70.

12. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising any combination of heavy chain (HC) and light chain (LC) as set forth in the table below: Table 3 An anti-FGFR2B antibody or an antigen-binding fragment thereof comprising:

13. 13. The isolated antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the antibody comprises a hypofucosylated or defucosylated Fc region.

14. 14. The isolated antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the antibody binds to FGFR2b but not to FGFR2c.

15. A nucleic acid molecule encoding the isolated antibody or antigen-binding fragment of any one of claims 1 to 14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A host cell expressing the nucleic acid molecule of claim 15 and / or the vector of claim 16.

18. 15. An immunoconjugate comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 14 conjugated to a payload.

19. 19. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 14, the nucleic acid molecule of claim 15, the vector of claim 16, the host cell of claim 17, or the immunoconjugate of claim 18.

20. 15. A method for preparing the isolated antibody or antigen-binding fragment of any one of claims 1 to 14, comprising: Culturing the host cell of claim 16 under conditions permissive for production of the antibody, antigen-binding fragment, or bispecific binding protein; Recovering the antibody or antigen-binding fragment from the culture. The method includes:

21. 20. A pharmaceutical combination comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 14, the nucleic acid of claim 15, the vector of claim 16, the host cell of claim 17, the immunoconjugate of claim 18, or the pharmaceutical composition of claim 19.

22. 21. A method for treating a disease associated with FGFR2b, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment of any one of claims 1 to 14, the nucleic acid of claim 15, the vector of claim 16, the host cell of claim 17, the immunoconjugate of claim 18, the pharmaceutical composition of claim 19, or the pharmaceutical combination of claim 21.

23. 23. The method of claim 22, wherein the subject is a human.

24. 24. The method of claim 23, wherein the disease is cancer, preferably selected from breast cancer (e.g., triple-negative breast cancer), gastric cancer, GEJ cancer, esophageal cancer, lung cancer (e.g., squamous cell NSCLC), ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, bile duct cancer, and pancreatic cancer, more preferably selected from breast cancer, gastric cancer, esophageal cancer, and lung cancer.

Images