Anti-FGFR2b antibodies and uses thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-FGFR2b antibody or an antigen binding fragment thereof and application of the antibody. In particular, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to FGFR2b without binding to FGFR2c; the antibody provided by the invention can block the binding between the FGFR2b protein and a receptor FGF7 and the binding between the FGFR2b protein and FGF10; in addition, the ADCC activity is relatively high, and tumor growth can be remarkably inhibited. The invention also relates to an application of the antibody in preparing a medicine for treating cancers, and the antibody plays an important role in treating human tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an antibody and its fragment that specifically binds to FGFR2b, and uses of such antibodies. Background Art

[0002] Members of the fibroblast growth factor (FGF) family bind to four known tyrosine kinase receptors, namely fibroblast growth factor receptors 1-4 (FGFR1-4) and their isoforms, where various FGFs bind to different FGFRs to varying degrees (Zhang et al., J. Biol. Chem. 281: 15694, 2006). The protein sequence of human FGFR2 is provided, for example, in GenBank locus AF487553. Each FGFR consists of an extracellular domain (ECD) including three immunoglobulin (Ig)-like domains (D1, D2, and D3), a single transmembrane helix, and an intracellular catalytic kinase domain (Mohammadi et al., Cytokine Growth Factor Revs, 16: 107, 2005). FGF binds to the receptor mainly through regions in D2 and D3 of the receptor. There is a stretch of consecutive acidic amino acids called the "acid box" (AB) in the linker between D1 and D2. The region containing D1 and AB is believed to be involved in auto-inhibition of the receptor, which is relieved upon ligand binding.

[0003] FGFR is characterized by multiple alternative splicing of its mRNA, resulting in multiple isoforms (Ornitz et al., J. Biol. Chem. 271: 15292, 1996); for sequences of FGFR2 and its isoforms, also see Swiss-Prot P21802 and isoforms P21802-1 to P21802-20. Notably, there are forms containing all three Ig domains (α isoform) or only two Ig domains, the D2 and D3 domains without D1 (β isoform). In FGFR1, FGFR2, and FGFR3, all forms contain the first half of D3 (designated IIIa), but two alternative exons are available for the second half of D3, resulting in IIIb and IIIc forms. For FGFR2, these are designated FGFR2-IIIb and FGFR2-IIIc (or simply FGFR2b and FGFR2c), respectively; the corresponding β forms are designated FGFR2(β)IIIb and FGFR2(β)IIIc. The FGFR2-IIIb form of FGFR2 (also known as K-sam-II) is a high-affinity receptor for members of the FGF1 and KGF families (FGF7, FGF10, and FGF22), while FGFR2-IIIc (also known as K-sam-I) binds both FGF1 and FGF2 well but does not bind members of the KGF family (Miki et al., Proc. Natl. Acad. Sci. USA 89: 246, 1992). Indeed, FGFR2-IIIb is the only receptor for members of the KGF family (Ornitz et al., 1996, supra) and is thus also designated KGFR.

[0004] FGFR and its isoforms are differentially expressed in various tissues. FGFR2-IIIb (and the IIIb forms of FGFR1 and FGFR3) are expressed in epithelial tissues, while FGFR2-IIIc is expressed in mesenchymal tissues (Duan et al., J. Biol. Chem. 267: 16076, 1992; Ornitz et al., 1996, supra). Certain FGF ligands for these receptors have opposite expression patterns. Thus, members of the KGF subfamily, including FGF7 (KGF), FGF10, and FGF22, bind only to FGFR2-IIIb (Zhang et al., supra) and are expressed in mesenchymal tissues, and perhaps also as paracrine effectors of epithelial cells (Ornitz et al., 1996, supra). In contrast, members of the FGF4 subfamily, FGF4-6, bind to FGFR2-IIIc and are expressed in both epithelial and mesenchymal lineages, and thus may have autocrine or paracrine functions. Because of the expression patterns of the isoforms of FGFR2 and its ligands, FGFR2 plays a role in epithelial-mesenchymal interactions (Finch et al., Dev. Dyn. 203: 223, 1995), and it is not surprising that knockout of FGFR2-IIIb in mice results in severe embryonic defects and lethality (De Moerlooze et al., Development 127: 483, 2000).

[0005] KGF (FGF7) and KGFR (FGFR2-IIIb) are overexpressed in many pancreatic cancers (Ishiwata et al., Am. J. Pathol. 153: 213, 1998), and their co-expression is associated with poor prognosis (Cho et al., Am. J. Pathol. 170: 1964, 2007). Somatic mutations of the FGFR2 gene are found in 12% of a large group of endometrial (uterine) cancers, and are required for tumor cell survival in several test cases (Dutt et al., Proc. Natl. Acad. Sci. USA 105: 8713, 2008). In both tumors, the FGFR2 mutations were the same S252W substitution associated with Apert syndrome. Amplification and overexpression of FGFR2 are associated with undifferentiated, diffuse gastric cancers with particularly poor prognosis, and inhibition of FGFR2 activity by small molecule compounds effectively inhibits the proliferation of such cancer cells (Kunii et al., Cancer Res. 68: 2340, 2008; Nakamura et al., Gastroenterol. 131: 1530, 2006). In gastric cancer, FGFR2 amplification results in high-level expression of the FGR2b receptor on the cell surface.

[0006] Gastric cancer is the fifth most common cancer worldwide and the third leading cause of cancer death, with a five-year survival rate for metastatic disease of 5%. In China, gastric cancer is the second most common cancer after lung cancer, with the number of new cases and deaths accounting for 44% and 50% of the global total, respectively. 80% of Chinese gastric cancer patients are diagnosed at an advanced stage, with limited treatment options. Approximately 80% of gastric cancers are HER2-negative, and FGFR2b is overexpressed in approximately 30% of HER2-negative gastric cancers globally. For patients with HER2-negative gastric cancer, the current first-line therapy remains the same systemic chemotherapy as in the 1990s. Researchers have also found that FGFR2b is overexpressed in many other cancers, including squamous non-small cell lung cancer, triple-negative breast cancer, ovarian cancer, pancreatic cancer, and intrahepatic cholangiocarcinoma. The potential of anti-FGFR2b antibodies as "broad-spectrum" anti-cancer drugs.

[0007] In addition, studies have found that patients with FGFR2b positivity tend to have higher PD-L1 expression, indicating that anti-FGFR2b antibodies may also be combined with immunotherapy in the future to exert greater efficacy.

[0008] It has been reported that inhibiting FGFR signaling can enhance anti-tumor immunity and impair metastasis in breast cancer. (See, for example, T. Ye et al., Breast Cancer Res. Treat. 143: 435-446 (2014).) For example, anti-FGFR2 antibodies have also been tested in gastric cancer models. Specific anti-FGFR2 antibodies are described, for example, in U.S. Patent No. 8,101,723 B2, including monoclonal antibodies that bind to human FGFR2-IIIb but poorly or not at all to FGFR2-IIIc, and vice versa.

[0009] In FGFR2b-overexpressing gastric and breast cancer cell lines, anti-FGFR2-IIIb inhibits FGF ligand-stimulated FGFR2b phosphorylation and cell proliferation. In FGFR2b-overexpressing gastric and breast xenografts, anti-FGFR2-IIIb also inhibits tumor growth. Therefore, three potential mechanisms of action of anti-FGFR2-IIIb include blocking ligand binding and downstream signaling, reducing the expression of FGFR2b-driven proteins, and enhancing ADCC.

[0010] Anti-FGFR2-IIIb produces complete and durable tumor growth inhibition in gastric cancer xenografts overexpressing FGFR2b and with FGFR2 gene amplification, where FGFR2b is regarded as the driving force of tumor growth (Gemo 2014). In addition, anti-FGFR2-IIIb confirmed the recruitment of NK cells and concomitant tumor growth inhibition in the 4T1 syngeneic tumor model with moderately expressed FGFR2b. These data suggest that ADCC may be effective in patients without FGFR2 gene amplification and with moderate FGFR2b overexpression, and ADCC activity may be a major contributor to the mechanism of action in these patients. SUMMARY OF THE INVENTION

[0011] In view of the above problems and / or other problems of the related art, an object of the present invention is to provide an antibody targeting FGFR2b with good binding specificity, high affinity and stability. The anti-FGFR2b antibody is an innovative antibody targeting FGFR2b of the FGF family, which can block the ligand signal binding of FGF and activate FGFR2b, without binding to FGFR2C, inhibit multiple downstream pathways, thereby inhibiting cancer growth. Moreover, it can also kill cancer cells through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0012] In a first aspect of the present invention, there is provided an anti-FGFR2b antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and / or a light chain variable region, and the heavy chain variable region comprises a heavy chain CDR selected from the amino acid sequences SEQ ID NO: 1-9, 32, 33 or variants thereof; the light chain variable region comprises a light chain CDR selected from the amino acid sequences SEQ ID NO: 10-17 or variants thereof.

[0013] In some embodiments, the present invention provides an anti-FGFR2b antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and / or a light chain variable region, and the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein:

[0014] (1) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; or

[0015] (2) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; or

[0016] (3) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or

[0017] (4) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:1, SEQ ID NO:32, and SEQ ID NO:3, respectively; or

[0018] (5) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:33, and SEQ ID NO:9, respectively;

[0019] And, the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0020] (6) The amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively: or

[0021] (7) The amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively: or

[0022] (8) The amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:17, respectively.

[0023] In some embodiments, for the antibody or its antigen-binding fragment, the amino acid sequences of the HCDR1, HCDR2, HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are selected from the following groups:

[0024] (1) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; or

[0025] (2) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 respectively; or

[0026] (3) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:17 respectively; or

[0027] (4) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:1, SEQ ID NO:32, and SEQ ID NO:3 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively; or

[0028] (5) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:33, and SEQ ID NO:9 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:17 respectively.

[0029] In some preferred embodiments, for the antibody or its antigen-binding fragment, the amino acid sequences of the HCDR1, HCDR2, HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are selected from the following groups:

[0030] (1) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively; or

[0031] (2) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:17 respectively; or

[0032] (3) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:1, SEQ ID NO:32, and SEQ ID NO:3 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively; or

[0033] (4) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:33, and SEQ ID NO:9 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:17 respectively.

[0034] In a second aspect of the present invention, there is provided an anti-FGFR2b antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL): the amino acid sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:18-20, SEQ ID NO:34-41, and the amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:21-23, SEQ ID NO:42-45; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO:18-20, SEQ ID NO:34-41, SEQ ID NO:21-23, SEQ ID NO:42-45.

[0035] In some embodiments, the antibody of the present invention includes an antibody that comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities:

[0036] (1) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 21; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0037] (2) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 22; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0038] (3) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 23; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0039] (4) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0040] (5) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0041] (6) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 43; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0042] (7) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 43; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0043] (8) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 38, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 44; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0044] (9) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 44; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0045] (10) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 40, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 45; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;

[0046] (11) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 45; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence.

[0047] In some preferred embodiments, the antibodies of the present invention are selected from the following groups:

[0048] (1) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 21; or

[0049] (2) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 23; or

[0050] (3) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; or

[0051] (4) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 45.

[0052] In the third aspect of the present invention, there is provided an anti-FGFR2b antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is murine, chimeric, or humanized; wherein the FR region sequences on the variable regions of the light chain and heavy chain of the humanized antibody are derived from the germline light chain and heavy chain of the human race or their mutant sequences; the antibody of the present invention further includes a heavy chain constant region and a light chain constant region.

[0053] Preferably, the antibody comprises a heavy chain constant domain of human IgG1, IgG2, IgG3, IgG4 and a light chain constant domain of human κ or λ type or a variant thereof, wherein the variant includes substitution of one or several amino acids.

[0054] Preferably, the antibody comprises a human IgG1 constant domain or a variant thereof, and a κ constant domain or a variant thereof, wherein the variant includes substitution of one or several amino acids.

[0055] More preferably, the antibody comprises a heavy chain constant region as shown in SEQ ID NO:30 and a light chain constant region as shown in SEQ ID NO:31.

[0056] More preferably, the antibody of the present invention comprises a complete structure having two light chains and two heavy chains, and the antibody is selected from the following groups:

[0057] (1) a heavy chain amino acid sequence as shown in SEQ ID NO:46 and a light chain amino acid sequence as shown in SEQ ID NO:47; or

[0058] (2) a heavy chain amino acid sequence as shown in SEQ ID NO:48 and a light chain amino acid sequence as shown in SEQ ID NO:49.

[0059] In the fourth aspect of the present invention, there is provided an isolated nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof of the present invention.

[0060] In some embodiments, the nucleic acid molecule has a heavy chain nucleotide sequence as shown in SEQ ID NO:24-26, SEQ ID NO:50, SEQ ID NO:52, and a light chain nucleotide sequence as shown in SEQ ID NO:27-29, SEQ ID NO:51, SEQ ID NO:53.

[0061] In some preferred embodiments, the nucleotide molecule encoding the antibody or an antigen-binding fragment thereof of the present invention is selected from the following groups:

[0062] (1) the nucleic acid molecule has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO:24 and a light chain variable region nucleotide sequence as shown in SEQ ID NO:27; or

[0063] (2) The nucleic acid molecule has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 25, and a light chain variable region nucleotide sequence as shown in SEQ ID NO: 28; or

[0064] (3) The nucleic acid molecule has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 26, and a light chain variable region nucleotide sequence as shown in SEQ ID NO: 29; or

[0065] (4) The nucleic acid molecule has a heavy chain nucleotide sequence as shown in SEQ ID NO: 50, and a light chain nucleotide sequence as shown in SEQ ID NO: 51; or

[0066] (5) The nucleic acid molecule has a heavy chain nucleotide sequence as shown in SEQ ID NO: 52, and a light chain nucleotide sequence as shown in SEQ ID NO: 53.

[0067] In the fifth aspect of the present invention, there is provided an expression vector, which comprises an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0068] In the sixth aspect of the present invention, there is provided a host cell, which comprises the above-mentioned expression vector. The host cell can be a prokaryotic cell or a eukaryotic cell. In a preferred example, the host cell is a HEK293 cell.

[0069] In the seventh aspect of the present invention, there is provided an antigen.

[0070] In the eighth aspect of the present invention, there is provided a method for preparing the isolated monoclonal antibody, the antibody specifically binding to FGFR2b or the antigen-binding fragment described in the first aspect. The antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). The somatic cell hybridization method is preferred. In principle, other techniques for producing monoclonal antibodies, such as virus or oncogene transformation of B lymphocytes or phage display techniques using antibody gene libraries, can be used to prepare the antibodies of the present invention. Chimeric or humanized antibodies are also well known in the art, such as U.S. Patent Nos. 4,816,567, 5,225,539, 5,530,101, 5,585,089, 5,693,762 or 6,180,370, etc.

[0071] The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the prior art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0072] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polet et al, Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0073] Another strategy for generating monoclonal antibodies is to directly isolate the genes encoding the antibodies from antibody-producing lymphocytes of a defined strategy, see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

[0074] In a ninth aspect of the present invention, there is provided the use of the antibody in the manufacture of a medicament for the treatment of cancer. The cancer includes but is not limited to hepatocellular carcinoma, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, squamous non-small cell lung cancer, colon cancer, prostate cancer, colorectal cancer, glioma, head and neck cancer, melanoma, gastric cancer, esophageal cancer, triple-negative breast cancer, intrahepatic cholangiocarcinoma, and renal cell carcinoma.

[0075] The technical solution of the present invention has achieved beneficial technical effects:

[0076] The present invention provides an antibody or antigen-binding fragment that specifically binds to FGFR2b and does not bind to FGFR2c; the antibody of the present invention can block the binding between the FGFR2b protein and the receptor FGF7 and between the FGFR2b protein and FGF10; and has strong ADCC activity and can significantly inhibit tumor growth. Brief Description of the Drawings

[0077] Figure 1 : FACS detection results of mouse hybridoma antibody and HEK293-huFGFR2b cells

[0078] Figure 2 : FACS detection results of chimeric antibody and HEK293-huFGFR2b cells

[0079] Figure 3 : FACS detection results of humanized antibody KA-1947 and SNU16 tumor cells

[0080] Figure 4 : FACS detection results of humanized antibody KA-1953 and SNU16 tumor cells

[0081] Figure 5 : Activity detection of humanized antibody competing with FGF7 protein for binding to FGFR2b

[0082] Figure 6 : Detection of the activity of humanized antibody competing with FGF10 protein for binding to FGFR2b

[0083] Figure 7 : ADCC activity results of humanized antibody KA-1947

[0084] Figure 8 : ADCC activity results of humanized antibody KA-1953

[0085] Figure 9 : Tumor volume changes of humanized antibody in subcutaneous xenograft tumor model of SNU16 cells in female NOG mice

[0086] Figure 10 : Mouse body weight changes of humanized antibody in subcutaneous xenograft tumor model of SNU16 cells in female NOG mice Detailed Description of the Invention

[0087] The present invention will be further explained and described below in conjunction with the embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the protection scope of the present invention.

[0088] In the following embodiments, the experimental materials used can be purchased or prepared with reference to the existing publicly disclosed technologies; those without indicating the source and specifications are commercially available; the various processes and methods not described in detail are conventional methods well known in the art.

[0089] The positive control antibody used in the examples was Bemarituzumab, which has a human IgG1 / κ constant region and was prepared according to the amino acid sequence in (WHO Drug Information, Vol. KA-1947, No. 2, 2017. Proposed INN: List 117); the positive control antibody FPA144-mIgG2a (the heavy chain amino acid sequence is shown in SEQ ID NO: 54 and the light chain amino acid sequence is shown in SEQ ID NO: 55) was prepared by replacing the human-derived Fc of Bemarituzumab with murine FC (mIgG2a).

[0090] The FGFR2b-mFc protein (the amino acid sequence is shown in SEQ ID NO: 56) was obtained by searching for the sequence of human FGFR2b protein (P21802-3) in Uniprot, adding a mouse Fc (mFc) tag in front of the amino acid sequence of the extracellular region protein of FGFR2b, constructing an expression vector, transfecting CHO cells, and obtaining the FGFR2b-mFC protein by conventional purification methods for protein immunization and detection.

[0091] Example 1 Mouse Immunization

[0092] 5 female BALB / C mice aged 6-8 weeks, numbered 1 # -5 # ,5 # mice were used as blank controls. The FGFR2b-mFc protein was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant at a volume ratio of 1:1 (Freund's complete adjuvant was used for the first immunization and Freund's incomplete adjuvant was used for the latter two immunizations), stirred on a stirrer for 1 hour, and the emulsified protein was used to immunize mice 1 # and 2 # subcutaneously and intraperitoneally at an immunization dose of 50 μg / mouse. The immunization interval was 14 days. Blood was collected one week after the three immunizations to detect the antibody titer, and the 2 # mice with the best immunization effect were selected for spleen boosting immunization. The FGFR2b-mFc protein used for immunization was mixed with the aqueous adjuvant at a volume ratio of 1:1, and 10 μg / mouse was used to immunize the hind leg muscles of mice 3 # and 4 # mice. The immunization interval was 14 days. Blood was collected from the tail vein after the two immunizations to detect the antibody titer, and the 4 # mice were selected for spleen boosting immunization.

[0093] Example 2 Cell Fusion and Screening

[0094] Two mice were sacrificed by cervical dislocation # and 4 #Immunized mice, spleens were extracted and homogenized to generate single-cell suspensions, and cell fusion was performed with mouse myeloma cells SP2 / 0 cells in the logarithmic growth phase at a ratio of 2:1 to obtain hybridoma cells. High-throughput ELISA binding assays were used to screen for hybridoma clones that bind to human and monkey FGFR2b but not to FGFR2C protein. The 96-well ELISA plates were coated with human FGFR2b-his (0.5 μg / ml), 100 μl per well, overnight at room temperature. The ELISA plates were washed 3 times with PBST (PBS + 0.05% Tween 20) solution, blocked with 200 μl of blocking solution (PBS + 2% non-fat milk powder + 0.05% Tween 20) at room temperature for 2 hours, and then washed 3 times with PBST solution. 50 μl of the supernatant of the hybridoma cell culture medium was added to the sample detection wells, the negative antibody was PBS, and the positive control antibody was mouse serum 1:5000. After incubation at room temperature for 1 hour, the plates were washed 3 times with PBST. 100 μl of goat anti-mouse Fc-HRP (1∶5000) was added to each well, incubated at room temperature for 1 hour, washed 3 times with PBST, and 50 μl of TMB was added for color development. After 5 - 10 minutes, 50 μl of 0.16 M sulfuric acid was added to terminate the color development, and the OD450 value was measured with an enzyme-linked immunosorbent assay reader. Hybridoma cell lines with an OD value greater than 5 times that of the negative control were selected, and 165 candidate hybridoma cell lines were obtained. The supernatants of the 165 hybridoma cell lines were taken for ELISA binding screening with human FGFR2C protein, and the operation method was the same as the ELISA binding assay of the supernatant of the hybridoma cell culture medium with human FGFR2b-his; the results showed that 82 clones did not bind to FGFR2C. The supernatants of the 82 hybridoma cell lines were taken for ELISA binding screening with monkey FGFR2b protein, and a total of 64 hybridoma cell clones that bind to human and monkey FGFR2b protein but do not bind to human FGFR2C were obtained.

[0095] The binding ability of the 64 selected hybridoma cell lines to human FGFR2b expressed by HEK293T-huFGFR2b cells and their binding to CHOK1-FGFR2C were further tested. First, 1×10 5The detection cells, HEK293T-huFGFR2b cells (Beijing Kangyuan Bochuang, KC-2051) and CHOK1-FGFR2C (Beijing Kangyuan Bochuang, KC-2149), were respectively added into the detection wells of different 96-well plates. The supernatant of the hybridoma culture was added into the sample detection wells, 50 μl per well, PBS was used as the negative control, and mouse serum at 1:50 was used as the positive control antibody. After incubation at 4°C for 1 hour, it was washed 3 times with FACS washing solution (PBS + 1% BSA + 0.01% Tween 20). Then, the cells were resuspended with FACS washing solution, and 500-fold diluted PE Goat anti-mouse IgG (Biolegend Cat: 405307) was added and incubated at 4°C for 1 hour. After the plate was washed 3 times with PBS, it was detected for cell fluorescence on a FACS detector (BD). The mean fluorescence intensity (MFI) value was analyzed and calculated using PRISM TM (GraphPad software). Clones with an MFI greater than or equal to 20 times that of the negative control antibody were used as positive clones, and mouse serum was used as the positive reference antibody.

[0096] Based on the above FACS screening, 45 hybridoma clones that bind to HEK293T-huFGFR2b cells and do not bind to CHOK1-FGFR2C were obtained.

[0097] The above 45 hybridoma clones were subcloned. During the subcloning process, multiple subclones (n > 3) of each clone were selected and characterized by the above ELISA / FACS detection. The subclones obtained through this step were determined as monoclonal hybridoma cell lines. Finally, 45 subclones with high binding affinity for human and monkey FGFR2b were obtained, and each subclone was derived from a different original parent clone.

[0098] Example 3 Hybridoma Antibody Expression and Purification

[0099] First, the monoclonal mouse antibodies of 45 selected clones were purified. Briefly, the hybridoma cells of 25 subclones were grown in T175 cell culture flasks, and each flask contained 100 mL of fresh serum-free hybridoma medium and 1% HT supplement. The cells were cultured in an incubator at 37°C and 5% CO2 for 10 days. The culture was collected, centrifuged at 4500 rpm for 5 minutes, and cell debris was removed by filtration through a 0.22 μm filter membrane. The monoclonal antibody was enriched and purified by a pre-equilibrated protein-A affinity column. Then, it was eluted with elution buffer (20 mM citric acid, pH 3.0 - 3.5). The antibody was stored in PBS (pH 7.0), and the antibody concentration was detected by NanoDrop.

[0100] Example 4 Determination of Antibody Binding Activity by FACS Method

[0101] Preparation of cell suspension: HEK293-huFGFR2b cells were resuspended in PBS, and the cell concentration was adjusted so that the number of cells per well was 1×10 5 cells.

[0102] The initial concentrations of the purified hybridoma antibody and the positive control FPA144-mIgG2a antibody, both at 20 μg / mL, were added to the first well of a 96-well plate, and 3-fold serial dilutions were made in the remaining 8 wells. Then, the prepared cell suspension was added to the corresponding 96-well plate (50 μL / well). After incubation on a shaker at 4°C for 50 minutes, the cells were washed twice by centrifugation with PBS. Fluorescent secondary antibody: PE Goat anti-mouse IgG (Biolegend Cat: 405307) at 3 μg / ml, 100 μL per well, was added. After incubation on a shaker at 4°C for 40 minutes, the cells were washed twice by centrifugation with PBS. The prepared samples were then analyzed using a flow cytometer. The signals were read using a FACS BD Calibur. The data were analyzed using non-linear regression with PRISM TM (GraphPad software), and the EC50 values were calculated.

[0103] Comparison of the EC50 values of 45 monoclonal antibodies revealed that 3 antibodies, namely 15E7-5, 30F2-7, and 48F1-6, had a better binding affinity to HEK293-huFGFR2b than the control antibody FPA144-mIgG2a. Their EC50 values and binding curve profiles are shown in Table 1 and Figure 1 .

[0104] Table 1 Binding activity of murine antibodies

[0105] Sample EC50 (μg / ml) FPA144-mIgG2a 0.2597 15E7-5 0.1892 30F2-7 0.2106 48F1-6 0.1761

[0106] Example 5 Antibody sequencing

[0107] Antibody subtype identification: The supernatant of hybridoma cell culture was taken, and the antibody subtype was identified using an IsoStripTM Mouse Monoclonal Antibody Subtype Identification Kit. Based on the subtype identification results, specific nested PCR primers were designed. The primer sequences used in this amplification reaction were complementary to the first framework region and the constant region of the antibody variable region. The PCR amplification products were directly subjected to TA cloning, and the products of TA cloning were directly sequenced. The CDRs and FRs of the antibody variable region were divided using the Kabat numbering rule. The CDR sequences of the heavy chain, the CDR of the light chain, and the amino acid sequences of the heavy and light chain variable regions of the antibody are shown in SEQ ID NO: 1-23, and the nucleotide sequences are shown in SEQ ID NO: 24-29, and are listed in Table 2 and Table 3.

[0108] Table 2 CDR sequences of murine antibodies

[0109] Antibody Name 15E7-5 30F2-7 48F1-6 HCDR1 SEQ ID NO: 1 SEQ ID NO: 4 SEQ ID NO: 7 HCDR2 SEQ ID NO: 2 SEQ ID NO: 5 SEQ ID NO: 8 HCDR3 SEQ ID NO: 3 SEQ ID NO: 6 SEQ ID NO: 9 LCDR1 SEQ ID NO: 10 SEQ ID NO: 13 SEQ ID NO: 16 LCDR2 SEQ ID NO: 11 SEQ ID NO: 14 SEQ ID NO: 11 LCDR3 SEQ ID NO: 12 SEQ ID NO: 15 SEQ ID NO: 17

[0110] Table 3 Variable Region Sequences of Murine Antibodies

[0111]

[0112]

[0113] Example 6 Construction, Expression and Purification of Chimeric Antibodies

[0114] The genes encoding the heavy and light chain variable regions of antibodies 15E7-5, 30F2-7 and 48F1-6 (shown as SEQ ID NO: 24 and SEQ ID NO: 27; SEQ ID NO: 25 and SEQ ID NO: 28; SEQ ID NO: 26 and SEQ ID NO: 29 respectively) and their respective human IgG1 / κ constant regions (shown in Table 4, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO: 30, and the sequence of the light chain constant region is shown as SEQ ID NO: 31) were designed and synthesized into target gene fragments by standard methods known to those skilled in the art and inserted into the pCDNA3.4 vector to construct murine-human chimeric expression plasmids.

[0115] Table 4 Amino Acids of Antibody Heavy and Light Chain Constant Regions

[0116] Name Description Serial Number H-IgG1-FC Heavy Chain Constant Region SEQ ID NO: 30 L-k-FC Light Chain Constant Region SEQ ID NO: 31

[0117] The plasmids obtained above were transfected into Expi-293F cells respectively. Specifically, Expi-293F cells were cultured in Expi-293F expression medium, and each expression plasmid was transfected into the cells using the Expi-293F transfection kit. The ratio of DNA to Expi-Fectamine was 1:3, and the amount of DNA added to each milliliter of cell culture medium was 1.5 μg. The transfected Expi-293F cells were cultured in an incubator at 37°C and 5% CO2 at a rotation speed of 100 RPM. After 5-7 days, the cell culture supernatant was collected, and the monoclonal antibody was purified according to the method steps of Example 3.

[0118] The chimeric antibody of antibody 15E7-5 is named antibody M15E7-5-hIgG1. The amino acid sequences of its heavy chain and light chain variable regions are shown in SEQ ID NO: 18 and 21 respectively, and the amino acid sequences of its heavy chain constant region and light chain constant region are shown in SEQ ID NO: 30 and 31 respectively; The murine-human chimeric antibody of antibody 30F2-7 is named antibody M30F2-7-hIgG1. The amino acid sequences of its heavy chain and light chain variable regions are shown in SEQ ID NO: 19 and 22 respectively, and the amino acid sequences of its heavy chain constant region and light chain constant region are shown in SEQ ID NO: 30 and 31 respectively; The murine-human chimeric antibody of antibody 48F1-6 is named antibody M48F1-6-hIgG1. The amino acid sequences of its heavy chain and light chain variable regions are shown in SEQ ID NO: 20 and 23 respectively, and the amino acid sequences of its heavy chain constant region and light chain constant region are shown in SEQ ID NO: 30 and 31 respectively.

[0119] Example 7 Binding assay of chimeric monoclonal antibodies to human FGFR2b expressed by HEK293T cells

[0120] The binding activities of chimeric antibodies M15E7-5-hIgG1, M30F2-7-hIgG1 and M48F1-6-hIgG1 to human FGFR2b expressed on 293T cells were further verified by FACS experiments on HEK293T-huFGFR2b cells. The specific experimental operations are shown in Example 4. The positive control antibody was Bemarituzumab, and the secondary antibody was a fluorescent secondary antibody against human FC. The measurement results are as Figure 2 shown in Table 5. The chimeric antibody M15E7-5-hIgG1 has high affinity for HEK293T-huFGFR2b, and its binding activity is better than that of the positive control antibody Bemarituzumab; The activity of M48F1-6-hIgG1 is comparable to that of the control antibody Bemarituzumab; The activity of M30F2-7-hIgG1 is weaker than that of the control antibody Bemarituzumab. Therefore, two antibodies, 15E7-5 and 48F1-6, were selected for humanization and further evaluation.

[0121] Table 5 FACS binding EC50 values of chimeric antibodies to HEK293T-HuFGFR2b cells

[0122] Sample EC50 (μg / ml) Bemarituzumab 0.07551 M15E7-5-hIgG1 0.0631 M30F2-7-hIgG1 0.1869 M48F1-6-hIgG1 0.07815

[0123] Example 8 Antibody humanization

[0124] Based on the sequencing results, the 15E7-5 and 48F1-6 antibodies (see U.S. Patents 4816567, 5225539, 5530101, 5585089, 5693762, and 6180370) were humanized by transplanting the CDRs onto the human germline framework to humanize the anti-FGFR2b antibodies. The specific method is as follows. To screen the receptor frameworks for humanized chimeric antibodies, the light and heavy chain variable region sequences of these chimeric antibodies were aligned with the NCBI human immunoglobulin gene database (http: / / www.ncbi.nlm.nih.gov / igblast / ), and the human germline IGVH and IGVK with the highest homology, relatively high expression levels, and also used in other approved drugs were selected as the frameworks for humanization. The human light chain germline receptor sequences selected for 15E7-5 and 48F1-6 were both IGKV1-5*01, and the germline receptor sequences of the heavy chain were both IGHV1-46*01.

[0125] For the variable domains of 15E7-5 and 48F1-6, the antibody crystal structure models with the closest sequences (structural resolution higher than 2.5 Å) were retrieved using PDB BLAST for three-dimensional structure simulation to determine the key framework amino acid residues that may be important for maintaining the CDR loop structures, thereby designing the back mutations of the humanized antibodies. Briefly, the selected structural templates have the same types of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3 loop structures as 15E7-5 and 48F1-6 respectively. Using the selected structural templates, the structural models of humanized 15E7-5 and 48F1-6 were constructed by replacing the murine frameworks with the human germline heavy and light chain framework sequences. Subsequently, three-dimensional structure modeling was performed to identify the key framework amino acid residues that may be important for maintaining the CDR loop structures or the heavy and light chain linkages. When the murine antibody framework and the human germline receptor framework have the same amino acid residue at a certain position, the human germline amino acid residue is retained. On the other hand, when the murine framework and the human germline receptor framework have different amino acid residues at a certain position, the importance of this residue is evaluated through structure simulation. If it is found that an amino acid residue within the human germline receptor framework interacts with the CDR region residues and affects the CDR residues, then this residue is back mutated to the murine residue. In addition, post-translational modifications were performed on the antibody CDR regions, and back mutations were made at the sites that may affect antibody stability and binding activity.

[0126] The 15E7-5 humanized antibodies are named KA-1946, KA-1947, KA-1948, and KA-1949 respectively; the 48F1-6 humanized antibodies are named KA-1950, KA-1951, KA-1952, and KA-1953 respectively. The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the above humanized antibodies are shown in Table 6.

[0127] Table 6 Amino Acid Sequences of the Heavy Chain Variable Region and the Light Chain Variable Region of Humanized Antibodies

[0128] Antibody Name Heavy Chain Variable Region (VH) Light Chain Variable Region (VL) KA-1946 SEQ ID NO: 34 SEQ ID NO: 42 KA-1947 SEQ ID NO: 35 SEQ ID NO: 42 KA-1948 SEQ ID NO: 36 SEQ ID NO: 43 KA-1949 SEQ ID NO: 37 SEQ ID NO: 43 KA-1950 SEQ ID NO: 38 SEQ ID NO: 44 KA-1951 SEQ ID NO: 39 SEQ ID NO: 44 KA-1952 SEQ ID NO: 40 SEQ ID NO: 45 KA-1953 SEQ ID NO: 41 SEQ ID NO: 45

[0129] Example 9 Expression and Purification of Humanized Antibodies

[0130] 9.1 Plasmid Preparation

[0131] The target gene fragment was designed and synthesized by standard methods known to those skilled in the art, and the sequence was constructed as follows: Leader sequence - the variable region of the target antibody - the constant region of human IgG1. The target gene fragment was subcloned into the pcDNA3.4 expression vector. The expression plasmid at the transfection level was prepared, and the plasmid was extracted in large quantities using the plasmid large-scale extraction kit of axygen. Aliquoted into 1.5 ml centrifuge tubes, labeled, and stored at -20 °C or -80 °C.

[0132] 9.2 Cell Culture and Transfection

[0133] Expi293F TM cells (Thermo Fisher Scientific) were cultured in serum-free medium, and the cells were inoculated in a shake flask and cultured on a shaker at 37 °C in an environment of 8% CO2. One day before plasmid transfection, the cell density was adjusted. On the day of transfection, DNA and ExpiFectamine TM 293 reagent were mixed in an appropriate ratio and added to the cell culture shake flask. 16 - 18 hours after transfection, ExpiFectamine TM 293 Transfection Enhancer 1 and ExpiFectamine TM 293 Transfection Enhancer 2 were added. The cells were cultured for 6 days and the supernatant was collected for purification.

[0134] 9.3 Antibody Purification

[0135] Centrifuge the cell culture supernatant and filter it. Load the filtered supernatant onto an affinity purification column and adjust the flow rate. After washing away impurities and eluting, perform buffer replacement on the fractionally collected eluates. Conduct SDS-PAGE purity analysis and A280 concentration determination on the final purified antibodies KA-1946, KA-1947, KA-1948, KA-1949, KA-1950, KA-1951, KA-1952, and KA-1953.

[0136] Example 10 Detection of the Affinity of Humanized Antibodies

[0137] Use SPR method with Biacore T200 to determine the affinity kinetics of the interaction between the antibody and FGFR2b protein. The specific operation is as follows:

[0138] (1) Couple Protein G to the CM5 chip covalently according to the instructions.

[0139] (2) Dilute the ligand FGFR2b-his protein with HBS-EP+ buffer solution respectively.

[0140] (3) Serial dilute the humanized antibody with HBS-EP+ buffer solution and set a zero concentration point.

[0141] (4) Set the program as shown in Table 7 below and run it.

[0142] Table 7 Affinity Running Parameters

[0143]

[0144] Biacore T200 Control Software collects the SPR signals and saves them. Then, use Biacore T200 Evaluation analysis software for data processing. Subtract the signal value of the corresponding reference channel (Fc1 or Fc3) from the signal value of the detection channel (Fc2 or Fc4) to obtain the corrected signal curve. For affinity evaluation, use double subtraction. Add a zero concentration cycle (Sample 0) with the same parameter settings before the sample cycle. Subtract the zero concentration cycle from the sample cycle (Sample - Sample0) to obtain the secondarily corrected signal curve. Fit the affinity kinetic curve according to the 1:1 Langmuir binding model, and fit the affinity curve according to the Steady State Affinity model, and calculate the KD value. The results are shown in Table 8. KA-1947 and KA-1953 have the best affinity, and these two antibody strains are selected for further study.

[0145] Table 8 Results of the Affinity of Humanized Antibodies

[0146]

[0147]

[0148] The corresponding CDR regions of the humanized antibodies KA-1947 and KA-1953 are shown in Table 9;

[0149] Table 9 Amino acid sequences of the CDR regions of humanized antibodies

[0150]

[0151] Exemplarily, the amino acid and nucleotide sequences of the heavy and light chains of the antibody are as follows:

[0152] Table 10 Sequences of the heavy and light chains of humanized antibodies

[0153]

[0154] Example 11 FACS binding assay of humanized antibodies

[0155] The FACS experiment was used to detect the binding activities of the humanized antibodies KA-1947, KA-1953 and the control antibody Bemarituzumab to the tumor cell SNU16. The specific operations were as follows:

[0156] (1) Collect SNU16 cells, adjust the cell density to 2×10 6 cells / mL, and inoculate 50 μl per well into a 96-well "U"-bottom plate;

[0157] (2) Prepare the samples to be tested: with an initial concentration of 15 μg / ml, and perform 3-fold serial dilution to obtain a total of 8 concentration gradients. Then inoculate 50 μl per well into a 96-well "U"-bottom plate and mix well, and let it stand at 2-8 °C for 1 h;

[0158] (3) Centrifuge the whole plate for 3 minutes, discard the supernatant, add 200 μl of PBS to each well, and wash the plate 3 times;

[0159] (4) Add 100 μl of fluorescent secondary antibody dilution (diluted 1:400 to a final concentration of 2.5 μg / ml) to each well and mix well; let it stand at 2-8 °C for 1 h;

[0160] (5) Repeat step (3);

[0161] (6) Centrifuge the whole plate at 2000 RPM for 3 minutes, discard the supernatant, add 100 μl of PBS to each well, mix well, and then detect the fluorescence intensity by machine;

[0162] (7) Analyze the data and make statistical graphs. Use the logarithm of the sample concentration as the X value and the fluorescence intensity value (APC) as the Y value to make a four-parameter equation, and plot the graph to analyze the cell binding activity of the sample. The results are asFigure 3 , Figure 4 as shown in Table 11.

[0163] Table 11 EC50 values of the binding of the humanized antibody to SNU16 cells

[0164] Sample Name EC50 (μg / ml) Bemarituzumab 0.2339 KA-1947 0.1565 KA-1953 0.1408

[0165] Blocking Activity of the Humanized Antibody in Example 12

[0166] The activity test of the humanized antibodies KA-1947 and KA-1953 and the control antibody Bemarituzumab competing for binding to the FGFR2b with FGF7 / FGF10 protein was performed as follows:

[0167] (1) Coating the plate: Dilute FGF7 protein to 1 μg / ml with the coating solution and FGF-10 protein to 2 μg / ml with the coating solution, and coat 100 μl / well in a 96-well plate, and place it in a refrigerator at 2-8 °C overnight.

[0168] (2) Blocking: Discard the liquid in the wells, pat dry, add the blocking solution, 320 μl / well, block at 25 °C for 2 h, wash the plate 3 times with PBST solution, and pat dry.

[0169] (3) Dilution process of the antibody to be tested: Gradually dilute with the dilution solution, the curve range is from 40 μg / ml to 2.441 ng / ml, 7 gradients of 4-fold dilution, add a zero concentration point, the volume ratio of FGF7 to FGFR2b (2 μg / ml) is 50 μL:50 μL, and add the sample to the enzyme-linked immunosorbent assay (ELISA) plate; the volume ratio of FGF10 to FGFR2b (4 μg / ml) is 50 μL:50 μL, and add the sample to the ELISA plate.

[0170] (4) Incubation: Add 100 μl / well of the diluted reference substance and test substance to the ELISA plate, incubate at 25 °C for 2 h, discard the liquid, wash the plate 4 times, and pat dry.

[0171] (5) Adding the secondary antibody: Dilute the Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, HRP 10,000 times, add 100 μl / well to the plate, incubate at 25 °C for 1 h, discard the solution in the wells, and wash 7 times with 300 μl / well of the washing solution.

[0172] (6) Color development: Add the TMB substrate, 100 μl / well, and develop color at room temperature in the dark for 2-4 min.

[0173] (7) Termination: Add the termination solution, 100 μl / well, to terminate the reaction. Read the absorbance at 450 nm.

[0174] The results are asFigure 5 , Figure 6 As shown in Figure 6 and Table 12, the blocking activities of KA-1947 and KA-1953 are both superior to the control.

[0175] Table 12 IC50 values of humanized antibodies competing with FGF7 / FGF10 protein for binding to FGFR2b

[0176] Sample Name IC50-FGF7 (μg / ml) IC50-FGF10 (μg / ml) KA-1947 0.3578 0.2452 KA-1953 0.2252 0.1517 Bemarituzuma 0.7327 0.4938

[0177] Example 13 Determination of ADCC Activity of Antibodies by Reporter Gene Assay

[0178] The ADCC activities of humanized antibodies KA-1947, KA-1953 and control antibody Bemarituzumab were detected by reporter gene assay. The specific experimental steps are as follows:

[0179] 1) Effector cell treatment: Collect Jurkat-ADCC-Luc cells and transfer them to a new sterile centrifuge tube for centrifugation. Discard the supernatant, resuspend the cells with 1640 culture medium containing 10% FBS and count them. Then adjust the cell density to 7.5x10 5 cells / ml for use.

[0180] 2) Target cell treatment: Collect HEK293T-huFGFR2b cells and transfer them to a new sterile centrifuge tube for centrifugation. Discard the supernatant, resuspend the cells with 1640 culture medium containing 10% FBS and count them. Then adjust the cell density to 3×10 5 cells / ml for use.

[0181] 3) Mix the cells from the above two steps in equal volume and add 100 μl per well to the experimental plate.

[0182] 4) Prepare the test samples with an initial concentration of 50 μg / ml and perform 5-fold serial dilution to obtain 8 gradients. Each concentration point has 3 replicates. Take 50 μl of each sample and add it to the culture plate containing cells.

[0183] 5) Incubate the culture plate in an incubator at 37 °C (±2 °C) and 5% (±1%) CO2 for 5 h.

[0184] 6) Add 50 μl of Bright-Glo-Luciferase detection reagent to each well and measure the RLU value on the machine.

[0185] 7) Use the logarithm of the sample concentration as the X value and the RLU value as the Y value to fit a four-parameter equation and plot a graph to analyze the ADCC activity of the sample.

[0186] The results are shown in Figure 7 , Figure 8As shown in Table 13, the ADCC EC50 values of KA-1947 and KA-1953 are superior to the control.

[0187] Table 13 ADCC Activity of Humanized Antibodies

[0188] Sample Name EC50 μg / ml) Top KA-1947 0.01961 171295 KA-1953 0.01978 171510 Bemarituzumab 0.2606 90269

[0189] Example 14 In Vivo Pharmacodynamic Experiment

[0190] Study on the Effects of Human Antibodies KA-1947, KA-1953 and Control Antibody Bemarituzumab on Subcutaneous Xenograft Tumor Models of SNU16 Cells in Female NOG Mice

[0191] Mix human gastric cancer SNU16 cells with Matrigel at a volume ratio of 1:1, and inoculate 0.1 mL per mouse subcutaneously in the right hypochondrium of female NOG mice. The amount of SNU16 cells inoculated per mouse is 5×10 6 cells. When the average tumor volume of the mice reaches 121 mm 3 , mice with too large or too small tumor volumes are excluded. According to the tumor volume and body weight of the mice, the selected mice are randomly divided into groups of 6 mice each. Administration starts on the day of grouping. The administration doses of humanized antibodies KA-1947, KA-1953 and control antibody Bemarituzumab are all 10 mg / kg, and the administration dose of the vehicle control group PBS is 10 ml / kg. The day of administration is recorded as day 0, and administration is given twice a week for four consecutive weeks, which are recorded as D0, D4, D7, D11, D14, D17, D20 and D23 respectively.

[0192] The administration route for all groups is intraperitoneal injection. During administration and observation, the body weight and tumor volume of the mice are measured 2 - 3 times a week, and the measured values are recorded. The maximum diameter (D) and minimum diameter (d) of the tumor are measured with an electronic vernier caliper every week, the tumor volume is calculated, and the tumor growth inhibition rate of each administration group is calculated according to the formula.

[0193] Tumor volume (mm 3 ) = [D×d 2 / 2;

[0194] Tumor growth inhibition rate TGI (%) = (1 - average tumor volume of the administration group / average tumor volume of the control group) × 100%.

[0195] The results are as Figure 9 shown in Table 14: On day D23 after administration, the average tumor volume of the vehicle group is 754.7 mm 3, the TGI values of the humanized antibodies KA-1947 and KA-1953 and the control antibody Bemarituzumab were 77%, 76% and 61% respectively; compared with the vehicle control group, the antibodies KA-1947, KA-1953 and the control antibody Bemarituzumab all had a very significant anti-tumor effect (P<0.01). The above results indicate that the anti-FGFR2b humanized antibodies KA-1947 and KA-1953 both showed good anti-tumor effects, and the tumor inhibitory effect was better than that of the control antibody Bemarituzumab. Compared with the vehicle control group PBS, there was no significant change in the body weight of the mice during the administration period in the administration group, and the mice had good activity and feeding status during the administration and observation periods, indicating that the animals had good tolerance to the test substance. The results were as Figure 10 shown.

[0196] Table 14 Tumor inhibitory effects of humanized antibodies in animals

[0197]

Claims

1. An anti-FGFR2b antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region. The heavy-chain variable region contains heavy-chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein: (1) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; or (2) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; or (3) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (4) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown as SEQ ID NO:1, SEQ ID NO:32, and SEQ ID NO:3; or (5) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown as SEQ ID NO:7, SEQ ID NO:33, and SEQ ID NO:9; And, the light-chain variable region contains light-chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein, (6) The amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; or (7) The amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively shown as SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; or (8) The amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:

17.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of the HCDR1, HCDR2, HCDR3 of the heavy-chain variable region and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region are selected from the following groups: (1) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; (2) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 respectively; (3) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:17 respectively; (4) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:1, SEQ ID NO:32, and SEQ ID NO:3 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively; (5) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:33, and SEQ ID NO:9 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:16, SEQ ID NO:11, and SEQ ID NO:17 respectively.

3. The anti-FGFR2b antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:18 - 20, SEQ ID NO:34 - 41, and the amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:21 - 23, SEQ ID NO:42 - 45; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences selected from the group consisting of SEQ ID NO:18 - 20, SEQ ID NO:34 - 41, SEQ ID NO:21 - 23, SEQ ID NO:42 - 45.

4. The anti-FGFR2b antibody or antigen-binding fragment thereof according to claim 3, wherein The amino acid sequences of the antibody heavy chain variable region and light chain variable region are selected from the following groups: (1) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:21; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence; (2) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 22; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (3) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 23; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (4) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (5) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (6) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 43; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (7) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 43; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (8) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 38, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 44; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (9) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 44; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequences; (10) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 40, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 45; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (11) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 45; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence.

5. The anti-FGFR2b antibody or antigen-binding fragment thereof according to claim 4, wherein The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are selected from the following groups: (1) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 21; (2) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 23; (3) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; (4) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:

45.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-human FGFR2b antibody or its antigen-binding fragment according to any one of claims 1-5.

7. An expression vector, characterized in that, The expression vector contains the nucleic acid molecule according to claim 6.

8. Use of the anti-FGFR2b antibody according to any one of claims 1-5, characterized in that, Use of the said anti-human FGFR2b antibody in the preparation of a medicament for preventing and / or treating tumors.

9. Use of the anti-FGFR2b antibody according to claim 8, characterized in that, The tumors are selected from hepatocellular carcinoma, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, squamous non-small cell lung cancer, colon cancer, prostate cancer, colorectal cancer, glioma, head and neck cancer, melanoma, gastric cancer, esophageal cancer, triple-negative breast cancer, intrahepatic cholangiocarcinoma and renal cell carcinoma.

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