Anti-FGFR2B antibodies and uses thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to antibodies, more particularly to anti-FGFR2B antibodies and antigen-binding fragments thereof, methods for preparing the antibodies, and uses thereof for treating or preventing FGFR2B-related diseases or symptoms. Background Art

[0002] Targeted therapies that interfere with oncogenic driver gene alterations have achieved great success in tumors such as 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 cancers with specific driver gene alterations 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). The development of new therapies targeting alterations in other cancer driver factors is extremely urgent to improve patient outcomes.

[0003] Receptor tyrosine kinases (RTKs) are single-pass transmembrane proteins whose overexpression is associated with breast cancer and other cancers and reduces disease-free survival (Templeton AJ, Diez-Gonzalez L, Ace O, Vera-Badillo F, Seruga B, Jordán J, Amir E, Pandiella A, A. Prognostic relevance of receptor tyrosinekinase 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, KunduGC. Receptor tyrosine kinases (RTKs) in breast cancer: signaling, therapeutic implications and challenges. Mol Cancer.2018Feb 19;17(1):34). Following ligand stimulation, RTKs activate several pathways, including mitogen-activated protein kinases (MAPKs), Janus kinases (JAKs) / signal transducers and activators of transcription (STATs), phospholipase Cγ (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 consists of proteins from fibroblasts, adipocytes, immune cells, and the extracellular matrix and extended vasculature.

[0004] Fibroblast growth factor receptor (FGFR) and their isoforms are known RTK.After being combined with FGF and specific cofactor, the dimerization induction tyrosine (Y) phosphorylation of FGFR kinase domain causes receptor to be fully activated and phosphorylated and the raising of adaptor protein.FGFR is characterized by the multiple alternative splicing of its mRNA producing various isoforms, these alternative splicings (Ornitz et al., J.Biol.Chem.271:15292,1996;On the sequence of FGFR2 and its isoform, see also Swiss-ProtP21802 and isoform P21802-1 to -20), wherein the FGFR2IIIb form (also known as K-sam-II) of FGFR2 is the high affinity receptor of FGF1 and FGF family member (FGF7, FGF10 and FGF22).

[0005] Due to the expression pattern of FGFR2 isoforms and their ligands, FGFR2 plays an important role in epithelial-mesenchymal interactions (Finchetal., Dev.Dyn.203:223,1995), such as possibly mediating the effects from the tumor microenvironment, promoting dysplasia or tumor progression. According to reports, KGF (FGF7) and KGFR (FGFR2IIIb) are overexpressed in many pancreatic cancers (Ishiwata et al., Am.J.Pathol.153:213,1998, and Francavilla C, O'Brien CS.Fibroblastgrowth factor receptor signalling dysregulation and targeting in breast cancer.Open Biol.2022Feb; 12 (2): 210373), and their co-expression is associated with poor prognosis (Cho et al., Am.J.Pathol.170:1964,2007). Somatic mutations in the FGFR2 gene are found in 12% of a large cohort of endometrial (uterine) carcinomas and are essential for tumor cell survival in several cases examined (Dutt et al., Proc. Natl. Acad. Sci. USA 105:8713, 2008). In both tumors, the FGFR2 mutation was found to be the same S252W substitution associated with Apert syndrome. Amplification and overexpression of FGFR2 are associated with undifferentiated diffuse gastric cancer, which carries a significantly poor prognosis (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al., Gastroenterol. 131:1530, 2006).

[0006] There is a need in the art for antibodies that can bind to FGFR2b with high affinity and block FGFR2b signaling. Summary of the Invention

[0007] The present invention provides anti-FGFR2B antibodies or antigen-binding fragments thereof, and methods for preparing and using the same, including methods for treating FGFR2B-related diseases or symptoms.

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

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

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

[0011] In some embodiments, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising three CDRs of a heavy chain variable region (VH), namely HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL), namely LCDR1, LCDR2 and LCDR3; wherein the VH and VL are selected from:

[0012] (1) VH comprises the amino acid sequence set forth in SEQ ID NO:43 and VL comprises the amino acid sequence set forth in SEQ ID NO:50;

[0013] (2) VH comprises the amino acid sequence set forth in SEQ ID NO:44 and VL comprises the amino acid sequence set forth in SEQ ID NO:51;

[0014] (3) VH comprises the amino acid sequence set forth in SEQ ID NO:45 and VL comprises the amino acid sequence set forth in SEQ ID NO:52;

[0015] (4) VH comprises the amino acid sequence set forth in SEQ ID NO:46 and VL comprises the amino acid sequence set forth in SEQ ID NO:53;

[0016] (5) VH comprises the amino acid sequence set forth in SEQ ID NO:47 and VL comprises the amino acid sequence set forth in SEQ ID NO:54;

[0017] (6) VH comprises the amino acid sequence set forth in SEQ ID NO: 48 and VL comprises the amino acid sequence set forth in SEQ ID NO: 55; or

[0018] (7) VH comprises the amino acid sequence shown in SEQ ID NO:49 and VL comprises the amino acid sequence shown in SEQ ID NO:56.

[0019] In one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of the heavy chain complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, wherein:

[0020] (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0021] (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 or 7, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5 or 8, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6;

[0022] (3) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11;

[0023] (4) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or 13, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14;

[0024] (5) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18; or

[0025] (6) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21.

[0026] In one aspect, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of the light chain complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, wherein:

[0027] (1) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24;

[0028] (2) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 25, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 27;

[0029] (3) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 30;

[0030] (4) LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 32, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 33;

[0031] (5) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 36;

[0032] (6) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 37, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 38, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 39; or

[0033] (7) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO:40, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:41, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:42.

[0034] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise heavy chain complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, wherein:

[0035] (1) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24;

[0036] (2) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 25, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 27;

[0037] (3) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 30;

[0038] (4) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 31, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 33;

[0039] (5) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or 13, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 36;

[0040] (6) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 37, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 38, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 39; or

[0041] (7) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 40, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 41, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 42.

[0042] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein that bind to FGFR2B comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any combination listed in Table 1 below:

[0043] Table 1

[0044]

[0045]

[0046] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 43-49.

[0047] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 50-56.

[0048] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 43, wherein the VL comprises an amino acid sequence identical to, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 50.

[0049] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 44, wherein the VL comprises an amino acid sequence identical to, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 51.

[0050] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 45, wherein the VL comprises an amino acid sequence identical to, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 52.

[0051] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 46, wherein the VL comprises an amino acid sequence identical to, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 53.

[0052] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 47, wherein the VL comprises an amino acid sequence identical to, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 54.

[0053] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 48, wherein the VL comprises an amino acid sequence identical to, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 55.

[0054] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 49, wherein the VL comprises an amino acid sequence identical to, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 56.

[0055] In some embodiments, the 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 as shown in any combination listed in Table 2 below:

[0056] Table 2

[0057]

[0058]

[0059] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 57, and the LC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 64.

[0060] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 58, wherein the LC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 65.

[0061] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 59, and the LC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 66.

[0062] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 60, wherein the LC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 67.

[0063] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 61, wherein the LC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 68.

[0064] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 62, wherein the LC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 69.

[0065] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided herein comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 63, and the LC comprises an amino acid sequence identical to, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with, the amino acid sequence shown in SEQ ID NO: 70.

[0066] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise an Fc region. In some embodiments, the Fc region is modified by mutating one or more amino acids (e.g., introducing one or more amino acid substitutions) to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or increase the affinity of the antibody for Fcγ receptors. In some preferred embodiments, the one or more amino acid mutations occur at the following sites 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, 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 some preferred embodiments, the one or more amino acid mutations occur at one or more of 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. In some preferred embodiments, the one or more amino acids mutated are selected from the following substitutions according to the EU numbering system: L235V, G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L. In some preferred embodiments, the mutated amino acid comprises the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) according to the EU numbering system. In some preferred embodiments, the mutated amino acid comprises the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL). The anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise an Fc region having the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) according to the EU numbering system.

[0067] In some preferred embodiments, 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 substitutions:

[0068] (1) L235V, F243L, R292P, Y300L, and P396L;

[0069] (2) S239D and I332E;

[0070] (3)S239D, A330L and I332E.

[0071] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain (HC) and a light chain (LC) as shown in any combination listed in Table 3 below:

[0072] Table 3

[0073]

[0074]

[0075] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise a constant region that is afucosylated or has reduced fucosylation.

[0076] In some embodiments, the isolated antibody or antigen-binding fragment thereof provided herein binds to FGFR2b but not to FGFR2c.

[0077] In some embodiments, the anti-FGFR2B antibodies provided herein are monoclonal antibodies.

[0078] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein are murine antibodies, chimeric antibodies, humanized antibodies, or human antibodies. In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein are full-length antibodies, single-domain antibodies (e.g., VHH), Fab, Fab', Fab'-SH, (Fab')2, single-chain antibodies (e.g., scFv), Fv, or dAb (domain antibodies).

[0079] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise an Fc region. In some embodiments, the amino acid sequence of the Fc region is identical to the Fc region sequence of human IgG1, IgG2, or IgG4, or is a variant thereof.

[0080] In another aspect, the present invention provides an anti-FGFR2B antibody that is antagonistic and comprises the CDRs of an antibody provided herein. In some embodiments, the antagonistic anti-FGFR2B antibody comprises an Fc region variant that enhances the effector function of the antibody. In some embodiments, the effector function is ADCC. In some embodiments, the antagonistic anti-FGFR2B antibody comprises an Fc region variant that is an IgG1 VLPYLL. In some embodiments, the antagonistic anti-FGFR2B antibody comprises an Fc region variant that is a hypofucosylated or afucosylated IgG1 or IgG4.

[0081] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

[0082] (1) Cross-reactivity with FGFR2B homologs from humans, cynomolgus monkeys, rats, and mice;

[0083] (2) with high affinity, for example, with a K of less than 100 nM, such as less than 50 nM, such as less than 30 nM, preferably less than 10 nM or 5 nM D value, binds to human FGFR2B, especially binds to the extracellular domain of human FGFR2B, wherein preferably K D The values ​​were measured using surface plasmon resonance assay;

[0084] (3) with high affinity, for example, with an EC of less than 100 nM, such as less than 50 nM, such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 or 5 nM 50 value, binds to human FGFR2B expressed on the surface of cells (such as tumor cells), wherein preferably EC 50 Values ​​were measured using FACS assay;

[0085] (4) blocking the binding of human FGFR2B and its ligands FGF7, FGF10 and / or FGF22, for example, as determined by ELISA, with an inhibition rate of at least 50%, for example, at least 60%, 70%, 80%, 85% or 90%, and preferably IC 50 The value is less than 10 nM, more preferably less than 1 nM;

[0086] (5) exhibiting the same or similar binding affinity and / or specificity as any of the antibodies listed in any of Tables 1-3;

[0087] (6) inhibiting (e.g., competitively inhibiting) the binding of any antibody molecule listed in any one of Tables 1-3 to FGFR2B;

[0088] (7) binds to the same or overlapping epitope as any of the antibodies listed in any of Tables 1-3;

[0089] (8) binds to a different epitope than any of the antibodies listed in any of Tables 1-3;

[0090] (9) having the same or similar biological activity as any of the antibodies listed in Tables 1-3.

[0091] In another aspect, the present invention provides an isolated nucleic acid encoding any of the antibodies or fragments thereof provided herein, preferably encoding a heavy chain or light chain, or a heavy chain variable region or a light chain variable region, of an antibody of the present invention. Preferably, the nucleic acid further comprises a signal peptide coding sequence.

[0092] On the other hand, the present invention provides a recombinant vector or expression vector comprising one or more nucleic acids provided by the present invention, wherein the vector is suitable for recombinantly producing any antibody or antigen-binding fragment thereof provided by the present invention. In some embodiments, the vector is an expression vector.

[0093] In another aspect, the present invention provides a host cell comprising one or more nucleic acids, recombinant vectors or expression vectors provided by the present invention.

[0094] In another aspect, the present invention provides a method for producing an anti-FGFR2B antibody or an antigen-binding fragment thereof, comprising culturing a host cell comprising an expression vector encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to cause the host cell to express the antibody or fragment capable of binding to FGFR2B, and optionally recovering the expressed antibody or fragment from the host cell.

[0095] In another aspect, the present invention provides an FGFR2B-targeting immunoconjugate or a pharmaceutically acceptable salt or solvate thereof, comprising the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention conjugated to a payload.

[0096] In some embodiments, the payload is a drug, such as a cytotoxic agent.

[0097] In another aspect, the present invention provides a pharmaceutical composition comprising the anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, or host cell provided by the present invention, and optionally comprising at least one pharmaceutically acceptable excipient (such as a pharmaceutical carrier or pharmaceutical excipient).

[0098] In another aspect, the present invention provides a pharmaceutical combination comprising the anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition provided by the present invention, and one or more additional therapeutic agents.

[0099] In another aspect, the present invention also provides the use of the anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell of the present invention in the preparation of a medicament for treating FGFR2B-related diseases or disorders.

[0100] In another aspect, the present invention provides a method for killing FGFR2B-positive tumor cells in vitro or in vivo, wherein the method comprises contacting an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, pharmaceutical composition or drug combination provided by the present invention with a cell population comprising FGFR2B-positive tumor cells, or administering the anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, pharmaceutical composition or drug combination provided by the present invention to a subject in need thereof.

[0101] In another aspect, the present invention also provides a use of an anti-FGFR2B antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer, preferably the tumor is selected from breast cancer (e.g., triple-negative breast cancer), gastric cancer, gastroesophageal junction cancer, esophageal cancer, lung cancer (e.g., squamous NSCLC), ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, bile duct cancer and pancreatic cancer.

[0102] On the other hand, the present invention also provides an anti-FGFR2B antibody or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a pharmaceutical composition or a drug combination for treating and / or preventing a tumor, preferably the tumor is selected from breast cancer (e.g., triple-negative breast cancer), gastric cancer, gastroesophageal junction cancer, esophageal cancer, lung cancer (e.g., squamous NSCLC), ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, bile duct cancer and pancreatic cancer.

[0103] In another aspect, the present invention provides a method for treating or preventing an FGFR2B-related disease or condition, comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof, or nucleic acid, vector, host cell, or pharmaceutical composition or combination comprising the same provided herein. In some embodiments, the FGFR2B-related disease or condition is cancer, such as breast cancer or gastric cancer.

[0104] The anti-FGFR2B antibodies or antigen-binding fragments thereof of the present invention can also be combined with other therapeutic agents or treatment modalities to treat or prevent FGFR2B-related diseases or symptoms.

[0105] In another aspect, the present invention also provides a method for detecting FGFR2B in a sample by using the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention. The method can be used to diagnose / detect FGFR2B-related diseases or symptoms.

[0106] The present invention also encompasses any combination of any of the embodiments described herein.Any embodiment described herein or any combination thereof applies to any and all anti-FGFR2B antibodies or fragments thereof, methods and uses of the invention described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Epitope binning of the humanized anti-FGFR2B monoclonal antibodies of the present invention is shown.

[0108] Figure 2 The ADCC effect of the chimeric anti-FGFR2B monoclonal antibody of the present invention on KATO-III cells was demonstrated.

[0109] Figure 3 The binding specificity of the humanized anti-FGFR2B monoclonal antibodies of the present invention was demonstrated, and their binding to other FGFR family members was tested.

[0110] Figure 4 The species cross-reactivity of the humanized anti-FGFR2B monoclonal antibodies of the present invention was shown.

[0111] Figure 5 ELISA assay shows the blocking effect of humanized monoclonal antibodies on the interaction between FGFR2b and FGF7.

[0112] Figure 6 Flow cytometry assay showing binding of FGFR2b antibodies to 293T_hFGFR2b (A) and 293T_hFGFR2c (B) cells.

[0113] Figure 7 The ADCC reporter gene bioactivity of humanized FGFR2b targeting KATO-III cells and KYSE-180 cells was shown.

[0114] Figure 8 The inhibitory effect of several FGFR2b antibodies on FGF7-induced MCF7 cell proliferation is shown. MCF7 cells were incubated in serum-free medium and left untreated (medium) or treated with 30 μg / mL of hIgG isotype or several FGFR2b antibodies for 72 hours in the absence or presence of FGF7 (25 ng / mL). Cell proliferation was assessed by luminescent cell viability assay.

[0115] Figure 9Shown are HTRF assays detecting FGF7- or FGF10-induced phosphorylation of FGFR2 and ERK1 / 2 proteins in SNU-16 cells. (AB) Several FGFR2b antibodies inhibited FGF7-mediated phosphorylation of FGFR2 (A) and ERK1 / 2 (B). (CD) Several FGFR2b antibodies inhibited FGF10-mediated phosphorylation of FGFR2 (C) and ERK1 / 2 (D).

[0116] Figure 10 Figure 2 shows the human PBMC-mediated ADCC activity of several FGFR2b antibodies targeting KATO-III cells. (A) Primary ADCC effect mediated by a donor with the FcγRIIIA genotype of 158V / V. (B) Primary ADCC effect mediated by a donor with the FcγRIIIA genotype of 158V / F.

[0117] Figure 11 Plasma concentration-time curves are shown.

[0118] Figure 12 Efficacy of humanized monoclonal antibodies against SNU16 tumor model in Balb / c nude mice Detailed Description of the Invention

[0120] The present invention provides anti-FGFR2B antibodies or antigen-binding fragments thereof, characterized by having unique CDR sequences, binding to human FGFR2B with high affinity and specificity, and preferably mediating ADCC more efficiently. The anti-FGFR2B antibodies or antigen-binding fragments provided herein can be used as stand-alone therapies or in combination with other therapies for the treatment of FGFR2B-related diseases or conditions, such as cancer, inflammation, or autoimmune diseases.

[0121] definition

[0122] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0123] In order to more easily understand the present invention, certain scientific and technical terms are specifically defined below. Unless otherwise expressly defined elsewhere herein, the scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. For definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard three-letter and / or one-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0124] As used herein (including the claims), the singular forms "a," "an," and "the" include the corresponding plural forms unless the context clearly dictates otherwise.

[0125] The term "about" refers to a value or integer that is within an acceptable error range of a particular value or integer as determined by one of ordinary skill in the art, which depends in part on how the value or integer is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, "about" can mean a range of up to 5%, 10%, or 20% (i.e., ±5%, ±10%, or ±20%).

[0126] The term "and / or" when used to link two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0127] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0128] The term "FGFR2B" herein belongs to the FGFR family, which includes four members (FGFR1-4), and its cognate ligand fibroblast growth factor (FGF) - a family comprising 22 members (FGF1-14 and FGF16-23). ​​As used herein, the term refers to any natural FGFR2B, derived from any vertebrate, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length", unprocessed FGFR2B and any form of FGFR2B or any fragment resulting from processing in the cell. The term also includes naturally occurring variants of FGFR2B, such as splice variants or allelic variants. In some embodiments, FGFR2B refers to full-length FGFR2B from humans or a fragment thereof (e.g., a mature fragment without a signal peptide). In some embodiments, human FGFR2B refers to a mature FGFR2B (amino acid residues 1-21 are a leader peptide) identical to the amino acid sequence set forth in Accession No. Uniprot#P21802, or a fragment thereof (such as an extracellular domain comprising AA 22-377). In some embodiments, the term also encompasses a fusion protein comprising FGFR2B or a fragment thereof (such as an extracellular domain thereof), such as a fusion protein comprising the extracellular domain of human FGFR2B and an Fc region.

[0129] The term "FGFR2B ligand" or "FGF7 / 10" herein refers to a natural ligand of FGFR2B, or a functional variant thereof.

[0130] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains), or any antigen-binding fragment, mutant, variant or derivative thereof that retains the necessary epitope binding characteristics of an Ig molecule. Such mutant, variant or derivative antibody forms are known in the art, and non-limiting embodiments are discussed below. As used herein, the term "antibody" may refer to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but 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.

[0131] The term "affinity" herein 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 indicated, as used herein, "binding affinity" refers to 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 can generally be expressed in terms of the dissociation constant (K D Examples of assays known in the art for determining binding affinity include surface plasmon resonance (eg, BIACORE) or similar techniques (eg, ForteBio).

[0132] The term "specific binding" or "specifically binds", when referring to the interaction of an antibody, binding protein, or peptide with a second chemical substance, means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the second chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. Generally, if the antibody is specific for epitope "A", the presence of molecules containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody. Consistent with the present disclosure, a specific binding protein with a K of 10 nM or less D Binds to the corresponding antigen, for example, at 1 nM or less.

[0133] As used herein, the term "k on "(also referred to as "Kon", "kon") refers to the association rate constant for the association of a binding protein (e.g., an antibody) with an antigen to form an association complex (e.g., an antibody / antigen complex) as known in the art. "k on” is also referred to as the term “association rate constant” or “ka”, as used interchangeably herein. This value represents the rate of binding of an antibody to its target antigen or the rate of complex formation between the antibody and the antigen, as shown in the following formula:

[0134] Antibody ("Ab") + antigen ("Ag") → Ab-Ag.

[0135] As used herein, the term "k off ” (also known as “Koff”, “koff”) refers to the rate constant for the dissociation of a binding protein (e.g., an antibody) from an associated complex (e.g., an antibody / antigen complex), or the “dissociation rate constant,” as known in the art. This value represents the rate of dissociation of an antibody from its target antigen, or the rate at which the Ab-Ag complex separates into free antibody and antigen over time, as shown in the following formula:

[0136] Ab+Ag←Ab-Ag.

[0137] As used herein, the term "K D ” (also known as “K d ”) is intended to mean the “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by the dissociation rate constant (k off ) divided by the binding rate constant (k on The binding rate constant (k on ), dissociation rate constant (k off ) and the equilibrium dissociation constant (K D ) is used to express the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Fluorescence-based techniques can be used, providing high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental methods and instruments can be used, such as (Biomolecular Interaction Analysis) assay (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). Using e.g. Biomembrane interferometry (BLI) using the RED96 system (Pall Forte Bio LLC) is another affinity determination technique. Alternatively, the RED96 system available from Sapidyne Instruments (Boise, Idaho) can be used. (Kinetic Exclusion Assay) determination.

[0138] The terms "antagonist anti-FGFR2B antibody," "FGFR2B inhibitor," "FGFR2B antagonist antibody," "antagonist FGFR2B antibody," and "FGFR2B antibody antagonist" are used interchangeably herein. These terms include antibodies that can inhibit and / or block FGFR2B-mediated biological signal transduction activity. In some embodiments, FGFR2B antagonist antibodies inhibit or reduce signal transduction pathways triggered by FGFR2B, and / or inhibit or reduce FGFR2B-mediated cellular responses such as cancer cell proliferation or cancer cell survival, for example, by blocking the binding of FGFR2B to a FGFR2B ligand or substantially reducing the binding of FGFR2B to a FGFR2B ligand.

[0139] The term "FGFR2B-related disease or condition" herein refers to a non-physiological condition associated with the expression, function, or activity of FGFR2B or associated with FGFR2B-mediated signal transduction activity, including but not limited to cancer, inflammation, and autoimmune diseases. In some preferred embodiments, the disease will benefit from blocking FGFR2B-mediated signal transduction.

[0140] The terms "immune response" or "immune reaction" are used interchangeably herein and refer to an effect produced by, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which results in selective damage, destruction, or removal of invading pathogens, cells or tissues infected with pathogens, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation from the human body. In some embodiments, the FGFR2B antibody antagonists of the present invention can inhibit or reduce the immune response, for example, reducing immune rejection in graft-versus-host disease. In some embodiments, the FGFR2B antibody agonists of the present invention can enhance anti-tumor immune responses.

[0141] Term " signal transduction " refers to usually by protein-protein interaction, such as the biochemical cause-effect relationship that FGF7 / 10 (ligand) starts in conjunction with FGFR2B (receptor), and described relationship causes signal to be transmitted to another part of cell from a part of cell.Usually, transmission comprises the specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in the series reaction that causes signal transduction.The penultimate process usually includes nuclear event, thus causes the variation of gene expression.

[0142] The terms "activity" or "biological activity" or "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, ability to neutralize or antagonize FGFR2B activity in vivo or in vitro, ability to enhance or activate FGFR2B in vivo or in vitro, IC50 activity to block FGFR2B binding to FGF7 / 10, and the ability to inhibit FGFR2B binding to FGF7 / 10. 50 ICs that inhibit FGFR2B-FGF7 / 10-mediated cell proliferation 50 , the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies well known in the art include, for example, (species) cross-reactivity (i.e., usually with the non-human homologues of the target peptide, or with other proteins or tissue cross-reactions), and the ability to maintain high expression levels of the antibody in mammalian cells. The aforementioned properties or characteristics can be observed, measured or assessed using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other source).

[0143] The terms "whole antibody," "full-length antibody," and "intact antibody" are used interchangeably herein to refer to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). "Complementarity determining regions" or "CDR regions" or "CDRs" are regions of the antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, starting from the N-terminus. Sequential numbering. The CDRs within the antibody heavy chain variable domain are sequentially referred to as HCDR1, HCDR2, and HCDR3, while the CDRs within the antibody light chain variable domain are sequentially referred to as LCDR1, LCDR2, and LCDR3. Each VH and VL constant region consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in antibody-antigen binding but exhibits various effector functions.

[0144] In a given VH or VL amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known schemes, including, for example, 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); 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 according to any one or a combination of schemes in the art and human evaluation.

[0145] The light chains of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The heavy chains of antibodies can be divided into five main different types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2.

[0146] An "IgG-type antibody" refers to an antibody whose heavy chain constant region belongs to the IgG type. For example, an IgG2-type antibody refers to an antibody whose heavy chain constant region is derived from IgG2.

[0147] The term "antigen-binding fragment" of an antibody herein includes fragments or derivatives of an antibody. Typically, the antigen-binding fragment comprises at least one fragment of the antigen-binding region or variable region of the 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 formed from antibody fragments. When the binding activity to the antigen is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of the antigen-binding activity of the antibody from which it is derived. Preferably, the 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.

[0148] It is contemplated that antibodies or antigen-binding fragments thereof may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "function-conservative variants" of antibodies). In a preferred aspect, conservative substitutions are from the conservative substitution residues shown in Table A below, preferably the preferred conservative amino acid substitution residues shown in Table A.

[0149] Table A

[0150]

[0151]

[0152] An epitope is a region of an antigen to which an antibody binds. An epitope can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0153] The term "isolated anti-FGFR2b antibody or antigen-binding fragment" herein refers to the purified state of the anti-FGFR2b antibody or antigen-binding fragment. For example, "isolated" can mean that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. However, as is known to those skilled in the art, the term "isolated" does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the antibodies described herein. In some embodiments, the isolated antibody or antigen-binding fragment can have a purity greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0154] The term "monoclonal antibody" herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, traditional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0155] As used herein, the term "chimeric antibody" refers to an antibody that has the variable domains of a first antibody and the constant domains of a second antibody, wherein the first and second antibodies are from different species. Typically, the variable domains are derived from antibodies of experimental animals such as rodents, while the constant domain sequences are derived from human antibodies. This makes the resulting chimeric antibody less likely to induce an adverse immune response in human subjects compared to antibodies from these experimental animals.

[0156] The term "humanized antibody" herein refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises at least one, usually two, variable domains, in which all or substantially all of the hypervariable loops are equivalent to those of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are those of human immunoglobulins. A humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc). In some cases, as is known to those skilled in the art, amino acid mutations may be introduced into a humanized antibody (e.g., variable domains, framework regions, and / or constant regions (if present)), for example to improve certain properties of the antibody; such an antibody form also falls within the scope of the present invention's "humanized antibody."

[0157] As is known to those skilled in the art, antibodies may have sugar chains in the form of the cells used to produce the antibody. For example, when produced in mice, in mouse cells, or in hybridomas derived from mouse cells, antibodies may contain mouse sugar chains. Alternatively, when produced in rats, in rat cells, or in hybridomas derived from rat cells, antibodies may contain rat sugar chains.

[0158] The term "Fc region" herein is used to define the C-terminal region containing at least a portion of a constant region in an immunoglobulin heavy chain. The term includes native sequence Fc regions and Fc region variants. Native sequence Fc regions encompass naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc regions of their allotypes (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, human IgG heavy chain Fc regions extend from Cys226, or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is 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 ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0159] The terms "Fc region variant" or "variant Fc region" are used interchangeably herein to refer to an Fc region polypeptide comprising modifications relative to a native sequence Fc region. The Fc region variants of the present invention are defined according to the amino acid modifications that comprise them. Thus, for example, L235V is an Fc region variant comprising a substitution of leucine with valine at position 235 relative to the parent polypeptide, where numbering is according to the EU index. Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. Variants may comprise non-natural amino acids.

[0160] The term "Fc receptor" or "FcR" herein describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is an FcγR (gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, as well as allelic variants and alternative splicing forms of those receptors. FcγRII includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA is an immunoreceptor that contains a tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB is an immunoreceptor that contains a tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Annu. Rev. Immunol. 15:203-234 (1997). For review of FcRs, see, e.g., Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and deHaas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" is used herein to encompass other FcRs, including those that will be identified in the future. The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulates the homeostasis of immunoglobulins. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Immunol. 24(13):591-597 (1998). Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.). Human FcRn high-affinity binding polypeptides can be assayed for binding to human FcRn in vivo and for serum half-life, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO 2000 / 42072 (Presta) describes antibody variants with improved or reduced FcR binding. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0161] The term "pharmaceutically acceptable excipient" refers to a diluent, an adjuvant (eg, Freund's adjuvant (complete and incomplete)), a pharmaceutical excipient, a pharmaceutical carrier, or a stabilizer, etc., which is administered together with an active substance.

[0162] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0163] As used herein, the term "therapeutic agent" encompasses any substance that is effective in preventing or treating a relevant disease, such as cancer.

[0164] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0165] "Chemotherapeutic agents" include small chemical molecule drugs useful in treating cancer or immune system disorders.

[0166] The term "small molecule drug" refers to a low molecular weight compound that is capable of modulating biological processes. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

[0167] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that regulates (e.g., suppresses or enhances) an immune response. The immune response can be a humoral response or a cellular response. In some embodiments, the immunomodulator comprises an immunosuppressant that suppresses the immune response, such as an immunosuppressant that is beneficial for suppressing the immune response in the treatment of inflammation and autoimmune diseases. In some embodiments, the immunomodulator comprises an agent or drug that enhances the immune response, such as an agent or drug that is beneficial for enhancing the anti-cancer immune response in the treatment of cancer.

[0168] The terms "cancer" and "cancer" refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. Included within this definition are benign and malignant tumors, as well as dormant tumors or micrometastases. Cancer includes, but is not limited to, solid tumors and hematologic cancers. Examples of various cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias.

[0169] The term "isolated nucleic acid" refers to a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that has been separated by human intervention from all or part of the polynucleotides with which it is associated in nature; is operably linked to a polynucleotide with which it is not naturally associated; or occurs as part of a larger sequence that is not found in nature.

[0170] The term "vector" herein refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively linked genes. Such vectors are referred to herein as "expression vectors," which refer to nucleic acids that can replicate and express a target gene when transformed, transfected, or transduced into a host cell. An expression vector comprises one or more phenotypic selection markers and a replication origin to ensure that the vector is maintained and to provide amplification in the host if necessary.

[0171] As used herein, "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation can be performed under natural or artificial conditions using various methods well known in the art. Transformation can rely on any known method for inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell to be transformed and may include, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replicating as an autonomously replicating plasmid or as part of the host chromosome. Cells that transiently express the inserted DNA or RNA for a limited time are also included.

[0172] The term "recombinant host cell" (or simply "host cell") means a cell into which exogenous DNA has been introduced. In one embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids encoding an antibody, for example, a host cell as described in U.S. Patent No. 7,262,028. These terms are intended to refer not only to a specific subject cell, but also to the offspring of such a cell. Because certain modifications may occur in offspring due to mutations or environmental influences, these offspring may actually be different from the parent cell, but are still included in the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotes and eukaryotic cells selected from any kingdom of life. In another embodiment, eukaryotic cells include protozoa, fungi, plants, and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line Escherichia coli; mammalian cell lines CHO, HEK 293, Jurkat, COS, NS0, SP2, and PER.C6; insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

[0173] The term "subject" or "patient" or "individual" herein includes any human or non-human animal. 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.

[0174] The terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" herein 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, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms, such as an amount to treat, cure, prevent, or ameliorate a related medical condition or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0175] As used herein, "treatment" includes 1) therapeutic measures that cure, slow down, alleviate the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder and 2) prophylactic or preventative measures that prevent and / or slow the development of a pathological condition or disorder. Thus, treatment includes individuals already suffering from a disorder, individuals susceptible to developing a disorder, and individuals in whom a disorder is to be prevented. In some embodiments, the present invention relates to the treatment of a disease or condition; in other embodiments, the present invention relates to the prevention of a disease or condition.

[0176] In some embodiments according to the present invention, " treatment " of disease or symptom refers to improving disease or symptom (that is, slowing down or preventing or reducing at least one of the progress of disease or its clinical symptoms). In other embodiments, " treatment " refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In other embodiments, " treatment " refers to regulating disease or symptom physically (for example, the stabilization of discernible symptoms), physiologically (for example, the stabilization of physical parameters) or in these two aspects. Unless clearly described in this article, the method for the treatment and / or prevention of the assessment of disease is generally known in the art.

[0177] In some further embodiments according to the present invention, "prevention" of a disease or symptom includes the inhibition of the occurrence or development of a disease or symptom or a symptom of a particular disease or symptom. In some embodiments, subjects with a family history of cancer are candidates for preventative regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly before they occur in subjects at risk for cancer.

[0178] In certain embodiments, a cancer is "treated" using the methods of the present invention and is considered successfully treated if the individual exhibits one or more of the following: a decrease in the number or complete disappearance of cancer cells; a decrease in tumor size; an inhibition or lack of cancer cell infiltration into peripheral organs, including, for example, spread of cancer to soft tissue and bone; an inhibition or lack of tumor metastasis; an inhibition or lack of tumor growth; relief of one or more symptoms associated with the particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in the tumorigenicity, frequency of tumorigenesis, or tumorigenic capacity of a tumor; a reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; or a combination of effects.

[0179] "Inhibiting tumor growth" refers to any mechanism by which tumor cell growth can be inhibited. In certain embodiments, tumor cell growth is inhibited by slowing tumor cell proliferation. In certain embodiments, tumor cell growth is inhibited by stopping tumor cell proliferation. In certain embodiments, tumor cell growth is inhibited by killing tumor cells. In certain embodiments, tumor cell growth is inhibited by inducing apoptosis in tumor cells. In certain embodiments, tumor cell growth is inhibited by inducing differentiation in tumor cells. In certain embodiments, tumor cell growth is inhibited by depriving tumor cells of nutrients. In certain embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In certain embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

[0180] As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. "(Percentage) Sequence Identity" can be calculated in the following manner: two optimally aligned sequences are compared in a comparison window, the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present in the two sequences to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to produce a percentage of sequence identity. Optimal alignment for determining percent sequence identity can be achieved in a variety of ways known in the art, for example, using 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 any algorithm required to achieve maximum alignment within the full-length sequence being compared or within the region of the target sequence. In the present invention, with respect to antibody sequences, percent amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred embodiment, optimally aligning according to the Kabat numbering convention.

[0181] Anti-FGFR2B antibodies and their production

[0182] The antibodies of the present invention can be produced using any suitable method for producing antibodies. Any suitable form of FGFR2B can be used as an immunogen (antigen) for producing antibodies. By way of example and not limitation, any FGFR2B variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing mouse-derived monoclonal anti-human FGFR2B antibodies can be produced by methods well known in the art. These methods include, but are not limited to, the hybridoma technology initially developed by Kohler et al. (1975) (Nature 256:495-497). Preferably, mouse splenocytes are isolated according to standard protocols and fused with a mouse myeloma cell line using PEG or by electrofusion. Hybridoma cells are then screened for secretory antibodies having FGFR2B binding activity. The DNA sequence of the hybridoma immunoglobulin variable region of the present invention can be determined using a degenerate primer PCR method.

[0183] Antibodies derived from rodents (such as mice) can cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use causes the human body to produce an immune response against the therapeutic antibody, which at least results in a loss of therapeutic efficacy, while severe cases result in potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which the mouse variable region is fused to the human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the retention of the intact rodent variable region in the chimeric antibody can still cause harmful immunogenicity in patients.

[0184] Grafting rodent variable region CDRs onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences. The humanized antibodies described herein can be constructed by inserting murine CDR regions into human germline framework regions using methods known in the art. See U.S. Patent No. 5,225,539 to Winter et al. and U.S. Patents Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0185] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the present invention can be determined using any of a number of well-known schemes (e.g., Kabat, Chothia, AbM, Contact, or North). It should be noted that the boundaries of the CDRs of the variable region of the same antibody obtained based on different definition systems may differ. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the specific CDR sequences, but whose claimed CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0186] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a subsection of a CDR. As will be appreciated by those skilled in the art, the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein. In some embodiments, in a variant of a CDR of an anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or IMGT can be replaced by conservative amino acid residues.

[0187] In some embodiments, the present invention also provides antibodies with altered effector functions. The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody class. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The effector functions of antibodies include, for example, 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; antibody cross-linking mediated by binding of the Fc region to cell surface FcR receptors. As will be appreciated by those skilled in the art, the appropriate antibody Fc region sequence can be selected based on factors such as whether it is necessary to recruit the immune system to kill target cells, whether it is necessary to induce antibody cross-linking through interaction with FcR, and the like. For example, when immune system recruitment and target cell killing are desired properties of the desired antibody, the Fc region of the antibody can be selected or further modified to provide enhanced binding to activated FcγR receptors and / or complement to promote, for example, ADCC or CDC effector functions. In addition, the Fc region can be selected or mutated to selectively provide the antibody with one or more Fc receptors while reducing or eliminating the binding to another one or more FcRs, thereby achieving the adjustment of the antibody effector function, such as enhancing antibody cross-linking while changing the strength 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; ShieldsRL, 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.2001Mar 2;276(9):6591-604.Epub 2000Nov 28.

[0188] In some embodiments, the Fc region of the antibodies provided herein may be modified with one or more amino acids to produce an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., an Fc region of human IgG1, IgG2, IgG3, or IgG4) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. For example, in Bruhns and In an article published in Immunol Rev. 2015 Nov; 268(1): 25-51, on page 44, a number of modifications to human IgG1 were summarized to enhance or reduce its binding to FcγR and enhance or reduce the corresponding functions.

[0189] In some embodiments, the present invention provides an isolated anti-FGFR2B antibody or an antigen-binding fragment thereof, which comprises one to three selected from HCDR1, HCDR2 and HCDR3 of the heavy chain variable region (VH), wherein the amino acid sequence of the VH is shown in any one of SEQ ID NOs: 43-49.

[0190] In some embodiments, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises one to three of LCDR1, LCDR2 and LCDR3 selected from the light chain variable region (VL), wherein the amino acid sequence of VL is shown in any one of SEQ ID NOs: 50-56.

[0191] In some embodiments, the present invention provides an isolated anti-FGFR2B antibody or an antigen-binding fragment thereof, which comprises three CDRs of a heavy chain variable region (VH), namely HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL), namely LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the VH is shown in any one of SEQ ID NOs: 43-49, and the amino acid sequence of the VL is shown in any one of SEQ ID NOs: 50-56.

[0192] In some embodiments, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising three CDRs of a heavy chain variable region (VH), namely HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL), namely LCDR1, LCDR2 and LCDR3; wherein the VH and VL are selected from:

[0193] (1) VH comprising the amino acid sequence of SEQ ID NO:43 and VL comprising the amino acid sequence of SEQ ID NO:50;

[0194] (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;

[0195] (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;

[0196] (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;

[0197] (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;

[0198] (6) VH comprising the amino acid sequence shown in SEQ ID NO: 48 and VL comprising the amino acid sequence shown in SEQ ID NO: 55; or

[0199] (7) VH comprising the amino acid sequence shown in SEQ ID NO: 49 and VL comprising the amino acid sequence shown in SEQ ID NO: 56.

[0200] In some embodiments, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of the heavy chain complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, wherein:

[0201] (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom;

[0202] (2) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or 7 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or 8 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom;

[0203] (3) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 11 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom;

[0204] (4) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or 13 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom;

[0205] (5) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom; or

[0206] (6) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0207] In some embodiments, the present invention provides an isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising one to three of the light chain complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, wherein:

[0208] (1) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 23 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto;

[0209] (2) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 25 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 27 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto;

[0210] (3) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 28 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 29 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 30 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto;

[0211] (4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 32 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 33 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto;

[0212] (5) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 34 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 36 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto;

[0213] (6) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 37 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 38 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 39 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto; or

[0214] (7) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 40 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 41 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 42 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared thereto.

[0215] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises heavy chain complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3 and light chain complementarity determining regions (LCDRs), LCDR1, LCDR2 and LCDR3, wherein:

[0216] (1) The HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 40 The amino acid sequence shown in NO:24, optionally further having at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared thereto;

[0217] (2) The HCDR1 comprises the amino acid sequence of SEQ ID NO: 4 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 5 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 25 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 26 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 10 The amino acid sequence shown in NO:27, optionally further having at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared thereto;

[0218] (3) The HCDR1 comprises the amino acid sequence of SEQ ID NO: 7 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 8 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 28 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 29 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 40 The amino acid sequence shown in NO:30, optionally further having at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared thereto;

[0219] (4) The HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 11 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 31 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 32 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 40 The amino acid sequence shown in NO:33, optionally further having at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared thereto;

[0220] (5) The HCDR1 comprises the amino acid sequence of SEQ ID NO: 12 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or 13 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 34 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 35 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR3 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence shown in NO:36, optionally further having at least one and no more than 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) compared thereto;

[0221] (6) The HCDR1 comprises the amino acid sequence of SEQ ID NO: 16 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 37 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 38 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 40 NO:39, and optionally further comprising at least one and no more than 3, 2 or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) compared thereto; or

[0222] (7) The HCDR1 comprises the amino acid sequence of SEQ ID NO: 19 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 20 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 21 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 40 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 41 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 52 and optionally further has at least one and no more than 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom, The amino acid sequence shown in NO:42, and optionally further has at least one and no more than 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) compared thereto.

[0223] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to FGFR2B, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any combination listed in Table 1 below:

[0224] Table 1

[0225]

[0226]

[0227] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 43-49.

[0228] In some embodiments, the anti-FGFR2B antibody or antigen-binding fragment thereof provided by the present invention comprises a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 50-56.

[0229] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 43, or an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, or an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0230] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 44, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 51.

[0231] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 45, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 52.

[0232] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 46, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 53.

[0233] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 47, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 54.

[0234] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 48, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 55.

[0235] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 49, wherein the VL comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56.

[0236] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to CCR8, comprising a heavy chain variable region VH and a light chain variable region VL as shown in any combination listed in Table 2 below:

[0237] Table 2

[0238]

[0239] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 57, wherein the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 64.

[0240] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 58, wherein the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 65.

[0241] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 59, wherein the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 66.

[0242] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 60, wherein the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 67.

[0243] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 61, wherein the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 68.

[0244] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 62, wherein the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 69.

[0245] In some embodiments, the present invention provides an anti-FGFR2B antibody or antigen-binding fragment thereof, which comprises a heavy chain (HC) and a light chain (LC), wherein the HC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 63, wherein the LC comprises an amino acid sequence identical to or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 70.

[0246] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise an Fc region. In some embodiments, the Fc region is modified by mutating one or more amino acids (e.g., introducing one or more amino acid substitutions) to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or increase the affinity of the antibody for Fcγ receptors. In some preferred embodiments, the one or more amino acid mutations occur at the following sites 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, 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 some preferred embodiments, the one or more amino acid mutations occur at one or more of 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. In some preferred embodiments, the one or more amino acids mutated are selected from the following substitutions according to the EU numbering system: L235V, G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L. In some preferred embodiments, the mutated amino acid comprises the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) according to the EU numbering system. In some preferred embodiments, the mutated amino acid comprises the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL). The anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise an Fc region having the following substitutions: L235V, F243L, R292P, Y300L, and P396L (VLPYLL) according to the EU numbering system.

[0247] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain (HC) and a light chain (LC) as shown in any combination listed in Table 3 below:

[0248] Table 3

[0249]

[0250] In some embodiments, the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein comprise a constant region that is afucosylated or has reduced fucosylation.

[0251] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0252] In preferred embodiments, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region. In some embodiments, the amino acid changes occur in the heavy chain constant region and / or the light chain constant region.

[0253] In some embodiments, antibodies of the invention comprising amino acid changes have comparable or similar properties as the specific antibodies disclosed herein.

[0254] In some embodiments, the anti-FGFR2B antibodies of the invention include post-translational modifications to the CDRs, light chain variable region, heavy chain variable region, light chain, or heavy chain.

[0255] In some embodiments, 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 bispecific (multispecific) antibody.

[0256] In some embodiments, the anti-FGFR2B antibodies provided by the present invention are antibodies of any IgG isotype, such as IgG1, IgG2, IgG3 or IgG4.

[0257] On the one hand, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites. Glycosylation can be changed to, for example, increase the affinity of the antibody for the "antigen". This carbohydrate modification can be accomplished by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the site. This aglycosylation-free modification can increase the affinity of the antibody for the antigen. Such methods are described, for example, in U.S. Patent No. 5,426,300. When the antibody comprises an Fc region, the carbohydrates attached thereto can be changed. In some applications, modifications to remove unwanted glycosylation sites can be useful, such as removing the fucose module to improve antibody-dependent cell-mediated cytotoxicity (ADCC) function. In other applications, galactosidation modification can be performed to modify complement-dependent cytotoxicity (CDC).

[0258] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0259] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0260] In some embodiments, the antibodies of the invention have one or more of the following properties:

[0261] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

[0262] (1) Cross-reactivity with FGFR2B homologs from humans, cynomolgus monkeys, rats, and mice;

[0263] (2) with high affinity, for example, with a K of less than 100 nM, such as less than 50 nM, such as less than 30 nM, preferably less than 10 nM or 5 nM Dvalue, binds to human FGFR2B, especially binds to the extracellular domain of human FGFR2B, wherein preferably K D The values ​​were measured using surface plasmon resonance assay;

[0264] (3) with high affinity, for example, with an EC of less than 100 nM, such as less than 50 nM, such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 or 5 nM 50 value, binds to human FGFR2B expressed on the surface of cells (such as T cells), wherein preferably EC 50 Values ​​were measured using FACS assay;

[0265] (4) blocking the binding of human FGFR2B and its ligand FGF7 / 10, for example, as determined by ELISA, with an inhibition rate of at least 50%, for example, at least 60%, 70%, 80%, 85% or 90%, and preferably IC 50 The value is less than 10 nM, more preferably less than 1 nM;

[0266] (5) exhibit the same or similar binding affinity and / or specificity as any of the antibodies listed in Table 2;

[0267] (6) inhibiting (e.g., competitively inhibiting) the binding of any antibody molecule listed in Table 2 to FGFR2B;

[0268] (7) bind to the same or overlapping epitope as any of the antibodies listed in Table 2;

[0269] (8) binds to a different epitope than any of the antibodies listed in Table 2;

[0270] (9) having the same or similar biological activity as any of the antibodies listed in Tables 1-3.

[0271] In some embodiments, the FGFR2B antibody of the present invention is an antagonist antibody comprising an Fc region that binds to an FcR, such as an FcγR, such as a human IgG1, IgG2 or IgG4 Fc region or a variant thereof (e.g., IgG4 S228P), preferably a human IgG1 Fc region or a variant thereof. The variant preferably has an FcγR binding affinity comparable to or stronger than that of the parent Fc region (e.g., a native sequence Fc region). Preferably, the antibody comprises a human IgG1 or IgG4 Fc region sequence having a constant region sequence as shown in SEQ ID NO: 74 or 75, or a human IgG1 or IgG4 Fc region variant having at least 95%, 96%, 97% or 99% identity to an Fc region sequence of a constant region sequence as shown in SEQ ID NO: 74 or 75, or having no more than 10, 5 or 1-3 amino acid modifications to an Fc region sequence of a constant region sequence as shown in SEQ ID NO: 74 or 75.

[0272] In some embodiments, the antibody comprises an Fc region variant, wherein the binding affinity of the Fc region variant to FcγR is enhanced by at least 10%, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more relative to a parent Fc region (e.g., a native sequence Fc region). In some embodiments, the antibody of the invention comprising an Fc region variant has enhanced effector function mediated by FcγR relative to a corresponding antibody comprising a 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). In some embodiments, the antibody comprises a human IgG1 Fc region sequence that is identical to the Fc region sequence of the constant region sequence as shown in SEQ ID NO: 21, or comprises a human IgG1 Fc region variant that is at least 95%, 96%, 97%, 98% or 99% identical to the Fc region sequence of the constant region sequence as shown in SEQ ID NO: 21, or has no more than 10, 5 or 1-3 amino acid modifications to the Fc region sequence of the constant region sequence as shown in SEQ ID NO: 21, and comprises a mutation that reduces the binding affinity of the Fc region to FcγR, preferably L235V, F243L, R292P, Y300L and P396L substitutions.

[0273] In some embodiments, the FGFR2B antagonist antibodies of the invention have one or more of the following characteristics:

[0274] (1) With high affinity, for example, less than 10 nM, more preferably less than 5 nM KD value, binding to human FGFR2B, wherein preferably K D The values ​​were measured using surface plasmon resonance assay;

[0275] (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, wherein the EC50 value is preferably measured using a FACS assay;

[0276] (3) blocking the binding of FGFR2B and its ligand FGF7 / 10, for example, as measured by ELISA, with an inhibition rate of at least 70%, preferably at least 80%, 85% or 90%, and preferably an IC50 value of less than 10 nM, more preferably less than 1 nM;

[0277] (4) blocking FGFR2B-mediated signaling activity;

[0278] (5) Inhibit the proliferation of cancer cells.

[0279] Antibody expression

[0280] The present invention relates to host cells comprising one or more expression vectors and methods for producing any of the antibodies or antigen-binding fragments thereof of the present invention, the methods comprising culturing the host cells, purifying and recovering the antibodies or antigen-binding fragments.

[0281] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications, or as commonly practiced in the art, or as described herein. The aforementioned techniques and procedures can generally be performed according to conventional methods well known in the art, which are also described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0282] In one aspect, the present invention provides nucleic acids encoding any of the above anti-FGFR2B antibodies or antigen-binding fragments thereof. For example, the present invention provides nucleic acids encoding segments comprising a heavy chain, light chain, variable region, or complementarity determining region as described herein. In some aspects, 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 some aspects, 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.

[0283] In one aspect, one or more vectors comprising the nucleic acid are provided. In some embodiments, the vector is an expression vector. The choice of expression vector depends on the intended host cell in which the vector is 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 its antigen-binding fragment. In some embodiments, the expression vector further comprises a sequence encoding the antibody constant region.

[0284] In one aspect, the present invention provides host cells for expressing the recombinant antibodies of the present invention, including prokaryotic or eukaryotic cells. In some embodiments, Escherichia coli is a prokaryotic host that can be used to clone and express the nucleic acids of the present invention. Other suitable microbial hosts include Bacillus, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas. In these prokaryotic hosts, expression vectors can also be prepared, which generally contain expression control sequences (e.g., replication origins) compatible with the host cell. In some embodiments, mammalian host cells are used to express and produce the anti-FGFR2B antibody polypeptides of the present invention. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes, or they can be mammalian cell lines with exogenous expression vectors, including normal human cells, or immortalized animal or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas.

[0285] In one aspect, the present invention provides a method for preparing an anti-FGFR2B antibody, wherein the method comprises introducing an expression vector into a mammalian host cell and producing the antibody by culturing the host cell for a sufficient period of time to allow expression of the antibody in the host cell, or more preferably secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods. The antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0286] Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation from one another. However, all antibodies encoded by the nucleic acids provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies.

[0287] Immunoconjugates

[0288] The present invention relates to immunoconjugates comprising any anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention conjugated to a payload. In some preferred embodiments, the immunoconjugate comprises one or more drugs (such as cytotoxic agents, small molecule compounds, immunostimulants, etc.) or markers as a payload.

[0289] Assay

[0290] The anti-FGFR2B antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. In one aspect, the antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blotting, etc. Binding to FGFR2B can be determined using methods known in the art, exemplary methods of which are disclosed herein.

[0291] The present invention also provides an assay for identifying anti-FGFR2B antibodies with biological activity. Biological activity can include, for example, binding to FGFR2B (e.g., in conjunction with human FGFR2B), improving FGFR2B-mediated signal transduction (e.g., improving NFkB-mediated transcription), enhancing T effector cell function (e.g., by improving effector T cell proliferation and / or improving cytokine production (e.g., interferon-gamma) by effector T cells), etc. Antibodies with such biological activity in vivo and / or in vitro are also provided.

[0292] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0293] Cells for use in any of the above in vitro assays include naturally expressing FGFR2B or engineered to express FGFR2B cell lines, such as tumor cell lines. Such cells also include cell lines that express FGFR2B and cell lines that are not normally expressing FGFR2B and are transfected with DNA encoding FGFR2B.

[0294] It will be appreciated that any of the above assays can be performed using an immunoconjugate or immunofusion of the invention in place of or in addition to an anti-FGFR2B antibody.

[0295] It will be appreciated that any of the above assays can be performed using a combination of an anti-FGFR2B antibody and an additional active agent.

[0296] Pharmaceutical composition

[0297] The pharmaceutical composition of the present invention may include the antibody of the present invention and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition of the present invention may be included in a drug box, and in other embodiments, the pharmaceutical composition of the present invention may be included in a kit, such as a diagnostic kit.

[0298] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0299] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use and application of excipients, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. The composition may also contain a small amount of wetting agent or emulsifier, or pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin, etc.

[0300] The present invention provides pharmaceutical compositions comprising one or more monoclonal antibodies, or antigen-binding fragments thereof, nucleic acids, vectors, host cells, or immunoconjugates or immunofusions that bind to FGFR2B. It should be understood that the anti-FGFR2B antibodies, or antigen-binding fragments thereof, nucleic acids, vectors, host cells, or immunoconjugates or fusions provided herein can be incorporated into pharmaceutical compositions with suitable pharmaceutical carriers, excipients, and other agents in the formulation for co-administration, thereby providing improved transfer, delivery, tolerance, and the like.

[0301] Pharmaceutical formulations comprising the anti-FGFR2B antibodies described herein can be prepared by mixing an anti-FGFR2B antibody or antigen-binding fragment thereof of the invention having the desired purity with one or more optional pharmaceutically acceptable excipients, preferably in the form of an aqueous solution or a lyophilized formulation. Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0302] The pharmaceutical compositions or formulations of the present invention may also include one or more other active ingredients that are required for treating a specific disease, preferably those having complementary activities that do not adversely affect each other. For example, it is desirable to further include other therapeutic agents. In some embodiments, the other therapeutic agents are chemotherapeutic agents, radiotherapeutic agents, cytokines, vaccines, other antibodies, immunomodulators, or other biomacromolecule drugs.

[0303] In some embodiments, the pharmaceutical composition of the present invention further comprises a composition of nucleic acids encoding anti-FGFR2B antibodies or antigen-binding fragments thereof.

[0304] Methods and uses

[0305] The present invention provides a method for preventing, diagnosing, or treating FGFR2B-related diseases or symptoms. The method comprises 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 or pharmaceutical composition comprising the same, or a nucleic acid, vector, or host cell described herein.

[0306] In one aspect, the present invention provides use of an anti-FGFR2B antibody or antigen-binding fragment thereof, or an immunoconjugate or immunofusion or pharmaceutical composition comprising the same, in the production or preparation of a medicament for preventing or treating a FGFR2B-related disease or symptom in a subject.

[0307] In one aspect, the anti-FGFR2B antibodies and antigen-binding fragments thereof provided herein and pharmaceutical compositions comprising the same can be used as therapeutic agents for preventing or treating FGFR2B-related diseases or symptoms in subjects. For FGFR2B-related diseases or symptoms in subjects identified by standard methods, the anti-FGFR2B antibodies and antigen-binding fragments thereof disclosed herein and pharmaceutical compositions or immunoconjugates or immunofusions comprising the same, or nucleic acids, vectors or host cells described herein can be administered.

[0308] 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 modality. In some embodiments, the therapeutic agent is, for example, a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, another antibody, an immunomodulator, or another biomacromolecule. In some embodiments, treatment modalities include surgery; radiation therapy, localized or focused irradiation, and the like.

[0309] The above-mentioned combination therapy includes combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, wherein the administration of the anti-FGFR2B antibody or antigen-binding fragment thereof of the present invention can occur before, simultaneously with and / or after the administration of the additional therapeutic agent and / or adjuvant and / or treatment method.

[0310] In some embodiments, the FGFR2B related diseases or symptoms described herein refer to diseases or symptoms associated with abnormal FGFR2B expression, activity and / or signal transduction in a subject, including but not limited to cancer, inflammation and autoimmune diseases. In some embodiments, in diseases or symptoms associated with FGFR2B, the nucleic acid (level or content) encoding FGFR2B increases, or FGFR2B expression increases, or FGFR2B protein levels or activity increases, or signal transduction mediated by FGFR2B is enhanced. In other embodiments, in diseases or symptoms associated with FGFR2B, the nucleic acid (level or content) encoding FGFR2B decreases, or FGFR2B expression decreases, or FGFR2B protein level activity decreases, or signal transduction mediated by FGFR2B decreases.

[0311] In some embodiments, treatment of the disease or condition would benefit from inhibition of FGFR2B at the nucleic acid or protein level, or from blocking the binding of FGFR2B to its ligand, or from inhibiting FGFR2B-mediated signaling.

[0312] In other embodiments, treatment of the disease or condition would benefit from increasing nucleic acid or protein levels of FGFR2B, or from enhancing FGFR2B-mediated signaling.

[0313] In some embodiments, the FGFR2B-related disease or condition is cancer. Specifically, cancer includes, but is not limited to, solid tumors, breast cancer, urothelial carcinoma, melanoma, renal 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, lymphoid leukemia, and sarcoma. Preferably, the antibody used to prevent, diagnose, or treat a cancer associated with FGFR2B is an FGFR2B agonist.

[0314] In some embodiments, FGFR2B related diseases or symptoms are inflammation and / or autoimmune diseases. In some embodiments, the inflammation and / or autoimmune diseases related to FGFR2B are selected from atopic dermatitis, rheumatoid arthritis, asthma (such as allergic asthma), COPD, autoimmune uveitis, multiple sclerosis, lupus (such as systemic lupus erythematosus), ulcerative colitis, scleroderma and graft-versus-host disease (GVHD). Preferably, the antibody for treating or preventing the inflammation and / or autoimmune diseases related to FGFR2B is a FGFR2B antagonist.

[0315] In some 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 certain embodiments, the subject receives or has received other treatments, e.g., chemotherapy and / or radiation therapy.

[0316] The antibodies or antigen-binding fragments of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, pharmaceutical combinations and kits comprising the invention) can be administered by any suitable means, including oral, parenteral, intrapulmonary and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration and pulse infusion.

[0317] The antibodies or antigen-binding fragments of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, pharmaceutical combinations and kits comprising the invention) will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include 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 drug delivery, the method of administration, the dosing schedule and other factors known to practitioners. Optionally, the antibody is formulated with one or more agents currently used to prevent or treat the disease. The effective amount of these other agents depends on the amount of antibody present in the formulation, the type of disease or treatment and the other factors discussed above.

[0318] For the prevention or treatment of disease, appropriate dosages of the antibodies or antigen-binding fragments of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, pharmaceutical combinations and kits comprising the invention) when used alone or in combination with one or more other additional therapeutic agents will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0319] In certain embodiments, any of the anti-FGFR2B antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of FGFR2B in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a lesion associated with a hyperproliferative or cancerous lesion.

[0320] In one embodiment, the present invention antibodies or their antigen-binding fragments can be used to diagnose diseases or symptoms associated with FGFR2B, such as cancer, for example, to evaluate (e.g., monitor) treatment or progression of a disease described herein in an individual, its diagnosis and / or staging. In certain embodiments, labeled anti-FGFR2B antibodies or their antigen-binding fragments are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected moieties, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions. In some embodiments, provided herein are kits for diagnosing diseases associated with FGFR2B, comprising antibodies or their antigen-binding fragments of the present invention.

[0321] In some embodiments provided herein, the sample is obtained before treatment with an anti-FGFR2B antibody or antigen-binding fragment thereof. In some embodiments, the sample is obtained before treatment with other therapies. In some embodiments, the sample is obtained during treatment with other therapies, or after treatment with other therapies.

[0322] The present invention includes any combination of the specific embodiments described herein. It should be understood that although specific content and examples are described to indicate preferred embodiments of the present invention, this is merely illustrative and for example, and the present invention also encompasses modifications to the preferred embodiments of the present invention that are obvious to those skilled in the art. For all purposes, all publications, patents, and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety. Example

[0323] Example 1: Generation of anti-FGFR2b monoclonal antibodies

[0324] Anti-FGFR2b monoclonal antibodies (mAbs) were generated by conventional hybridoma fusion technology. Monoclonal antibodies with FGFR2b binding specificity in enzyme-linked immunosorbent assay (ELISA) were selected for further characterization.

[0325] Construction of the expression vector pcDNA3.1-human FGFR2b

[0326] DNA encoding full-length human FGFR2b isoform (Uniprot accession number P21802-2) was inserted into pcDNA3.1(+) vector (Synbio Technologies) by seamless cloning and the construct was confirmed by DNA sequencing. Large-scale DNA was prepared for immunization using the Plasmid Maxiprep System from Qiagen.

[0327] Immunization, hybridoma fusion and cloning

[0328] Mice were immunized with 100 μg of the pcDNA3.1-human FGFR2b vector prepared above via intramuscular injection, and booster immunization was performed three times with 10 μg of recombinant human FGFR2b-extracellular domain (ECD) (SinoBiological, human FGFR2b-Fc, Cat: 16485-H02H) fused to its C-terminus with a human Fc moiety via intramuscular injection. Antibody immune responses were observed by FGFR2b-specific ELISA. After 10 days of serum screening, mice with the highest anti-FGFR2b antibody serum titers were booster immunized with 10 μg of FGFR2b-Fc via intravenous injection. Three days after booster immunization, spleen cells were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Supernatants from mouse hybridoma clones were used to screen for FGFR2b-specific antibodies, and hybridoma cell lines that produced FGFR2b-specific antibodies that only bound to FGFR2b and not to FGFR2c were selected. ELISA and flow cytometry assays showed that several antibodies, including 55D6, 38D4, 39C2, 35B11, 52E2, 61B7, and 30C7, showed strong affinity for human FGFR2-IIIb, but no detectable binding to human FGFR2c. No binding of these antibodies to human FGFR1, FGFR3c, FGFR3b, or FGFR4 was detected. Interestingly, all of these FGFR2b-specific antibodies blocked the interaction between FGFR7 and FGFR2b, as determined by ELISA.

[0329] Example 2: V gene cloning and production of chimeric antibodies

[0330] 1. Cloning and sequencing of hybridoma antibody V genes

[0331] Lead antibodies with the desired characteristics were selected for V gene cloning. Sequences of the mouse anti-human FGFR2b light and heavy chain variable regions were obtained by polymerase chain reaction (PCR) amplification 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 from positive hybridoma cells was isolated using the MiniBest Universal RNA Extraction Kit (TaKaRa), and cDNA was synthesized using the 1st Strand cDNA Synthesis Kit (TaKaRa) with Oligo(dT) primers. The variable regions of the mouse IgG gene were amplified by PCR using primers for different isotypes of the heavy chain variable region and kappa chain primers for the light chain variable region. The PCR products were subcloned into the TA cloning vector. For each variable gene construct, more than 10 single clones were used for DNA sequencing by Synbio Technologies (Suzhou, China). The amino acid sequences of Vh and Vk were derived from the DNA sequencing results.

[0332] 2. Construction of Chimeric Antibodies

[0333] Seven kinds of antibodies, 55D6, 38D4, 39C2, 35B11, 52E2, 61B7 and 30C7, were selected as lead antibodies to produce chimeric antibodies using human IgG1 as constant region, and their SEQ are listed in Table 4. After sequencing analysis and confirmation, the cDNA of heavy chain and light chain variable region was synthesized and fused with the constant region sequence chain sequence of human IgG1 and people kappa. In order to enhance the ADCC of antibody, the Fc domains of these chimeric monoclonal antibodies were engineered with L235V, F243L, R292P, Y300L and P396L (VLPYLL). In order to enhance the secretion of antibody, signal peptide sequences (MEFGLSWVFLVALFRGVQC and MDMRVPAQLLGLLLLWLRGARC) were added to the N-terminal of heavy chain and light chain respectively. The chimeric antibody gene obtained was cloned into an expression vector. Large-scale DNA was prepared by using the Plasmid Maxi-prep System from Qiagen.

[0334] 3. Expression and Purification of Chimeric Antibodies

[0335] The heavy and light chains were co-transfected using Invitrogen's ExpiFectamine TM CHO Reagent was performed according to the manufacturer's instructions. 5-6 × 10 6 cell / ml ExpiCHO-S cells were grown using ExpiFectamine TM CHO Reagent was used to transfect 5-6×10 cells in ExpiCHO Expression Medium with equal amounts of heavy chain vector and light chain vector DNA at a final concentration of 0.8 μg / ml. 6 cell / ml ExpiCHO-S cells. TM Dilute plasmid DNA or ExpiFectamine in culture medium TM CHO Reagent, then mix by vortexing and / or inversion. TM The CHO / plasmid DNA mixture was incubated at room temperature for 1-5 minutes and then slowly transferred to the shake flask containing cells. The transfected cells were incubated on a shaker (125 rpm) at 37°C and 5% CO2 in a humidified atmosphere. 18-22 hours after transfection, the ExpiCHO TM Feed, and obtain conditioned medium on the 10th day. The supernatant was centrifuged at 4000rpm for 20 minutes, and then filtered with a 0.22μm filter to remove cell debris. The filtered supernatant was loaded into a pre-equilibrated Protein-A affinity column. The Protein-A resin was washed with equilibrium buffer (PBS), and the antibody was then eluted using 25mM citrate (pH3.5). The purified antibody solution was adjusted to pH 6.0-7.0 using 1MTris base (pH 9.0). Endotoxin was controlled below 1EU / mg. Finally, the purified antibody was characterized by SDS-PAGE.

[0336] An anti-human FGFR2b specific antibody Bema_VLPYLL (VH and VL sequences are from patent WO2015 / 017600A1, FIVE PRIME THERAPEUTICS) was also expressed as a positive control, and its Fc segment was human IgG1_VLPYLL.

[0337] Table 4 Variable region sequences of murine antibodies or chimeric antibodies

[0338]

[0339]

[0340] Note: Kabat-defined CDR regions are bold and underlined.

[0341] Example 3: Epitope Characterization Analysis of Chimeric Anti-FGFR2b Monoclonal Antibodies

[0342] Epitope characterization of the chimeric monoclonal antibody and Bema_VLPYLL was performed using BLI. 100 nM human FGFR2b-biotin protein (Katcus, Cat: FGF-HM4ABB) was loaded onto the SA biosensor. After a wash step, the biosensor was immersed in a primary antibody solution and bound to the first antibody for 90 seconds, resulting in a clear binding signal. The biosensor was washed with KD buffer and then incubated with a second antibody; this signal indicates whether the two antibodies compete for the epitope.

[0343] According to epitope competition assay ( Figure 1 ), we found that 55D6, 38D4, 39C2, and Bema_VLPYLL recognized the same epitope, and any of these antibodies could 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 competed for 52E2 binding to FGFR2b, but also for Bema or other antibodies binding to this antigen. Two benchmark antibodies, 2-10 (Daiichi Sankyo) and GP369 (Aveo Thereapeutic), were also expressed and tested for their epitopes. Both 2-10 and GP369 competed for Bema_VLPYLL binding, but they did not compete for 52E2 binding to FGFR2b; the results are not shown here.

[0344] Example 4 ADCC activity of anti-FGFR2b chimeric monoclonal antibody

[0345] The ADCC reporter gene bioassay is a bioluminescent reporter gene assay used to quantify antibody bioactivity via FcγRIIIa-mediated pathway activation and was used to evaluate the ADCC activity of FGFR2b antibodies.

[0346] Jurkat-NFAT Luc-FcγRIIIa-V176 cells (Jurkat cells (Shanghai Institutes for Biological Sciences, Cat# SCSP-513) were transfected with PGL4.30-Luc / NFAT-RE / Hygro plasmid (Promega) and then selected with hygromycin. The cell line Jurkat-NFAT-Luc was stably expressed. The sequence of FcγRIIIA-V176 (SEQ ID NO:37) was constructed into the vector pVitro-neo (InvivoGen) to obtain the plasmid pVitro-neo-pcDNa3.1-FcγRIIIA-V176. The cell line obtained was transfected with the pVitro-neo-FcγriiA-V176 plasmid, and the stable expression cell line Jurkat-nFAT-Luc-FcγRIIIA-V176 was screened with the antibiotic G418. Jurkat cells (Shanghai Institutes for Biological Sciences, Cat# SCSP-513) were transfected with the PGL4.30-Luc / NFAT-RE / Hygro plasmid (Promega) and then screened with hygromycin. The cell line Jurkat-nfat-Luc was stably expressed. The sequence of FcγRIIIA-V176 (SEQ ID NO:37) was constructed into the vector pVitro-neo (InvivoGen) to obtain the plasmid pVitro-neo-pcDNa3.1-FcγRIIIA-V176. The resulting cell line was transfected with the pVitro-neo-FcγRIIA-V176 plasmid, and the stably expressing cell line (Jurkat-nFAT-Luc-FcγRIIIA-V176) was selected with the antibiotic G418 and used as effector cells. The 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.

[0347] Human FGFR2 gene-amplified cancer cell lines (KATO-III and KYSE-180) were used as target cells for the ADCC reporter gene bioassay. KATO-III is a gastric cancer cell line maintained in IMDM medium supplemented with 20% FBS and 1× penicillin-streptomycin; KYSE-180 is an esophageal squamous cell carcinoma cell line maintained in RPMI-1640 medium supplemented with 10% FBS and 1× penicillin-streptomycin.

[0348] Target cells were seeded at 20,000 per well in a 96-well white-bottom assay plate and then incubated with serially diluted antibodies. After incubation at 37°C for 30 minutes, 1.2 × 10 5Jurkat-NFAT Luc-FcγRIIIa-V176 reporter cells were seeded into the assay plate and incubated at 37°C for 5 hours. TM Luminescence was measured using Luciferase Assay Reagent (Promega, Cat. no. E6120) using a SpectraMax M5 microplate reader. Samples and controls were assayed in duplicate, and the average reporter gene signal of the sample solutions was plotted as relative luminescence units (RLU) versus antibody concentration. Dose-response curves were fitted using Prism Graphpad statistical software using a four-parameter model.

[0349] ADCC reporter gene bioassays were used to confirm the ADCC activity of chimeric monoclonal antibodies. Figure 2 As shown, 55D6, 38D4, 39C2, 35B11, 52E2, 61B7, and 30C7 exhibited potent ADCC activity against KATO-III cells (FGFR2b amplified and highly overexpressed).

[0350] Example 5: Humanization of mouse antibodies

[0351] 55D6, 38D4, 39C2, 35B11, and 52E2 were selected for humanization. The humanization process, which produces highly optimized monoclonal antibodies, involves five steps. The first step is to select an acceptor human framework (FR) suitable for its antigen-binding activity, immunogenicity, expression, stability, and pharmacokinetics. The selected germline sequences include IGHV1-46*01 (for the VH of 55D6, 39C2, and 38D4), IGHV1-69-2*02 (for the VH of 35B11), IGHV1-2*02 (for the VH of 52E2), IGKV3-11*01 (for the VL of 55D6), IGKV1-39*01 (for the VL of 39C2, 38D4, and 52E2), and IGKV2-30*02 (for the VL of 35B11). The second step is to transplant the CDR region of the mouse antibody onto the FR of the human antibody described in the first step to generate a CDR-transplanted monoclonal antibody, and then simulate the three-dimensional (3D) Fv structure model of the parent mouse antibody and the CDR-transplanted monoclonal antibody. Finally, based on the guidance of the structure, the amino acids located at the VH and VL interface and the framework region near the CDR region inside the structure are reversely mutated to the corresponding amino acids in the mouse antibody FR. The third step is to prepare expression vectors for multiple versions of humanized antibodies. The fourth step is to express and purify the humanized antibodies. The last step is to perform a multi-dimensional evaluation of the humanized antibodies.

[0352] Using this humanization procedure, we obtained humanized antibodies 55D6, 38D4, 39C2, 35B11, and 52E2, and named them Hu55D6, Hu38D4, Hu39C2, Hu35B11, and Hu52E2, respectively. The constant regions of the humanized monoclonal antibodies are human IgG1 and kappa. To enhance ADCC, these humanized antibodies were expressed in a low-fucose form by adding the fucose analog 2-deoxy-2-fluoro-L-fucose (Biosynth, W-203582) to the expression medium. To compare the biological activities of the five humanized monoclonal antibodies and the positive control, Bema (with human IgG1 and kappa constant regions) was also expressed in a low-fucose form. The following table lists the VH and VL sequences of the humanized monoclonal antibodies.

[0353] Table 5 Variable region sequences of humanized antibodies

[0354]

[0355]

[0356] Note: Kabat-defined CDR regions are bold and underlined.

[0357] Example 6: Kinetic Binding of Humanized Monoclonal Antibodies to Human FGFR2b

[0358] The kinetic binding of humanized monoclonal antibodies to human FGFR2b (SinoBiological, Cat: 16485-H08H) was determined by using biolayer interferometry (BLI). 100 nM humanized monoclonal antibodies in 1× Kinetics buffer (1× PBS, pH 7.4, 0.02% Tween 20, 0.1% BSA) were loaded onto four pre-wetted Protein A biosensors and incubated with solutions of human FGFR2b at varying concentrations. All binding data were collected at 30°C. The experiment consisted of five steps: 1. Baseline acquisition (60 seconds); 2. Antibody loading onto Protein A biosensor (60 seconds); 3. Second baseline acquisition (60 seconds); 4. Antigen association to measure binding k on ((120 seconds); 5. Antigen dissociation to measure k off ((180 s). Four different concentrations of antigen diluted in 1× Kinetics buffer were used, including 100 nM, 33.3 nM, 11.1 nM, and 0 nM. Baseline and dissociation steps were performed in 1× Kinetics buffer. off With k on The ratio of the two determines the equilibrium dissociation constant K DThe biosensor was regenerated in regeneration buffer (10 mM glycine-HCl, pH 1.7) for 5 seconds and then neutralized in neutralization buffer (1×PBS, pH 7.4, 0.02% Tween 20, 0.1% BSA) for 5 seconds. This process was repeated three times.

[0359] As shown in Table 6 below, Hu55D6, Hu38D4, Hu39C2, Hu35B11, and Hu52E2 bind to human FGFR2b with high affinity, and the K values ​​of these humanized monoclonal antibodies are D The values ​​are better than Bema's K D value.

[0360] Table 6 Summary of kinetic binding of humanized monoclonal antibodies to human FGFR2b

[0361] Sample ID Sample ID <![CDATA[K D (M)]]> <![CDATA[k on (1 / Ms)]]> <![CDATA[k dis (1 / s)]]> human FGFR2b Bema 7.90E-09 2.91E+05 2.30E-03 human FGFR2b Hu38D4 2.23E-09 2.10E+05 4.69E-04 human FGFR2b Hu52E2 1.80E-09 1.63E+05 2.92E-04 human FGFR2b Hu55D6 1.48E-09 1.83E+05 2.71E-04 human FGFR2b Hu39C2 2.05E-09 1.87E+05 3.85E-04 human FGFR2b Hu35B11 3.71E-09 2.44E+05 9.07E-04

[0362] Example 7: Binding specificity of humanized monoclonal antibodies to human FGFR2b

[0363] The ELISA method was used to determine the binding specificity of humanized antibodies to FGFR family members to avoid side effects caused by off-target binding. Briefly, human FGFR2b (Sino Biological, Cat: 16485-H08H), FGFR2c (Sino Biological, Cat: 10824-H08H), FGFR1 (Sino Biological, Cat: 10616-H08H), FGFR3b (Sino Biological, Cat: 10648-H08H), FGFR3c (Sino Biological, Cat: 10644-H08H), or FGFR4 (Sino Biological, Cat: 10538-H08H) were immobilized on a plate. Humanized monoclonal antibodies were serially diluted in PBS and added for incubation for 1 hour. HRP-conjugated goat anti-human IgG polyclonal antibody and TMB were then added, and binding was detected at an OD of 450 nm. Data were analyzed using GraphPad Prism.

[0364] According to the results of ELISA analysis, Hu55D6, Hu38D4, Hu39C2, Hu35B11 and Hu52E2 specifically bind to FGFR2b but not to other FGFR family members (see Figure 3 ).

[0365] Example 8: Species Cross-Reactivity of Humanized Monoclonal Antibodies

[0366] ELISA was used to test whether our candidate antibodies could bind to mouse, rat, or monkey FGFR2b, providing a basis for the subsequent selection of animal models. Briefly, human FGFR2b-His (Sino Biological, Cat: 16485-H08H), mouse FGFR2b-His ((Sino Biological, Cat: 51128-M08H), cynomolgus monkey FGFR2b-His ((Sino Biological, Cat: FGF-CM1BB)), or rat FGFR2b-mFc (self-expressed, sequence from Uniprot, F1LSG7) were immobilized on a plate, and humanized monoclonal antibodies were added after serial dilution in PBS and incubated for 1 hour. Then, HRP-labeled goat anti-human IgG polyclonal antibody and TMB were added, and binding was detected at an OD of 450 nm. Finally, data were analyzed using GraphPad Prism.

[0367] According to ELISA analysis, Hu55D6, Hu38D4, Hu39C2, Hu35B11 and Hu52E2 can not only bind to human FGFR2b, but also cross-bind to FGFR2b of mouse, rat and cynomolgus monkey. These results support that mouse models can be used to evaluate the efficacy of these antibodies, and rats and monkeys can be used to evaluate the preclinical toxicology of these humanized antibodies (see Figure 4 ).

[0368] Example 9: Blocking ELISA Assay for Binding of FGFR2b to FGF7

[0369] 0.5 μg / ml hFGFR2b-Fc (Sino Biological, cat#16485-H02H, lot#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. After washing, 50 μl of diluted humanized monoclonal antibody and 50 μl of biotinylated FGF7-Fc (prepared in-house, FGF7-Fc was expressed and purified by EZ-Link) were added. TMBiotin-labeled using the Sulfo-NHS-LC-Biotin Kit (ThermoFisher, Catalog number: A39257) was added to each well and incubated at 37°C for 1 hour. The plate was washed 6 times, and then 100 μl / well of 1:5000 HRP-conjugated streptavidin (Abcam, cat#ab7403, lot#GR3259274-11) was added. After incubation at room temperature for 1 hour, the mixed TMB substrate reagent was added and incubated at room temperature for 5 minutes, then terminated by adding 0.1 M H2SO4. The OD450 nm was recorded using a microplate reader, and the IC value for blocking the binding of the humanized anti-FGFR2b monoclonal antibody to the ligand FGF7 was calculated. 50 value.

[0370] The results of the blocking ELISA test showed that all five humanized monoclonal antibodies could effectively block the interaction between FGFR2b and FGF7, and the IC 50 Equivalent to Bema (see Figure 5 ).

[0371] The blocking effect of all five humanized monoclonal antibodies on the interaction between FGFR2b and FGF10 was determined using a BLI assay. Briefly, FGFR2b-biotin was loaded onto SA biosensors, followed by incubation with the test antibody or isotype control, and then the sensors were immersed in an FGF10 solution. All five humanized monoclonal antibodies also blocked the interaction between FGFR2b and FGF10; data not shown here.

[0372] Example 10: Flow cytometric binding analysis of FGFR2b antibodies on HEK293T cells stably expressing human FGFR2b and FGFR2c

[0373] HEK293T cells (Cell Bank, Chinese Academy of Sciences, Cat: 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. After washing, the cells were plated at 5×10 4% 4% 4% 5% 2% 1% 1% 2 ... 4Cells / well were seeded in a 96-well plate and then incubated with several antibody solutions at 4°C for 60 minutes. After washing twice with cold wash buffer, 0.5 μg / mL FITC-conjugated goat anti-human IgG1 polyclonal antibody (Abcam, cat#ab98623, lot#GR3319406) was immediately added to the cells and incubated at 4°C for 30 minutes. After washing twice with cold wash buffer, the cells were resuspended in 120 μL of cold PBS and analyzed by flow cytometry. The median fluorescence intensity (MFI) value of FITC was fitted with the antibody concentration, and the binding EC values ​​of several antibodies were calculated using Prism6.02 statistical software. 50 The results are as follows Figure 6 As shown, it was demonstrated that all five humanized FGFR2b antibodies could bind to 293T-hFGFR2b cells but not to 293T-hFGFR2c cells.

[0374] ·

[0375] Example 11: ADCC reporter assay of humanized monoclonal antibodies

[0376] The ADCC reporter gene bioassay described in Example 3 was also used to determine the ADCC activity of humanized FGFR2b antibodies. All humanized FGFR2b antibodies showed strong ADCC activity against KATO-III cells (ATCC, Cat#HTB-103) and KYSE-180 cells (FGFR2b low expression) (Cobioer, Cat: CBP60456), and the maximum RLU value of Hu52E2 was higher than that of Bema and other FGFR2b antibodies (see Figure 7 ).

[0377] Example 12: FGFR2b antibody inhibits FGF7-induced MCF7 cell proliferation

[0378] MCF7 cells (Cobioer, Cat: CBP60380) from patients with triple-negative breast cancer (TNBC) were cultured in EMEM medium containing 10% FBS and 1× penicillin-streptomycin. 10,000 cells were seeded per well in a 96-well plate in complete growth medium and cultured overnight to allow attachment. The cells were then cultured in serum-free medium for 24 hours and then treated with 30 μg / mL human IgG isotype or several FGFR2b antibodies for 72 hours in the presence or absence of FGF7 (25 ng / mL). The cells were cloned and expressed in the presence of a 5-well plate (Cat. no. G7571). Luminescent cell viability assay was used to assess cell proliferation. Samples and controls were tested in duplicate, and Prism 6.02 statistical software was used to plot the average reporter gene signal in the sample solution in the form of relative luminescence units versus antibody concentration. Figure 8 As shown, MCF7 cells were stimulated by FGF7 protein, and Bema and several FGFR2b antibodies effectively blocked FGF7-induced MCF7 cell proliferation.

[0379] Example 13: HTRF assay to detect FGFR2 and ERK1 / 2 protein phosphorylation induced by FGF7 or FGF10 in SNU-16 cells

[0380] FGFR2b is a receptor tyrosine kinase that is primarily involved in cell survival, proliferation, migration, and angiogenesis via the RAS-MAPK signaling pathway. To determine whether FGFR2b antibodies could block downstream signals induced by FGF7 and FGF10, we used a cell-based fluorescence resonance energy transfer (HTRF) assay to detect the endogenous levels of phosphorylated ERK1 / 2 (Thr202 / Tyr204) and phosphorylated FGFR2 (Tyr653 / 654) induced by ligands FGF7 and FGF10 in SNU-16 cells (Cobioer, Cat: CBP60502) (gastric cancer cells with FGFR2b amplification). Briefly, in the presence of 10% SNU-16 cells were cultured in RPMI-1640 medium with FBS and 1× penicillin-streptomycin until the confluence reached 90%. The cells were plated into 96-well plates with RPMI-1640 medium without FBS and incubated overnight at 37°C, and then treated with 15μg / mL or 0.15μg / mL human IgG isotype or several FGFR2b antibodies for 1.5 hours. The cells were then treated with ligand-induced complexes containing 30ng / ml FGF7 / 10 and 20ug / ml heparin for 5 minutes at 37°C. After activation, the cells were immediately incubated with lysis buffer at room temperature for at least 30 minutes with shaking. After lysis was complete, 16μL of cell lysate was transferred to a 96 half-well white plate and 4μL of premixed antibody solution prepared in detection buffer was added. The incubation complex system was incubated at room temperature for 4 hours or at 4°C overnight in a compatible Phospho-ERK1 / 2 (Thr202 / Tyr204) and phosphorylated-FGFR2 (Tyr653 / 654) were read on a reader via fluorescence emission at two different wavelengths (665 nm and 620 nm). The fluorescence intensity was calculated by ((665 nm signal / 620 nm signal)*10 4 The HTRF ratio was calculated, which reflects the cellular phosphorylation level induced by the corresponding ligand.

[0381] like Figure 9As shown, all of the several FGFR2b antibodies and Bema were able to significantly inhibit the phosphorylation of FGFR2 and ERK1 / 2 induced by FGF7 or FGF10.

[0382] Example 14: ADCC activity of FGFR2b antibody targeting KATO-III cells mediated by PBMCs

[0383] Use flow cytometry analysis to carry out the in vitro test of the ADCC activity of determining FGFR2b antibody. Briefly, KATO-III cells (ATCC, Cat#HTB-103) are maintained in IMDM culture medium containing 20% ​​FBS and 1× penicillin-streptomycin, and freshly separated peripheral blood mononuclear cells (PBMCs) from healthy donors are obtained from SailyBio.lnc. (Shanghai, China). The main ADCC determination is carried out on two different days using effector cells from two independent donors. The ADCC determination test uses freshly separated human PBMC as effector cells, with a ratio of effector cells to target cells (E / T) of 40:1. First, use CellTrace TM Target cells were labeled with Far Red staining solution and then incubated for 16 hours in the presence of effector cells and increasing concentrations of several antibodies. Target cell lysis was analyzed by flow cytometry as a double-positive stain (propidium iodide and Far Red). Maximum lysis was determined in the presence of 5% Triton X-100, and spontaneous release was determined in the absence of antibody. Specific lysis was calculated as the percentage of maximum lysis minus spontaneous release:

[0384] (Specific cell lysis rate) = (experimental - spontaneous release) / (maximum - spontaneous release) × 100

[0385] The results are as follows Figure 10 As shown, several humanized antibodies against FGFR2b induced similar specific KATO-III cell lysis mediated by PBMCs from two donors with the 158V / V and 158V / F genotypes of FcγRIIIA.

[0386] Example 15: Pharmacokinetic evaluation of humanized monoclonal antibodies

[0387] The PK curves of the anti-FGFR2b lead molecule and Bema as a benchmark were characterized and compared head to head after a single intravenous injection at a dose of 30 mg / kg in Sprague Dawley (SD) rats. 18 female rats were randomly divided into 6 groups (3 animals per group) and administered once with 30 mg / kg Bema or Hu39C2, Hu55D6, Hu52E2, Hu35B11 and Hu38D4 via slow bolus injection (bolus injection) with a dose volume of 10 mL / kg. Plasma was harvested from each group before administration (0 minutes), 30 minutes, 2 hours, 8 hours, 24 hours, 48 ​​hours, D4, D7, D10, D14, D21, and D28, and PK analysis was performed using a partially validated ELISA method with a detection range of 0.156 to 20 ng / mL. The microplate was pre-coated with human FGFR2b protein with a His tag (Acrobiosystems, FGB-H5223). After blocking, the standard (STD), quality control sample (QC), matrix blank sample and test sample were added to the wells. After washing, biotinylated mouse anti-human IgG4 (BD Pharmingen) was added to the wells of the microplate. TM , 555879), then add streptavidin labeled with horseradish peroxidase (HRP). Tetramethylbenzidine (TMB) is added to the microwells and color (blue) is developed in the presence of HRP. After color development, stop solution is added to each well to stop the reaction. Optical density (OD) is measured using a microplate reader set to 450nm and 620nm. The optical density (OD) values ​​of quality control (QC) samples and test samples are converted to concentrations by comparison with the standard curve of the 4-parameter logistic model regression analyzed simultaneously. Figure 11 The mean values ​​and mean plasma concentration-time curves are described in

[14] . Non-compartmental analysis (NCA) was performed using Phoenix software to calculate and estimate relevant PK parameters (Table 7).

[0388] Table 7 PK parameters after single injection in SD rats

[0389]

[0390]

[0391] Example 16 Anti-tumor effect in nude mouse SNU16 xenograft model

[0392] In vitro studies have shown that humanized FGFR2b monoclonal antibody can induce ADCC effect on SNU16 (Cobioer, Cat: CBP60502). Therefore, an in vivo model was established and used to evaluate antitumor activity. Briefly, SNU16 tumor pieces were cut into 3-5 mm3 Each female Balb / c nude mouse was subcutaneously inoculated with SNU16 tumor tissue fragments immersed in matri-gel (Nova) using a trocar in the right flank. 16 days after inoculation, the tumor size was about 80 mm. 3 Thirty-five mice were randomly divided into seven groups (n=5). Mice were then treated with either an isotype control or a humanized FGFR2b antibody at a dose of 10 mg / kg via intraperitoneal injection twice weekly for four weeks. At the end of the study, mice were sacrificed by carbon dioxide inhalation. Tumor size and volume were measured twice weekly. Results were analyzed using Prism GraphPad and expressed as mean ± SEM.

[0393] like Figure 12 As shown in Table 8, Hu55D6, Hu38D4, Hu39C2, Hu35B11, and Hu52E2 showed significant inhibition of tumor growth. Table 8 summarizes the tumor size and TGI of the treatment groups.

[0394] Table 8 Tumor growth inhibition (TGI) of humanized FGFR2b antibodies in the SNU16 xenograft model (mean ± SEM, n = 5)

[0395]

[0396] Sequence Listing

[0397]

[0398]

[0399]

[0400]

[0401]

Claims

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

56.

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

42.

3. 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, wherein: (1) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24; (2) the HCDR1 comprises the amino acid sequence of SEQ ID NO:4, the HCDR2 comprises the amino acid sequence of SEQ ID NO:5, the HCDR3 comprises the amino acid sequence of SEQ ID NO:6, and the LCDR1 comprises the amino acid sequence of SEQ ID NO:25, the LCDR2 comprises the amino acid sequence of SEQ ID NO:26, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:27; (3) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 30; (4) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 10, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 31, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 32, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 33; (5) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 15 or 13, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 14, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 36; (6) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 18, and the LCDR1 comprises the amino acid sequence of SEQ ID NO: 37, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 38, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 39; or (7) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 40, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 41, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

42.

4. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in any combination listed in Table 1 below:

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

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

56.

7. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL as shown in any combination listed in the following table:

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

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

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

70.

12. An isolated anti-FGFR2B antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) and a light chain (LC) as shown in any combination listed in the following table:

13. The isolated antibody or antigen-binding fragment of any one of claims 1-12, wherein the antibody comprises an Fc region that is afucosylated or has reduced fucosylation.

14. The isolated antibody or antigen-binding fragment of any one of claims 1-13, 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 1-14.

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

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. 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 sufficient to produce the antibody, antigen-binding fragment, or bispecific binding protein; and The antibody or antigen-binding fragment is recovered from the culture.

21. A pharmaceutical combination 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, the immunoconjugate of claim 18, or the pharmaceutical composition of claim 19.

22. A method for treating a disease associated with FGFR2b, comprising administering to a subject in need thereof an effective amount of 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, the immunoconjugate of claim 18, the pharmaceutical composition of claim 19, or the pharmaceutical combination of claim 21.

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

24. The method of claim 23, wherein the disease is cancer, preferably selected from breast cancer (e.g., triple-negative breast cancer), gastric cancer, gastroesophageal junction cancer, esophageal cancer, lung cancer (e.g., squamous 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