TW202442685A - Anti-FGFR2b mAb - Google Patents

Abstract

The present invention provides an anti-FGFR2Ⅲb antibody. Specifically, the anti-FGFR2Ⅲb antibody of the present invention can specifically bind to FGFR2Ⅲb, has high affinity and selectivity, and can effectively block the binding of FGFR2Ⅲb to its ligands FGF1, FGF7 or FGF10. In addition, the antibody can also mediate the specific toxicity of FGFR2Ⅲb-overexpressing cancer cells by immune cells through ADCC. The antibody of the present invention has excellent binding activity to FGFR2Ⅲb from various species. In vivo experiments show that it has a significant tumor-suppressing effect on a gastric cancer-bearing mouse model expressing FGFR2Ⅲb, and has a prospect of application in the field of tumor treatment.

Description

Anti-FGFR2b mAb

The present invention relates to the field of biomedicine. Specifically, the present invention relates to an anti-FGFR2IIIb monoclonal antibody.

Fibroblast Growth Factor Receptor (FGFR) belongs to a subfamily of the tyrosine kinase receptor superfamily. There are four FGFR genes ( FGFR1-4 ) in the human genome.

The signal transduction pathways mediated by FGFR are necessary for normal cell growth and differentiation. They are involved in physiological processes such as angiogenesis, cell proliferation and migration, regulation of organ development, and wound healing. However, when FGFR mutates or is overexpressed, it will cause overactivation of the FGFR signaling pathway and further induce normal cell carcinogenesis. Among them, overactivation of RAS-RAF-MAPK can stimulate cell proliferation and differentiation; overactivation of PI3K-AKT can inhibit cell apoptosis; SATA is closely related to promoting tumor invasion and metastasis and enhancing the ability of tumors to escape immune system; and the PLCγ signaling pathway is an important pathway for regulating tumor cell metastasis.

FGFRs contain three extracellular immunoglobulin-like domains (D1-D3), a hydrophobic single-span membrane helix, and an intracellular tyrosine kinase domain with an eight-residue acid box between D1 and D2. In FGFR1-3, ligand binding specificity is largely determined by alternative splicing at the C-terminus of the D3 domain. There are two alternative splices at the C-terminus of the D3 domain, which are encoded by exons 8 or 9 to generate FGFRb or FGFRc isoforms. These b and c isoforms are usually restricted to epithelial and mesenchymal tissues, respectively. In this way, alternative splicing of the receptor allows ligands to activate the receptor in adjacent mesenchymal or epithelial tissues without activating autocrine signaling.

The main ligands of the FGFR2Ⅲb (or FGFR2b) form of FGFR2 are FGF1, FGF7, FGF10 and FGF22. FGFR2b can be highly expressed in tumors through FGFR2 gene amplification or transcriptional upregulation of the FGFR2b isoform, of which FGFR2 amplification is the most common FGFR2 gene aberration. In the immunohistochemical section analysis of gastric cancer patients, FGFR2b expression can be detected in approximately 60% of tumor samples, of which 2%-9% of samples will be detected with FGFR2b overexpression, and the overexpression level ranges from 31% to 61%. These gastric cancer samples often show diffuse type and are associated with the aggressive characteristics of gastric cancer, including higher T stage, more frequent lymph node metastasis, and lower overall survival rate. With the deepening of research, it has been determined that the overexpression of FGFR2b receptor or the expansion of FGFR2 gene is directly related to the poor prognosis of gastric cancer patients. In addition to gastric cancer, abnormal activation of FGF/FGFR2 signaling pathways has also been observed in other cancers, including but not limited to esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, lung cancer (such as non-small cell lung cancer), cholangiocarcinoma, etc. Therefore, inhibiting FGFR2 signaling may be an effective mechanism for treating a variety of cancers.

The purpose of the present invention is to provide an anti-FGFR2IIIb monoclonal antibody.

In the first aspect of the present invention, an anti-FGFR2Ⅲb antibody is provided, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementary determining region (CDR) selected from the following group: (1)  VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 7, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (2)  VH-CDR1 shown in SEQ ID NO: 15, VH-CDR2 shown in SEQ ID NO: 16, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (3)  VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 19, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (4)  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, the CDRs are defined according to the Kabat rule; (5)  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:21, and VH-CDR3 shown in SEQ ID NO:8, the CDRs are defined according to the Kabat rule; and (6)  VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11, the CDRs are defined according to the IMGT rule; and the variable region of the light chain has a complementary determining region (CDR) selected from the following group: (1)  VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:21, and VH-CDR3 shown in SEQ ID NO:8, the CDRs are defined according to the Kabat rule; and (2)  VL-CDR1 shown in SEQ ID NO:17, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:14, said CDRs are defined according to the Kabat rule; (3)  VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, said CDRs are defined according to the Kabat rule; and (4)  VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, said CDRs are defined according to the IMGT rule; Furthermore, any one of the amino acid sequences in the above CDR sequences also includes a derivative sequence that has been optionally supplemented, deleted, modified and/or substituted with 1-2 amino acids, and enables the derivative antibody composed of the heavy chain and light chain containing the derivative CDR sequence to retain the binding affinity of FGFR2Ⅲb or its derivative protein.

In the first aspect of the present invention, an anti-FGFR2Ⅲb antibody is provided, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementary determining region (CDR) selected from the following group: (1)  VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 7, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (2)  VH-CDR1 shown in SEQ ID NO: 15, VH-CDR2 shown in SEQ ID NO: 16, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (3)  VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 19, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (4)  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, the CDRs are defined according to the Kabat rule; (5)  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:21, and VH-CDR3 shown in SEQ ID NO:8, the CDRs are defined according to the Kabat rule; and (6)  VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11, the CDRs are defined according to the IMGT rule; and the variable region of the light chain has a complementary determining region (CDR) selected from the following group: (1)  VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:21, and VH-CDR3 shown in SEQ ID NO:8, the CDRs are defined according to the Kabat rule; and (2)  VL-CDR1 shown in SEQ ID NO:17, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:14, said CDRs are defined according to the Kabat rule; (3)  VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, said CDRs are defined according to the Kabat rule; and (4)  VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, said CDRs are defined according to the IMGT rule.

In another preferred embodiment, the antibody has a heavy chain variable region CDR (VH-CDR) and a light chain variable region CDR (VL-CDR) selected from the following groups: (1) VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 7, VH-CDR3 shown in SEQ ID NO: 8, VL-CDR1 shown in SEQ ID NO: 12, VL-CDR2 shown in SEQ ID NO: 13, and VL-CDR3 shown in SEQ ID NO: 14, wherein the CDRs are defined according to the Kabat rule; (2) VH-CDR1 shown in SEQ ID NO: 15, VH-CDR2 shown in SEQ ID NO: 16, VH-CDR3 shown in SEQ ID NO: 8, VL-CDR1 shown in SEQ ID NO: 17, VL-CDR2 shown in SEQ ID NO: 18, and VL-CDR3 shown in SEQ ID NO: 19. NO:14, the VL-CDR3, the CDRs are defined according to the Kabat rule; (3)  VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:19, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, the CDRs are defined according to the Kabat rule; (4)  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, the CDRs are defined according to the Kabat rule; (5)  SEQ ID NO:15, VH-CDR1, VH-CDR2, VH-CDR3, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the CDRs are defined according to the Kabat rule; and (6)  VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the CDRs are defined according to the Kabat rule; and (7)  VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the CDRs are defined according to the Kabat rule; and (8)  VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, wherein the CDRs are defined according to the Kabat rule; and VL-CDR1 shown in ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, the CDRs are defined according to IMGT rules.

In another preferred embodiment, the antibody specifically binds to FGFR2IIIb or a protein derived therefrom.

In another preferred example, the antibody is capable of specifically binding to FGFR2IIIb from human, mouse and cynomolgus monkey.

In another preferred embodiment, the FGFR2IIIb is FGFR2IIIb on the cell surface or soluble FGFR2IIIb.

In another preferred example, the NCBI accession number of FGFR2IIIb is NP_075259.

In another preferred embodiment, the FGFR2IIIb derivative protein is a FGFR2IIIb S252W mutant.

In another preferred example, the affinity KD value of the antibody binding to FGFR2IIIb is ≤1×10 ^-7 M, preferably ≤1×10 ^-8 M, and more preferably ≤5×10 ^-9 M.

In another preferred example, the antibody blocks the binding of FGFR2IIIb or its derivative protein to FGF1, FGF7 or FGF10.

In another preferred example, the blocking refers to reducing the binding rate of the FGF1, FGF7 or FGF10 to FGFR2IIIb by 50%, preferably by 70%, and more preferably by 90%.

In another preferred example, the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30 or 31.

In another preferred example, the heavy chain further includes a heavy chain constant region.

In another preferred example, the heavy chain constant region is of human or mouse origin.

In another preferred example, the variable region of the light chain has the amino acid sequence shown in SEQ ID NO: 25, 27 or 29.

In another preferred example, the light chain further includes a light chain constant region.

In another preferred example, the light chain constant region is of human or mouse origin.

In another preferred example, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30 or 31; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 25, 27 or 29.

In another preferred example, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 31 or 4 in the sequence listing.

In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO: 25, 27, 29 or 5 in the sequence listing.

In another preferred embodiment, the antibody has: (1) a heavy chain variable region as shown in SEQ ID NO:24 and a light chain variable region as shown in SEQ ID NO:25; (2) a heavy chain variable region as shown in SEQ ID NO:26 and a light chain variable region as shown in SEQ ID NO:27; (3) a heavy chain variable region as shown in SEQ ID NO:28 and a light chain variable region as shown in SEQ ID NO:29; (4) a heavy chain variable region as shown in SEQ ID NO:30 and a light chain variable region as shown in SEQ ID NO:29; (5) a heavy chain variable region as shown in SEQ ID NO:31 and a light chain variable region as shown in SEQ ID NO:29; (6) a heavy chain variable region as shown in SEQ ID NO:26 and a light chain variable region as shown in SEQ ID NO:29; or (7) a heavy chain variable region as shown in SEQ ID NO:28 and a light chain variable region as shown in SEQ ID NO:29. The heavy chain variable region shown in NO:4 and the light chain variable region shown in SEQ ID NO:5.

In another preferred example, the heavy chain variable region of the antibody further includes a human framework region, and/or the light chain variable region of the antibody further includes a human framework region.

In another preferred example, the heavy chain variable region of the antibody further includes a murine framework region, and/or the light chain variable region of the antibody further includes a murine framework region.

In another preferred embodiment, the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

In another preferred embodiment, the amino acid sequence of the heavy chain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO: 32, 34, 36, 38, 39, or 22.

In another preferred embodiment, the amino acid sequence of the light chain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO: 33, 35, 37 or 23.

In another preferred embodiment, the antibody has: (1) a heavy chain as shown in SEQ ID NO:32 and a light chain as shown in SEQ ID NO:33; (2) a heavy chain as shown in SEQ ID NO:34 and a light chain as shown in SEQ ID NO:35; (3) a heavy chain as shown in SEQ ID NO:36 and a light chain as shown in SEQ ID NO:37; (4) a heavy chain as shown in SEQ ID NO:38 and a light chain as shown in SEQ ID NO:37; (5) a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:37; (6) a heavy chain as shown in SEQ ID NO:34 and a light chain as shown in SEQ ID NO:37; or (7) a heavy chain as shown in SEQ ID NO:22 and a light chain as shown in SEQ ID NO:23.

In the second aspect of the present invention, a recombinant protein is provided, the recombinant protein comprising: (i) the antibody as described in the first aspect of the present invention; and (ii) an optional tag sequence that assists expression and/or purification.

In another preferred example, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

In another preferred embodiment, the recombinant protein is specific for anti-FGFR2IIIb.

In another preferred embodiment, the recombinant protein is a fusion protein.

In another preferred embodiment, the fusion protein is a monospecific antibody (ie, a monospecific antibody against FGFR2IIIb), a bispecific antibody, or a multispecific antibody (eg, a trispecific antibody).

In another preferred embodiment, the bispecific antibody or multispecific antibody is not only directed against FGFR2IIIb, but also specifically binds to additional target antigens (such as other tumor antigens, such as other antigens of gastric cancer or other tumor antigens).

In the third aspect of the present invention, a chimeric antigen receptor (CAR) construct is provided, wherein the antigen binding region of the chimeric antigen receptor construct includes a single chain variable fragment (scFv) that specifically binds to FGFR2Ⅲb or its derivative protein, and the scFv has the heavy chain variable region and light chain variable region of the antibody described in the first aspect of the present invention.

In the fourth aspect of the present invention, a recombinant immune cell is provided, wherein the immune cell expresses an exogenous CAR construct as described in the third aspect of the present invention.

In another preferred embodiment, the immune cells are selected from the following group: NK cells, T cells.

In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

In the fifth aspect of the present invention, a use of an active ingredient is provided, wherein the active ingredient is selected from the following group: the antibody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the immune cell as described in the fourth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for: (a) preparing a diagnostic reagent, a test plate or a reagent kit; and/or (b) preparing a drug for preventing and/or treating a disease associated with abnormal expression or function of FGFR2Ⅲb or its derivative protein.

In another preferred example, the reagent is used to detect FGFR2IIIb or its derivative protein.

In another preferred embodiment, the reagent, test plate or test kit is used to detect diseases associated with abnormal expression or function of FGFR2IIIb or its derivative proteins.

In another preferred embodiment, the reagent, test plate or kit is used to predict the risk of tumor or cancer.

In another preferred embodiment, the medicament is used for preventing and/or treating tumors or cancer.

In another preferred embodiment, the tumor includes solid tumors and blood cancers.

In another preferred embodiment, the tumor is a tumor that over-expresses FGFR2IIIb or its S252W mutant.

In another preferred embodiment, the tumor is selected from: gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid carcinoma, bile duct cancer, lung cancer, and non-small cell lung cancer.

In the sixth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (i) an active ingredient, the active ingredient being selected from the following group: an antibody as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, an immune cell as described in the fourth aspect of the present invention, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In another preferred example, the drug composition comprises 0.01 to 99.99% of the antibody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the immune cell as described in the fourth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentages are the mass percentages of the drug composition.

In another preferred embodiment, the pharmaceutical composition is used for preventing and/or treating tumors or cancer.

In the seventh aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes a polypeptide selected from the following groups: (1)  The antibody as described in the first aspect of the present invention; (2)  The recombinant protein as described in the second aspect of the present invention; or (3)  The chimeric antigen receptor (CAR) construct as described in the third aspect of the present invention.

In the eighth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide as described in the seventh aspect of the present invention.

In another preferred embodiment, the vector includes: bacterioplasm, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus.

In another preferred embodiment, the vector is an adenovirus, a retrovirus, or other vectors.

In the ninth aspect of the present invention, an engineered host cell is provided, wherein the host cell contains the vector as described in the eighth aspect of the present invention or the polynucleotide as described in the seventh aspect of the present invention is integrated into its genome.

In the tenth aspect of the present invention, a method for detecting FGFR2Ⅲb or a protein derived therefrom in a sample is provided, the method comprising the steps of: (1) contacting the sample with the antibody as described in the first aspect of the present invention; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of FGFR2Ⅲb or a protein derived therefrom in the sample.

In another preferred embodiment, the detection is for in vitro non-therapeutic and non-diagnostic purposes.

In the eleventh aspect of the present invention, a composition for detecting FGFR2IIIb or its derivative protein in an in vitro sample is provided, which comprises the antibody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the immune cell as described in the fourth aspect of the present invention or a combination thereof as an active ingredient.

In the twelfth aspect of the present invention, a test plate is provided, comprising: a substrate (support plate) and a test strip, wherein the test strip contains the antibody as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, or a combination thereof.

In the thirteenth aspect of the present invention, a reagent kit is provided, the reagent kit comprising: (1) a first container, the first container containing the antibody as described in the first aspect of the present invention; and/or (2) a second container, the second container containing a secondary antibody against the antibody as described in the first aspect of the present invention; or, the reagent kit contains the detection plate as described in the twelfth aspect of the present invention.

In the fourteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising: (a) culturing the host cell as described in the ninth aspect of the present invention under conditions suitable for expression; (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody as described in the first aspect of the present invention or the recombinant protein as described in the second aspect of the present invention.

In the fifteenth aspect of the present invention, there is provided the use of the antibody as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the immune cell as described in the fourth aspect of the present invention and/or the drug composition as described in the sixth aspect of the present invention in the preparation of a drug for treating a disease associated with abnormal FGFR2Ⅲb expression or function.

In another preferred embodiment, the abnormal expression of FGFR2IIIb refers to overexpression of FGFR2b.

In another preferred embodiment, the overexpression refers to a ratio of the expression level of FGFR2IIIb (F1) to the expression level under physiological conditions (F0) (ie, F1/F0) of ≥1.5, preferably ≥2, and more preferably ≥2.5.

In another preferred embodiment, the medicament is used for preventing and/or treating tumors.

In another preferred embodiment, the medicament is used to prevent and/or treat tumor occurrence, growth and/or metastasis.

In another preferred embodiment, the tumor includes solid tumors and blood cancers.

In another preferred embodiment, the tumor is a tumor that over-expresses FGFR2IIIb or its S252W mutant.

In another preferred embodiment, the tumor is selected from: gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid carcinoma, bile duct cancer, lung cancer, and non-small cell lung cancer.

In the sixteenth aspect of the present invention, a method for treating a disease associated with abnormal expression or function of FGFR2Ⅲb or its derivative protein is provided, which comprises administering to a subject in need thereof an effective amount of the antibody as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the immune cell as described in the fourth aspect of the present invention, or the drug composition as described in the sixth aspect of the present invention, or a combination thereof.

In another preferred embodiment, the disease associated with abnormal expression or function of FGFR2IIIb or its derivative protein is a tumor or cancer, preferably gastric cancer.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions.

After extensive and in-depth research, the inventors have obtained a class of monoclonal antibodies against FGFR2Ⅲb. The antibodies of the present invention can specifically bind to FGFR2Ⅲb, have high affinity and can effectively block the binding of FGFR2Ⅲb to its ligands FGF1, FGF7 and FGF10. The antibodies of the present invention have excellent binding activity to FGFR2Ⅲb from various species. In addition, the antibodies of the present invention can also mediate the specific cytotoxicity of FGFR2Ⅲb-overexpressing cancer cells by immune cells through ADCC. In vivo experiments have shown that it has a significant tumor-suppressing effect in a gastric cancer-bearing mouse model. On this basis, the present invention was completed. Terminology

In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be varied. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the scope of the attached patent application.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. As used herein, the term "about" when used in reference to a specific enumerated numerical value means that the value may vary by no more than 1% from the enumerated value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including an antibody against FGFR2IIIb of the present invention and a composition thereof, to a patient who has one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the amount of the therapeutic agent that is effective in alleviating one or more disease symptoms (therapeutically effective amount) is administered to the patient.

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may occur but does not have to occur.

"Sequence identity" as used herein refers to the degree of identity between two nucleic acid sequences or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions or deletions. The sequence identity between the sequences described herein and sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. FGFR2IIIb

FGFR2Ⅲb is a transmembrane protein encoded by the FGFR2 gene (Fibroblast Growth Factor Receptor 2), with the NCBI number NP_075259. The protein consists of an extracellular domain, a hydrophobic single transmembrane helical structure, and a tyrosine kinase domain. The extracellular domain contains three relatively independent immunoglobulin-like domains (D1-D3). The ligand binds to the D2 domain, and the D3 domain is responsible for regulating the binding activity of the receptor protein and the ligand. The FGFR2 gene exhibits variable splicing in different tissues, and the D3 domain has two different splicing isomers. When epithelial cells express D3, they select exon 7 and exon 8 to form FGFR2Ⅲb protein, and when mesenchymal cells express D3, they select exon 7 and exon 9 to form FGFR2Ⅲc protein.

There are multiple pathogenic mutations in the FGFR2 gene in somatic cells and cancer cells, among which S252W (amino acid mutation from serine to tryptophan at position 252) is the most common and widely studied mutation site. The S252 site is located in exon 7, between the D2 domain and the D3 domain. When it mutates to tryptophan, it changes the affinity between the ligand and the receptor, promotes ligand-independent FGFR2 dimerization and constitutive activation of FGFR2 kinase, and induces tumors or other diseases. Endometrioid adenocarcinoma is the most common tumor type with FGFR2 S252W mutations. 12.52% of patients have FGFR2 mutations, and FGFR2 S252W mutations account for 4.71% of all endometrioid adenocarcinoma patients. Apert syndrome is one of the most serious types of human craniosynostosis and is an autosomal dominant genetic disease. Two-thirds of patients have FGFR2 S252W mutations. Antibodies

As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. At one end of each heavy chain is a variable region (VH), followed by multiple constant regions. At one end of each light chain is a variable region (VL) and at the other end is a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form the interface between the light and heavy chain variable regions.

As used herein, the term "variable" means that certain parts of the variable region of an antibody differ in sequence, which contributes to the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-fold configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-fold structure. The CDRs in each chain are closely connected through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit various effector functions, such as participating in the antibody-dependent cellular toxicity.

The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those in the art.

As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a class of essentially homogeneous populations, i.e., the individual antibodies contained in the population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized through fusion tumor culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a essentially homogeneous antibody population, and should not be interpreted as requiring any special method to produce the antibody.

Generally, the antigen binding properties of antibodies can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs), which are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, which is close to each other in space through the β-fold formed by the FRs in between. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge on the chain region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody. The Fv antibody contains the antibody heavy chain variable region, light chain variable region, but no constant region, and is the smallest antibody fragment that has all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the required structure for antigen binding.

The present invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity or fusion proteins formed by antibodies and other sequences, such as Fab or (Fab') ₂ fragments; antibody heavy chains; antibody light chains. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. An epitope may be a non-continuous three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the present invention.

The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of less than about ^10-7 M, such as less than about ^10-8 M, ^10-9 M or ^10-10 M or less.

In the present invention, antibodies include mouse, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombination techniques, and they are all useful antibodies. Chimeric antibodies are molecules in which different parts are from different animal species, such as chimeric antibodies with variable regions from mouse monoclonal antibodies and constant regions from human immunoglobulins (see, for example, U.S. Patent Nos. 4,816,567 and 4,816,397, which are hereby incorporated by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species, having one or more complementary determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant technology well known in the art.

In the present invention, the antibody can be monospecific, bispecific, trispecific, or more specific.

As used herein, the terms "recombinant variable region" and "VH" are used interchangeably.

As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

The term "CDR" refers to the hypervariable region within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of CDR is provided by Kabat E.A et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). In addition, IMGT (Lefranc, 2003) and Chothia (Al-Lazikani, 1997) both provide CDR definition rules, which are well known to those skilled in the art.

In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and a light chain constant region, and the light chain constant region can be of mouse or human origin.

In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A. Table A The initial residue Representative replacement Preferred replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu

The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" as used herein includes cell-mediated reactions in which non-specific cytotoxic cells expressing FcγRs recognize bound antibodies on target cells, causing lysis of the target cells. In various aspects, enhancing ADCC effector function can mean enhanced potency or enhanced efficacy. "Potency" as used in an experimental context means the concentration of the antibody when a specific therapeutic efficacy _EC50 is observed (half-maximal effective concentration). "Efficacy" as used in an experimental context means the maximum possible effector function of the antibody at a saturation level. Anti- FGFR2IIIb Antibodies

As used herein, the terms "antibody of the present invention", "anti-FGFR2IIIb antibody of the present invention" and "FGFR2IIIb antibody of the present invention" are used interchangeably and all refer to the antibody against FGFR2IIIb and its derivative protein (S252W mutant) described in the first aspect of the present invention.

The function of the antibody of the present invention is determined by the specific gene sequence of the variable region gene of the light chain and heavy chain of the antibody. The antibody of the present invention can specifically bind to FGFR2IIIb, has high affinity and can effectively block the binding of FGFR2IIIb with its ligands FGF1, FGF7 and FGF10. Using the variable region gene or complementary determining region (CDR) gene of the antibody of the present invention, different forms of genetically engineered antibodies can be transformed and produced in any expression system using prokaryotic and eukaryotic cells.

The present invention provides an anti-FGFR2Ⅲb antibody, the antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementary determining region (CDR) selected from the following group: (1) VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 7, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (2) VH-CDR1 shown in SEQ ID NO: 15, VH-CDR2 shown in SEQ ID NO: 16, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (3) VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 19, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (4) NO:15, VH-CDR1, VH-CDR2, and VH-CDR3, SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule; (5)  VH-CDR1, VH-CDR2, and VH-CDR3, SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule; and (6)  VH-CDR1, VH-CDR2, and VH-CDR3, SEQ ID NO:9, wherein the CDRs are defined according to the IMGT rule; and the variable region of the light chain has a complementary determining region (CDR) selected from the following groups: (1)  VL-CDR1, VL-CDR2, and VL-CDR3, SEQ ID NO:12, wherein the CDRs are defined according to the Kabat rule; (2)  VL-CDR1 shown in SEQ ID NO:17, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:14, said CDRs are defined according to the Kabat rule; (3)  VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, said CDRs are defined according to the Kabat rule; and (4)  VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, said CDRs are defined according to the IMGT rule. Furthermore, the amino acid sequence also includes a sequence formed by adding, deleting, modifying and/or replacing at least one amino acid sequence, preferably an amino acid sequence with a homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

Methods for determining sequence homology or identity that are well known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton, NY. Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Preferably, the antibody described herein is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody (sdAb) and a single-domain antibody (Signle-domain antibody), and a monoclonal antibody or a multiclonal antibody prepared from the above antibodies. The monoclonal antibody can be developed by a variety of approaches and technologies, including fusion tumor technology, phage display technology, single lymphocyte gene selection technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or gene-transfected mice through fusion tumor technology.

The full-length antibody protein is a conventional full-length antibody protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, or a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably a fully humanized antibody.

The antibody derivatives of the present invention may be single-chain antibodies and/or antibody fragments, such as Fab, Fab', (Fab') ₂ or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

Wherein, the animal is preferably a mammal, such as a mouse.

The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and/or modified antibody targeting FGFR2IIIb (eg, human FGFR2IIIb, mouse FGFR2IIIb or cynomolgus monkey FGFR2IIIb).

In the above content of the present invention, the amount of the added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total amount of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

In the above content of the present invention, more preferably, the number of amino acids added, deleted, modified and/or substituted can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2. Recombinant protein

The present invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2) and heavy chain CDR3 (VH-CDR3) of the antibody of the present invention, and/or one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2) and light chain CDR3 (VL-CDR3) of the antibody of the present invention.

Preferably, the recombinant protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region, the antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.

In another preferred embodiment, the recombinant protein comprises the antibody of the present invention.

The recombinant protein is a conventional protein in the art, preferably, it is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (scFv), a single domain antibody (sdAb) and a single-domain antibody (Signle-domain antibody), as well as a single antibody or multiple antibodies prepared from the above antibodies.

The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.

The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, which includes a light chain variable region, a light chain constant region and an Fd segment of a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

The single-domain antibody is a conventional single-domain antibody in the art, which only includes a heavy chain variable region.

Among them, the preparation method of the recombinant protein is a conventional preparation method in the field. The preparation method is preferably: isolating from a transformant strain that recombinantly expresses the protein or obtaining through artificial synthesis of the protein sequence. The preferred method for isolating from a transformant strain that recombinantly expresses the protein is as follows: a polynucleotide molecule encoding the protein and having a point mutation is cloned into a recombinant vector, the obtained recombinant vector is transformed into a transformant strain to obtain a recombinant expression transformant strain, and the recombinant protein can be isolated and purified by culturing the obtained recombinant expression transformant strain. Polynucleotide

The present invention also provides a polynucleotide encoding the above-mentioned antibody or recombinant protein of the present invention or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

The method for preparing the polynucleotide is a conventional method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above protein by gene selection technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

Those skilled in the art know that the base sequence of the amino acid sequence encoding the above-mentioned protein can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

The present invention also provides a recombinant expression vector comprising the polynucleotide.

The recombinant expression vector can be obtained by conventional methods in the art, that is, by linking the polynucleotide molecules described in the present invention to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned polynucleotide molecules. The vector preferably includes: various plastids, plastids, phages or virus vectors, etc.

The present invention also provides a recombinant expression transformant strain comprising the above recombinant expression vector.

Wherein, the preparation method of the recombinant expression transformant strain is a conventional preparation method in the art, preferably: the above-mentioned recombinant expression vector is transformed into a host cell to obtain the same. The host cell is any conventional host cell in the art, as long as the above-mentioned recombinant expression vector can stably replicate itself and the polynucleotide carried can be effectively expressed. Preferably, the host cell is E. coli TG1 or E. coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody). By transforming the aforementioned recombinant expression plasmid into a host cell, the preferred recombinant expression transformant strain of the present invention can be obtained. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method. Preparation of Antibodies

The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic sequence library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by selecting it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Usually, a long sequence fragment can be obtained by synthesizing multiple small fragments first and then connecting them.

At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely through chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.

The present invention also relates to a vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell; or a lower eukaryotic cell; or a higher eukaryotic cell, such as a mammalian cell.

Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional separation and purification methods well known to those skilled in the art to obtain the antibodies of the present invention.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

The antibodies of the present invention can be expressed inside cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Application

The present invention also provides uses of the antibodies, recombinant proteins, chimeric antigen receptor (CAR) constructs and/or immune cells of the present invention, for example, for preparing diagnostic preparations or preparing drugs.

Preferably, the drug is a drug for preventing and/or treating a disease associated with abnormal expression or function of FGFR2IIIb.

In the present invention, the disease associated with abnormal expression or function of FGFR2IIIb is a conventional disease associated with abnormal expression or function of FGFR2IIIb in the art. Preferably, the disease associated with abnormal expression or function of FGFR2IIIb is cancer.

In the present invention, the cancer is a conventional cancer in the art, preferably gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid cancer, bile duct cancer, lung cancer, and non-small cell lung cancer. Detection Use and Reagent Kit

The antibodies of the present invention can be used in detection applications, such as for detecting samples to provide diagnostic information.

In the present invention, the specimens (samples) used include cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, tumor resection specimens, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

Samples used in the present invention include fixed or preserved cell or tissue samples.

The present invention also provides a reagent kit containing the antibody (or its fragment) of the present invention. In a preferred embodiment of the present invention, the reagent kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed on a test plate. Drug composition

The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or corresponding immune cells, and a pharmaceutically acceptable carrier.

The antibody of the present invention may also be expressed in cells by nucleotide sequences and used for cell therapy.

The drug composition of the present invention is a drug composition for preventing and/or treating diseases associated with abnormal expression or function of FGFR2Ⅲb or its derivative protein.

The drug composition of the present invention contains a safe and effective amount of the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier or formulation. The drug formulation should match the administration method. The dosage of the active ingredient is a therapeutically effective amount. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

In one embodiment of the present invention, the polypeptide of the present invention can be used in combination with other therapeutic agents for treating and/or preventing tumors.

In the present invention, preferably, the drug composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable drug adjuvant. The drug adjuvant is a conventional drug adjuvant in the art, and preferably comprises a pharmaceutically acceptable excipient, filler or diluent, etc.

In the present invention, preferably, the amount of the drug composition administered is an effective amount, which is an amount that can alleviate or delay the progression of a disease, degenerative or destructive condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. A person skilled in the art can determine the effective amount by using the above factors on an individual basis and using no more than routine experiments.

The present invention provides the use of the above-mentioned drug composition in the preparation of a drug for preventing and/or treating a disease associated with abnormal expression or function of FGFR2Ⅲb . Preferably, the disease associated with abnormal expression or function of FGFR2Ⅲb is cancer. More preferably, the disease associated with abnormal expression or function of FGFR2Ⅲb is gastric cancer. Method and composition for detecting FGFR2Ⅲb in a sample

The present invention also provides a method for detecting FGFR2Ⅲb in a sample (e.g., detecting cells expressing FGFR2Ⅲb), comprising the following steps: contacting the above-mentioned antibody with the sample to be tested in vitro, and detecting whether the above-mentioned antibody and the sample to be tested bind to form an antigen-antibody complex.

The meaning of overexpression is common in the art, and refers to the overexpression of FGFR2Ⅲb RNA or protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and altered protein degradation), as well as local overexpression and increased functional activity (such as in the case of increased enzymatic hydrolysis of the substrate) due to changes in protein transport patterns (increased cell membrane localization).

In the present invention, the detection method of whether the above-mentioned binding forms an antigen-antibody complex is a conventional detection method in the art, preferably a flow cytometry (FACS) detection.

The present invention provides a composition for detecting FGFR2IIIb in a sample, which comprises the above-mentioned antibody, recombinant protein, immune cell, or a combination thereof as an active ingredient. Preferably, it also comprises a compound composed of a functional fragment of the above-mentioned antibody as an active ingredient.

The main advantages of the present invention include: 1) The anti-FGFR2Ⅲb antibody of the present invention can bind to FGFR2Ⅲb with high affinity and can effectively block the binding of FGFR2Ⅲb to its ligands FGF1, FGF7 and FGF10. 2) The antibody of the present invention has excellent binding activity to FGFR2Ⅲb from various species, and at the same time has excellent specificity, and basically does not bind to FGFR2Ⅲc; the antibody of the present invention has better selectivity for FGFR2Ⅲb relative to FGFR2Ⅲc. 3) The antibody of the present invention can mediate the specific toxicity of immune cells to FGFR2Ⅲb-overexpressing cancer cells through ADCC. 4) The antibody of the present invention has a significant tumor-suppressing effect in a tumor-bearing mouse model, and the antibody of the present invention has a significant tumor-suppressing effect at a low dose.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. Example 1 : Acquisition of anti-human FGFR2 III b antibodies

The present invention uses FGFR2Ⅲb (D2+D3) (SEQ ID NO: 1) as an antigen to immunize female Balb/c mice aged 6-8 weeks. The spleen cells of the immunized mice are fused with SP2/0-AG14 cells, and an appropriate amount of the fused cells are plated and cultured in a 96-well plate. The supernatant of each well is taken on the 7th to 10th day after fusion, and the binding activity of the mouse antibodies secreted by the fusion tumor cells to human FGFR2Ⅲb-his (ECD) (SEQ ID NO: 2) and FGFR2Ⅲc-his (ECD) (SEQ ID NO: 3) is detected by ELISA, and the inhibitory activity of the binding of FGF7/FGFR2Ⅲb and FGF10/FGFR2Ⅲb (FGF7 and FGF10 were purchased from Nearshore Protein, with the catalog numbers: CM88 and CR11, respectively). Finally, multiple fusion tumor cells that specifically bind to FGFR2Ⅲb-his (ECD) but not to FGFR2Ⅲc-his (ECD) were obtained. Among them, the fusion tumor cell with the best inhibition of FGF7/FGFR2Ⅲb and FGF10/FGFR2Ⅲb binding activity was 27E9A11, and its secreted antibody was obtained by sequencing. The antibody was named 27E9A11 antibody. The heavy chain variable region cDNA sequence and light chain variable region cDNA sequence corresponding to the 27E9A11 antibody, the heavy chain variable region sequence encoded by it is shown in SEQ ID NO: 4; the light chain variable region sequence encoded by it is shown in SEQ ID NO: 5. The antigen complementation determining cluster (CDR) sequence of the 27E9A11 antibody is shown in Table 1. Table 1 Antigenic complement determinants (CDRs) of mouse antibody 27E9A11 Coding rules VHCDR1 VHCDR2 VHCDR3 Kabat NYNMH (SEQ ID NO:6) AIYPGNGDTSYNQKFKG (SEQ ID NO:7) GDFGY (SEQ ID NO:8) IMGT GYTFANYN (SEQ ID NO:9) IYPGNGDT (SEQ ID NO: 10) ARGDFGY (SEQ ID NO: 11) VLCDR1 VLCDR2 VLCDR3 Kabat KASQDVNIDVA (SEQ ID NO: 12) SASYRYT (SEQ ID NO:13) QQHYSTPYT (SEQ ID NO: 14) IMGT QDVNID (SEQ ID NO:40) SAS QQHYSTPYT (SEQ ID NO: 14) Binding activity detection of 27E9A11 antibody and human FGFR2 Ⅲ b protein

A 96-well high affinity plate was coated with a 1 μg/mL human FGFR2Ⅲb-his protein solution at 100 μL/well (100 microliters per well) and shaken overnight at 4°C. The next day, the plate was washed three times with 300 μL PBST (Tween20: 0.5‰), then blocked with 100 μL/well 5% BSA/PBS for 2 hours and shaken at room temperature. Washed three times with 300 μL PBST. A gradient dilution solution of the 27E9A11 antibody sample was prepared with PBS. The solution was added to a 96-well plate at 100 μL/well and shaken at room temperature for 1 hour. Washed three times with 300 μL PBST. Prepare secondary antibody goat anti-mouse IgG HRP solution (Thermo Fisher, catalog number A16090, the same below), add 100 μL/well to a 96-well plate, and shake at room temperature for 30 minutes. Wash 4 times with 300 μL PBST. Add 100 μL/well TMB (tetramethylbenzidine) and color for 3 minutes. Add 100 μL/well 0.6NH ₂ SO ₄ to stop color development and detect OD ₄₅₀ nm. 27E9A11 antibody blocks the binding of human FGF factor (FGF7 or FGF10) to human FGFR2 Ⅲ b protein

1 μg/mL human FGF and 5 μg/mL sodium heparin solution (Sino-pharmaceuticals, catalog number 63007131, the same below) were coated on a 96-well high affinity plate at 100 μL/well and shaken overnight at 4°C. The next day, the plate was washed three times with 300 μL PBST (Tween20: 0.5‰), then blocked with 100 μL/well 5% BSA/PBS for 2 hours and shaken at room temperature. 300 μL PBST was washed three times. A gradient dilution solution of the 27E9A11 antibody sample was prepared with PBS, and 1 μg/mL FGFR2Ⅲb-huFc was added at a ratio of 1:1 to pre-mix. 100 μL/well was added to the 96-well plate and shaken at room temperature for 2 hours. Wash 3 times with 300 μL PBST. Prepare secondary antibody goat anti-human IgG HRP solution (Abcam, catalog number ab6858, the same below), add 100 μL/well to the 96-well plate, and shake at room temperature for 1 hour. Wash 4 times with 300 μL PBST. Add 100 μL/well TMB (tetramethylbenzidine) and color for 5 min. Add 100 μL/well 0.6NH ₂ SO ₄ to stop color development and detect OD ₄₅₀ nm.

Results: The binding activity of 27E9A11 antibody to FGFR2Ⅲb-his is shown in Figure 1, and the EC ₅₀ is shown in Table 2; the inhibitory activity of 27E9A11 antibody on FGF7/FGFR2Ⅲb binding is shown in Figure 2, and the IC ₅₀ is shown in Table 2; the inhibitory activity of 27E9A11 antibody on FGF10/FGFR2Ⅲb binding is shown in Figure 3, and the IC ₅₀ is shown in Table 2. Table 2 Binding and blocking ability of antibody 27E9A11 antibody _EC50 (FGFR2b) IC ₅₀ (FGF7/FGFR2b) IC ₅₀ (FGF10/FGFR2b) 27E9A11 0.050 μg/mL 0.417 μg/mL 0.545 μg/mL Example 2 : Binding of chimeric antibody III-0 to human FGFR2 III b protein

The heavy chain variable region and light chain variable region of mouse antibody 27E9A11 were respectively linked to the constant region of the heavy chain and the constant region of the κ chain of human IgG1 to obtain human-mouse chimeric antibody III-0, whose heavy chain sequence is shown in SEQ ID NO: 22 and the light chain sequence is shown in SEQ ID NO: 23.

The binding activity of III-0 to human FGFR2Ⅲb protein was studied. The detection method is shown in Example 1 (the secondary antibody was changed to goat anti-human IgG HRP). The results are shown in Figure 4. III-0 can effectively bind to human FGFR2Ⅲb protein with an EC ₅₀ of 40.42 ng / mL. Example 3 : III-0 blocks the binding of human FGF factors (FGF1/FGF7/FGF10) to human FGFR2Ⅲb protein

1 μg/mL human FGF and 5 μg/mL sodium heparin solution were coated on a 96-well high affinity plate at 100 μL/well and shaken overnight at 4°C. The next day, the plate was washed three times with 300 μL PBST (Tween20: 0.5‰), then blocked with 100 μL/well 5% BSA/PBS for 2 hours and shaken at room temperature. 300 μL PBST was used to wash three times. A gradient dilution solution of the antibody sample was prepared with PBS, and 1 μg/mL FGFR2Ⅲb-mFc was added at a ratio of 1:1 to pre-mix. 100 μL/well was added to the 96-well plate and shaken at room temperature for 2 hours. 300 μL PBST was used to wash three times. Prepare secondary antibody goat anti-mouse IgG HRP solution, add 100 μL/well to 96-well plate, shake at room temperature for 1 hour. Wash 4 times with 300 μL PBST. Add 100 μL/well TMB (tetramethylbenzidine) and color for 5 min. Add 100 μL/well 0.6NH ₂ SO ₄ to stop color development, and detect OD ₄₅₀ nm.

The results are shown in Figure 5. III-0 can effectively block the binding of FGF1/7/10 to FGFR2Ⅲb. Its IC ₅₀ is shown in Table 3. Table 3 Blocking ability of antibody III-0 antibody IC ₅₀ (FGF1/FGFR2b) IC ₅₀ (FGF7/FGFR2b) IC ₅₀ (FGF10/FGFR2b) III-0 0.904 μg/mL 0.694 μg/mL 0.840 μg/mL Example 4 : Binding of III-0 to FGFR2 IIIb protein on SNU-16 cells

SNU-16 is a cell derived from human gastric cancer that naturally overexpresses human FGFR2Ⅲb protein. Digest and centrifuge SNU-16 cells, resuspend in PBS solution at a density of 2x10 ⁶ cells/mL, transfer 100 μL to a centrifuge tube after mixing, centrifuge and discard the supernatant. Prepare gradient dilution solutions of antibody samples with PBS. Add 100 μL to the centrifuge tube and shake at 4℃ for 1 hour. Centrifuge the cells, discard the supernatant, and rinse with 400 μL PBS by aspiration to mix, repeat 3 times. Prepare secondary antibody goat anti-human IgG (H+L) flow cytometry detection antibody (Thermo Fisher, catalog number A21091, the same below), add 100 μL/well to the centrifuge tube, and shake at 4℃ for 30 minutes. The cells were centrifuged at 2000 rpm for 3 minutes, the supernatant was discarded, and 400 μL PBS was used to mix, and the mixture was repeated twice. The cells were transferred to a flow cytometer for detection (Beckman, Cytoflex). The results are shown in Figure 6. III-0 can effectively identify and bind to SNU-16 cells. The EC ₅₀ of binding is shown in Table 4. Example 5 : Binding of chimeric antibodies to FGFR2 III b protein on CHO-FGFR2 III b cells

The inventors constructed a CHO-K1 cell line that overexpresses FGFR2Ⅲb protein. CHO-FGFR2Ⅲb cells were digested and centrifuged, and resuspended in PBS solution at a density of 2x10 ⁶ cells/mL. After mixing, 100 μL was transferred to a centrifuge tube, centrifuged and discarded the supernatant. A gradient dilution solution of the antibody sample was prepared with PBS. 100 μL was added to the centrifuge tube respectively, and shaken at 4°C for 1 hour. The cells were centrifuged, the supernatant was discarded, and 400 μL PBS was aspirated and mixed, and repeated 3 times. The secondary antibody goat anti-human IgG (H+L) flow cytometry detection antibody was prepared, and 100 μL/well was added to the centrifuge tube, and shaken at 4°C for 30 minutes. Centrifuge the cells at 2000 rpm for 3 minutes, discard the supernatant, and rinse with 400 μL PBS to mix, repeat twice. Transfer to flow cytometry (Beckman, Cytoflex). The results are shown in Figure 7. III-0 can effectively identify and bind to CHO-FGFR2Ⅲb cells. The EC ₅₀ of binding is shown in Table 4. Table 4 Binding ability of antibody III-0 to different cells antibody EC ₅₀ (SNU-16) _EC50 (CHO-FGFR2b) III-0 1.641 μg/mL 1.010 μg/mL Example 6 : Binding of humanized antibodies to human FGFR2 III b and FGFR2 III b (S252W) proteins

The chimeric antibody III-0 was humanized using the CDR transplantation method to obtain the humanized antibody III-10. Then, III-10 was subjected to affinity maturation. Specifically, each amino acid site in the CDR region of the III-10 antibody was subjected to single-point saturation mutation, and the mutation hotspots with the ability to specifically bind to the antigen were screened using the ELISA method, and then these hotspots were combined to obtain candidate antibody mutation sequences. The SPR method was used to detect the affinity of the candidate antibodies to the antigen, and finally 5 antibodies were obtained, namely III-11, III-12, III-13, III-14, and III-15. The sequences of these antibodies are shown in the table below. Table 5 Affinity-modified antibody variable region sequences (CDR sequences defined by the Kabat rule) Name of selected strain CDR1 (SEQ ID NO) CDR2 (SEQ ID NO) CDR3 (SEQ ID NO) Variable region (SEQ ID NO) Full length (SEQ ID NO) III-10 heavy chain NYNMH (6) AIYPGNGDTSYNQKFKG (7) GDFGY (8) twenty four 32 III-10 Light Chain KASQDVNIDVA (12) SASYRYT (13) QQHYSTPYT (14) 25 33 III-11 Heavy Chain IYNMH (15) AIYPDNGDTFYNQKFKG (16) GDFGY (8) 26 34 III-11 Light Chain KASQDVNIDPA (17) LASYRYT (18) QQHYSTPYT (14) 27 35 III-12 Heavy Chain NYNMH (6) AIYPDNGDTSYNQKFKG(19) GDFGY (8) 28 36 III-12 Light Chain KASQDVNIDVA (12) LASYRYT (18) QQTYSTPYT (20) 29 37 III-13 Heavy Chain IYNMH (15) AIYPDNGDTSYNQKFKG(19) GDFGY (8) 30 38 III-13 Light Chain KASQDVNIDVA (12) LASYRYT (18) QQTYSTPYT (20) 29 37 III-14 Heavy Chain IYNMH (15) AIYPGNGDTFYNQKFKG(21) GDFGY (8) 31 39 III-14 Light Chain KASQDVNIDVA (12) LASYRYT (18) QQTYSTPYT (20) 29 37 III-15 Heavy Chain IYNMH (15) AIYPDNGDTFYNQKFKG (16) GDFGY (8) 26 34 III-15 Light Chain KASQDVNIDVA (12) LASYRYT (18) QQTYSTPYT (20) 29 37

S252W is a common mutation of FGFR2Ⅲb on tumors. The binding activity of a series of humanized antibodies of III-0 to FGFR2Ⅲb and FGFR2Ⅲb(S252W) was tested. See Example 2 for the detection method. The detection results are shown in Figure 8. The binding activity of the humanized antibody of III-0 to human FGFR2Ⅲb and FGFR2Ⅲb(S252W) protein is equivalent to that of III-0. The EC ₅₀ of each antibody is shown in Table 6. Table 6 Binding ability of affinity modified antibodies to FGFR2 Ⅲ b/FGFR2 Ⅲ b (S252W) protein _EC50 (μg/mL) III-0 III-10 III-11 III-14 III-15 FGFR2IIIb 0.048 0.052 0.051 0.058 0.042 FGFR2IIIb(S252W) 0.050 0.055 0.056 0.066 0.048 Example 7 : Binding of anti-human FGFR2 III b antibody to human FGFR2 III c protein

The human FGFR2Ⅲc protein solution with a concentration of 1 μg/mL was coated on a 96-well high affinity plate at 100 μL/well and shaken overnight at 4°C. The next day, the plate was washed three times with 300 μL PBST (Tween20: 0.5‰), and then blocked with 100 μL/well of 5% BSA/PBS for 2 hours at room temperature. Washed three times with 300 μL PBST. A gradient dilution solution of the antibody sample was prepared with PBS. The solution was added to the 96-well plate at 100 μL/well and shaken at room temperature for 1 hour. Washed three times with 300 μL PBST. A secondary antibody goat anti-human IgG HRP solution was prepared and added to the 96-well plate at 100 μL/well and shaken at room temperature for 30 minutes. Wash 4 times with 300 μL PBST. Add 100 μL/well TMB (tetramethylbenzidine) and color develop for 3 minutes. Add 100 μL/well 0.6NH ₂ SO ₄ to stop color development and detect OD ₄₅₀ nm.

The test results are shown in Figure 9. III-0, III-10, and III-11 bind very weakly or not at all to human FGFR2Ⅲc protein. However, III-12, III-13, III-14, and III-15 have relatively weak binding activity to human FGFR2Ⅲc protein. This result confirms that the antibodies of the present invention have high specificity, can reduce unnecessary binding, reduce potential side effects, and improve drug safety in clinical applications. Example 8 : Humanized antibodies block the binding of human FGF factors (FGF1/FGF7/FGF10) to human FGFR2 Ⅲ b protein

The specific detection method is shown in Example 3. The detection results are shown in Figure 10. Both III-10 and III-0 can block FGF1/FGFR2Ⅲb binding. The activity of III-10 in blocking FGF10/FGFR2Ⅲb binding is equivalent to that of III-0. The IC ₅₀ is shown in Table 7. Table 7 Blocking ability of antibodies III-0 and III-10 antibody IC ₅₀ (FGF1/FGFR2b) IC ₅₀ (FGF7/FGFR2b) IC ₅₀ (FGF10/FGFR2b) III-0 0.692 μg/mL 0.603 μg/mL 0.745 μg/mL III-10 0.581 μg/mL 0.565 μg/mL 0.724 μg/mL Example 9 : Humanized Antibodies ( Affinity Maturation ) Block the Binding of Human FGF Factors ( FGF1 /FGF10) to Human FGFR2 IIIb Protein

The specific detection method is shown in Example 3. The detection results are shown in Figure 11. III-11, III-12, III-13, III-14, and III-15 can all block the binding of FGF1/10 to FGFR2Ⅲb, and the blocking ability is equivalent, as shown in Table 8. Table 8 Blocking ability of affinity matured antibodies antibody IC ₅₀ (FGF1/FGFR2b) IC ₅₀ (FGF10/FGFR2b) III-11 1.179 μg/mL 0.976 μg/mL III-12 1.083 μg/mL 0.999 μg/mL III-13 0.966 μg/mL 0.905 μg/mL III-14 0.974 μg/mL 0.937 μg/mL III-15 0.941 μg/mL 0.805 μg/mL Example 10 : Binding of humanized antibodies to FGFR2 III b protein on CHO-FGFR2 III b cells

The specific detection method is shown in Example 5. The results are shown in Figure 12. The activity of III-10, III-11, III-14, and III-15 in binding to CHO- FGFR2Ⅲb is equivalent to that of III-0. The EC ₅₀ of each antibody is shown in Table 9. Example 11 : Binding of humanized antibodies to FGFR2Ⅲb protein on SNU-16 cells

The specific detection method is shown in Example 4. The results are shown in Figure 13. The activity of III-10, III-11, III-14, and III-15 in binding to SNU-16 is equivalent to that of III-0. The EC ₅₀ of each antibody is shown in Table 9. Table 9 Binding ability of humanized antibodies to different cells antibody _EC50 (CHO-FGFR2b) EC ₅₀ (SNU-16) III-0 0.342 μg/mL 0.740 μg/mL III-10 0.257 μg/mL 0.471 μg/mL III-11 0.190 μg/mL 0.319 μg/mL III-14 0.263 μg/mL 0.526 μg/mL III-15 0.260 μg/mL 0.378 μg/mL Example 12 : ADCC effect of humanized antibodies

The inventors constructed a Jurkat-NFAT-Luc-CD16A stable expression cell line that expresses CD16 receptor and NFAT (Nuclear Factor of Activated T-cells) reaction elements. CHO-FGFR2Ⅲb was used as the target cell. CHO-FGFR2Ⅲb cells were digested and centrifuged, and resuspended in culture medium at a density of 1.3E+06 cells/mL. After mixing, 60 μL was transferred to a 384-well plate and cultured overnight. A gradient dilution solution of the antibody sample was prepared with complete culture medium. The supernatant was discarded from the 384-well plate, and 15 μL of the antibody solution was added to the plate and pre-incubated at 37°C for 1 hour. Resuspend Jurkat-NFAT-Luc-CD16A cells in complete medium, add 15 μL of cell suspension to each well, and place in a 37°C incubator for 4 hours. Prepare ONE-Glo™ Luciferase detection solution (Promega, catalog number E6110), add 30 μL/well to a 384-well plate, and react for 1-3 minutes. Transfer to a multifunctional enzyme immunoassay instrument (Tecan Spark 20M) for detection. The results are shown in Figure 14. The EC50 of each antibody is roughly the same. In terms of activation effect, each antibody can activate immune cells, among which III-14 has the strongest activation effect. Table 10 ADCC effect of humanized antibodies antibody _EC50 (μg/mL) E _max (RFU) III-10 0.383 1170 III-11 0.419 1082 III-12 0.396 1007 III-13 0.404 983 III-14 0.352 1433 III-15 0.388 749 Example 13 : Binding of humanized antibodies to mouse / cynomolgus monkey FGFR2 III b protein

Apply 1 μg/mL mouse/cynomolgus monkey FGFR2Ⅲb protein solution to a 96-well high affinity plate at 100 μL/well and shake overnight at 4°C. The next day, wash three times with 300 μL PBST (Tween20: 0.5‰), then block with 100 μL/well 5% BSA/PBS for 2 hours and shake at room temperature. Wash three times with 300 μL PBST. Prepare gradient dilution solution of antibody samples with PBS. Add 100 μL/well to the 96-well plate and shake at room temperature for 1 hour. Wash three times with 300 μL PBST. Prepare secondary antibody goat anti-human IgG HRP solution and add 100 μL/well to the 96-well plate and shake at room temperature for 30 minutes. Wash 4 times with 300 μL PBST. Add 100 μL/well TMB (tetramethylbenzidine) and color development for 3 minutes. Add 100 μL/well 0.6 NH ₂ SO ₄ to stop color development and detect OD ₄₅₀ nm.

The results are shown in Figure 15. III-10 can bind to mouse and cynomolgus monkey FGFR2IIIb. The EC ₅₀ is shown in Table 11. Table 11 Binding ability of humanized antibodies to mouse / cynomolgus monkey FGFR2IIIb antibody EC ₅₀ (mouse FGFR2b) EC ₅₀ (Cynomolgus FGFR2b) III-10 0.127 μg/mL 0.128 μg/mL Example 14 : Detection of Antibody Affinity

Surface plasmon resonance (SPR) was used to detect antigen-antibody affinity. Using FGFR2Ⅲb-his as the antigen, a certain concentration of antibody was incubated with a protein A sensor chip for antibody capture. In the antigen binding phase, gradient-diluted FGFR2Ⅲb-his protein was used as the mobile phase to bind to the antibody captured on the sensor chip. In the dissociation phase, HBS-EP buffer was used for continuous elution. The binding of the antibody to FGFR2Ⅲb-his on the sensor chip was quantitatively detected using Biacore 8k (GE Healthcare). The test results are shown in Table 12. The affinity of III-11, III-12, III-13, III-14, and III-15 to FGFR2Ⅲb-his is significantly higher than that of III-0 and III-10. Table 12 Affinity of anti-human FGFR2 Ⅲ b antibody to FGFR2 Ⅲ b-his Sample ka (1/Ms) kd (1/s) KD (M) III-0 1.06E+05 8.61E-03 8.13E-08 III-10 1.39E+05 9.44E-03 6.81E-08 III-11 1.82E+05 2.25E-04 1.24E-09 III-12 1.58E+05 2.09E-04 1.32E-09 III-13 1.41E+05 1.73E-04 1.23E-09 III-14 1.06E+05 1.33E-04 1.26E-09 III-15 1.49E+05 1.55E-04 1.04E-09 Example 15 : Efficacy study of III-10 in the mouse SNU-16 tumor model

For the SNU16 tumor model, SNU16 gastric cancer cells (5.0×10 ⁶ cells) in serum-free medium were inoculated subcutaneously into the right upper abdomen of SCID female mice. When the tumor volume reached 170 mm ³ (5 days after inoculation), the mice were randomly divided into groups (8 per group) and began to be intraperitoneally injected with III-10 antibody and human albumin control twice a week for 3 consecutive weeks. The injection concentration of III-10 antibody and human albumin control was 5 mg/kg. During this period, the tumor volume and mouse weight and tumor volume were measured and recorded every 2-3 days.

The results are shown in Figure 16. III-10 showed a significant ability to inhibit the growth of SNU16 tumors. Further experimental results showed that the antibody of the present invention can also significantly inhibit SNU16 tumors at low doses.

The sequences of the present invention are shown in Table 13 below. Table 13 Sequence Listing SEQ ID NO: sequence Name 1 RAPYWTNTEKMEKRLHAVPAANTVKFRCPAGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESVVPSDKGNYTCVVENEYGSINHTYHLDVVERSPHRPILQAGLPANAST VVGGDVEFVCKVYSDAQPHIQWIKHVEKNGSKYGPDGLPYLKVLKHSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQSAWLTVLPKQQAPGREKEITASPDYLEHHHHHHHH FGFR2Ⅲb (D2+D3) 2 RPSFSLVEDTTLEPEEPPTKYQISQPEVYVAAPGESLEVRCLLKDAAVISWTKDGVHLGPNNRTVLIGEYLQIKGATPRDSGLYACTASRTVDSETWYFMVNVTDAISSGDDEDDTDGAEDFVSENSNNKRAPYWTNTEKMEKRLHAVPAANTVKFRCPAGGNPMPTMRWLKNGKEFKQEHR IGGYKVRNQHWSLIMESVVPSDKGNYTCVVENEYGSINHTYHLDVVERSPHRPILQAGLPANASTVVGGDVEFVCKVYSDAQPHIQWIKHVEKNGSKYGPDGLPYLKVLKHSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQSAWLTVLPKQQAPGREKEITASPDYLEHHHHHHHH FGFR2Ⅲb-his (ECD) 3 RPSFSLVEDTTLEPEEPPTKYQISQPEVYVAAPGESLEVRCLLKDAAVISWTKDGVHLGPNNRTVLIGEYLQIKGATPRDSGLYACTASRTVDSETWYFMVNVTDAISSGDDEDDTDGAEDFVSENSNNKRAPYWTNTEKMEKRLHAVPAANTVKFRCPAGGNPMPTMRWLKNGKEFKQEHR IGGYKVRNQHWSLIMESVVPSDKGNYTCVVENEYGSINHTYHLDVVERSPHRPILQAGLPANASTVVGGDVEFVCKVYSDAQPHIQWIKHVEKNGSKYGPDGLPYLKVLKAAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFHSAWLTVLPAPGREKEITASPDYLEHHHHHHHH FGFR2Ⅲc-his (ECD) 4 QVQLQQPGAELVRPGASVKMSCKASGYTFANYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARGDFGYWGQGTTLTVSS 27E9A11 HCVR 5 DIVMTQSHKFMSTSVGDRVTITCKASQDVNIDVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPYTFGGGTKLEIK 27E9A11 LCVR 6 NYNMH 27E9A11 VHCDR1 (Kabat) 7 AIYPGNGDTSYNQKFKG 27E9A11 VHCDR2 (Kabat) 8 GDJ 27E9A11 VHCDR3 (Kabat) 9 GYTFANYN 27E9A11 VHCDR1 (IMGT) 10 IYPGNGDT 27E9A11 VHCDR2 (IMGT) 11 ARGDFGY 27E9A11 VHCDR3 (IMGT) 12 KASQDVNIDVA VLCDR1 (Kabat) 13 SASYRYT VLCDR2 (Kabat) 14 QQHYSTPYT VLCDR3 (Kabat/IMGT) 15 IYN VHCDR1 (Kabat) 16 AIYPDNGDTFYNQKFKG VHCDR2 (Kabat) 17 KASQDVNIDPA VLCDR1 (Kabat) 18 LASYRYT VLCDR2 (Kabat) 19 AIYPDNGDTSYNQKFKG VHCDR2 (Kabat) 20 QQTYSTPYT VLCDR3 (Kabat) twenty one AIYPGNGDTFYNQKFKG VHCDR2 (Kabat) twenty two QVQLQQPGAELVRPGASVKMSCKASGYTFANYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARGDFGYWGQGTTLT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK III-0 HC twenty three DIVMTQSHKFMSTSVGDRVTITCKASQDVNIDVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC III-0 LC twenty four QVQLVQSGAEVKKPGSSVKVSCKASGYTFANYNMHWVRQAPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVTVSS III-10 HCVR 25 DIVMTQSPSSSLSASVGDRVTITCKASQDVNIDVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYSTPYTFGQGTKLEIK III-10 LCVR 26 QVQLVQSGAEVKKPGSSVKVSCKASGYTFAIYNMHWVRQAPGQGLEWIGAIYPDNGDTFYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVTVSS III-11, III-15 HCVR 27 DIVMTQSPSSSLSASVGDRVTITCKASQDVNIDPAWYQQKPGKAPKLLIYLASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYSTPYTFGQGTKLEIK III-11 LCVR 28 QVQLVQSGAEVKKPGSSVKVSCKASGYTFANYNMHWVRQAPGQGLEWIGAIYPDNGDTSYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVTVSS III-12 HCVR 29 DIVMTQSPSSSLSASVGDRVTITCKASQDVNIDVAWYQQKPGKAPKLLIYLASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQTYSTPYTFGQGTKLEIK III-12, III-13, III-14, III-15 LCVR 30 QVQLVQSGAEVKKPGSSVKVSCKASGYTFAIYNMHWVRQAPGQGLEWIGAIYPDNGDTSYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVTVSS III-13 HCVR 31 QVQLVQSGAEVKKPGSSVKVSCKASGYTFAIYNMHWVRQAPGQGLEWIGAIYPGNGDTFYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVTVSS III-14 HCVR 32 QVQLVQSGAEVKKPGSSVKVSCKASGYTFANYNMHWVRQAPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK III-10 HC 33 DIVMTQSPSSSLSASVGDRVTITCKASQDVNIDVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYSTPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC III-10 LC 34 QVQLVQSGAEVKKPGSSVKVSCKASGYTFAIYNMHWVRQAPGQGLEWIGAIYPDNGDTFYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK III-11, III-15 HC 35 DIVMTQSPSSSLSASVGDRVTITCKASQDVNIDPAWYQQKPGKAPKLLIYLASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQHYSTPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC III-11 LC 36 QVQLVQSGAEVKKPGSSVKVSCKASGYTFANYNMHWVRQAPGQGLEWIGAIYPDNGDTSYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK III-12 HC 37 DIVMTQSPSSSLSASVGDRVTITCKASQDVNIDVAWYQQKPGKAPKLLIYLASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQTYSTPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC III-12, III-13, III-14, III-15 LC 38 QVQLVQSGAEVKKPGSSVKVSCKASGYTFAIYNMHWVRQAPGQGLEWIGAIYPDNGDTSYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK III-13 HC 39 QVQLVQSGAEVKKPGSSVKVSCKASGYTFAIYNMHWVRQAPGQGLEWIGAIYPGNGDTFYNQKFKGKATLTADKSTSTAYMELSSLRSEDTAVYYCARGDFGYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK III-14 HC 40 QDVNID 27E9A11 VLCDR1 (IMGT)

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teaching content of the present invention, a person skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the scope of the patent application attached to this application.

The following drawings are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the present invention as defined by the scope of the patent application.

Figure 1 shows the binding activity of 27E9A11 to FGFR2Ⅲb-his.

Figure 2 shows the inhibitory activity of 27E9A11 on FGF7/FGFR2Ⅲb binding.

Figure 3 shows the inhibitory activity of 27E9A11 on FGF10/FGFR2Ⅲb binding.

Figure 4 shows the binding activity of III-0 to human FGFR2Ⅲb protein.

FIG5 shows the activity of III-0 in blocking FGF1/FGFR2IIIb binding, the activity in blocking FGF7/FGFR2IIIb binding, and the activity in blocking FGF10/FGFR2IIIb binding.

FIG6 shows the activity of III-0 binding to SNU-16 cells.

FIG7 shows the activity of III-0 binding to CHO-FGFR2IIIb cells.

Figure 8 shows the binding activity of III-0, III-10, III-11, III-14, and III-15 to human FGFR2Ⅲb and FGFR2Ⅲb (S252W) proteins.

Figure 9 shows the binding of III-0, III-10, III-11, III-12, III-13, III-14, and III-15 to human FGFR2IIIc protein.

FIG10 shows the FGF1/FGFR2IIIb binding blocking activity, the FGF7/FGFR2IIIb binding blocking activity, and the FGF10/FGFR2IIIb binding blocking activity of III-0 and III-10.

FIG11 shows the activities of III-11, III-12, III-13, III-14, and III-15 in blocking FGF1/FGFR2IIIb binding and the activities of III-11, III-12, III-13, III-14, and III-15 in blocking FGF10/FGFR2IIIb binding.

Figure 12 shows the binding activity of III-0, III-10, III-11, III-14, and III-15 to CHO-FGFR2IIIb.

Figure 13 shows the activities of III-0, III-10, III-11, III-14, and III-15 binding to SNU-16.

Figure 14 shows the ADCC activities of III-10, III-11, III-12, III-13, III-14, and III-15.

Figure 15 shows the activity of III-10 binding to mouse and cynomolgus monkey FGFR2IIIb.

Figure 16 shows the ability of III-10 to inhibit SNU16 tumor growth.

TW202442685A_113115842_SEQL.xml

Claims (17)

An antibody against FGFR2Ⅲb, the antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementary determining region (CDR) selected from the following group: (1) VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 7, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (2) VH-CDR1 shown in SEQ ID NO: 15, VH-CDR2 shown in SEQ ID NO: 16, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (3) VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 19, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; (4) VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 19, and VH-CDR3 shown in SEQ ID NO: 8, wherein the CDRs are defined according to the Kabat rule; NO:15, VH-CDR1, VH-CDR2, and VH-CDR3, SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule; (5)  VH-CDR1, VH-CDR2, and VH-CDR3, SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule; and (6)  VH-CDR1, VH-CDR2, and VH-CDR3, SEQ ID NO:9, wherein the CDRs are defined according to the IMGT rule; and the variable region of the light chain has a complementary determining region (CDR) selected from the following groups: (1)  VL-CDR1, VL-CDR2, and VL-CDR3, SEQ ID NO:12, wherein the CDRs are defined according to the Kabat rule; NO:14, the CDRs are defined according to the Kabat rules; (2)  VL-CDR1 shown in SEQ ID NO:17, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:14, the CDRs are defined according to the Kabat rules; (3)  VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, the CDRs are defined according to the Kabat rules; and (4)  VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, the CDRs are defined according to the IMGT rules; Furthermore, any one of the amino acid sequences in the above CDR sequences also includes a derivative sequence that has been optionally supplemented, deleted, modified and/or substituted with 1-2 amino acids, and enables the derivative antibody composed of the heavy chain and light chain containing the derivative CDR sequence to retain the binding affinity of FGFR2Ⅲb or its derivative protein. The antibody as described in claim 1 is characterized in that the antibody has a heavy chain variable region CDR (VH-CDR) and a light chain variable region CDR (VL-CDR) selected from the following groups: (1) VH-CDR1 shown in SEQ ID NO: 6, VH-CDR2 shown in SEQ ID NO: 7, VH-CDR3 shown in SEQ ID NO: 8, VL-CDR1 shown in SEQ ID NO: 12, VL-CDR2 shown in SEQ ID NO: 13, and VL-CDR3 shown in SEQ ID NO: 14, wherein the CDRs are defined according to the Kabat rule; (2) VH-CDR1 shown in SEQ ID NO: 15, VH-CDR2 shown in SEQ ID NO: 16, VH-CDR3 shown in SEQ ID NO: 8, VL-CDR1 shown in SEQ ID NO: 17, VL-CDR2 shown in SEQ ID NO: 18, and VL-CDR3 shown in SEQ ID NO: 19. NO:14, the VL-CDR3, the CDRs are defined according to the Kabat rule; (3)  VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:19, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, the CDRs are defined according to the Kabat rule; (4)  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, the CDRs are defined according to the Kabat rule; (5)  SEQ ID :  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:21, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, said CDRs are defined according to the Kabat rule; (6)  VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, said CDRs are defined according to the Kabat rule; and (7)  VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, VH-CDR3 shown in SEQ ID NO:11, VL-CDR2 shown in SEQ ID NO:12, and VL-CDR3 shown in SEQ ID NO:20, said CDRs are defined according to the Kabat rule. VL-CDR1 shown in NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to the IMGT rules. The antibody as described in claim 1 is characterized in that the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 31 or 4, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity thereto; and/or the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 25, 27, 29 or 5, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity thereto. The antibody as described in claim 1 is characterized in that the antibody has: (1) a heavy chain variable region as shown in SEQ ID NO:24 and a light chain variable region as shown in SEQ ID NO:25; (2) a heavy chain variable region as shown in SEQ ID NO:26 and a light chain variable region as shown in SEQ ID NO:27; (3) a heavy chain variable region as shown in SEQ ID NO:28 and a light chain variable region as shown in SEQ ID NO:29; (4) a heavy chain variable region as shown in SEQ ID NO:30 and a light chain variable region as shown in SEQ ID NO:29; (5) a heavy chain variable region as shown in SEQ ID NO:31 and a light chain variable region as shown in SEQ ID NO:29; (6) a heavy chain variable region as shown in SEQ ID NO:26 and a light chain variable region as shown in SEQ ID NO:29; or (7)  The heavy chain variable region shown in SEQ ID NO:4 and the light chain variable region shown in SEQ ID NO:5. The antibody as described in claim 1 is characterized in that the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 32, 34, 36, 38, 39, or 22, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity thereto; and/or the amino acid sequence of the light chain is as shown in SEQ ID NO: 33, 35, 37 or 23, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity thereto. The antibody as described in claim 1 is characterized in that the antibody has: (1) a heavy chain as shown in SEQ ID NO:32 and a light chain as shown in SEQ ID NO:33; (2) a heavy chain as shown in SEQ ID NO:34 and a light chain as shown in SEQ ID NO:35; (3) a heavy chain as shown in SEQ ID NO:36 and a light chain as shown in SEQ ID NO:37; (4) a heavy chain as shown in SEQ ID NO:38 and a light chain as shown in SEQ ID NO:37; (5) a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:37; (6) a heavy chain as shown in SEQ ID NO:34 and a light chain as shown in SEQ ID NO:37; or (7) a heavy chain as shown in SEQ ID NO:22 and a light chain as shown in SEQ ID NO:23. A recombinant protein, comprising: (i) the antibody as described in claim 1; and (ii) an optional tag sequence that assists expression and/or purification. A chimeric antigen receptor (CAR) construct, wherein the antigen binding region of the chimeric antigen receptor construct includes a single-chain variable fragment (scFv) that specifically binds to FGFR2Ⅲb or its derivative protein, and the scFv has the heavy chain variable region and light chain variable region of the antibody as described in claim 1. A recombinant immune cell, wherein the immune cell expresses an exogenous CAR construct as described in claim 8. A pharmaceutical composition, characterized in that the pharmaceutical composition contains: (i) an active ingredient, wherein the active ingredient is selected from the following group: the antibody described in claim 1, the recombinant protein described in claim 7, the immune cell described in claim 9, or a combination thereof; and (ii) a pharmaceutically acceptable carrier. Use of the antibody described in claim 1, the recombinant protein described in claim 7, the immune cell described in claim 9, or the pharmaceutical composition described in claim 10 in the preparation of a drug for preventing and/or treating a disease. The use as described in claim 11 is characterized in that the disease is cancer. The use as described in claim 12 is characterized in that the cancer is selected from: gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid carcinoma, bile duct cancer, lung cancer, and non-small cell lung cancer. A polynucleotide, characterized in that the polynucleotide encodes a polypeptide selected from the following group: (1) the antibody as described in claim 1; (2) the recombinant protein as described in claim 7; or (3) the chimeric antigen receptor (CAR) construct as described in claim 8. A vector, characterized in that the vector contains the polynucleotide as described in claim 14. An engineered host cell, characterized in that the host cell contains the vector as described in claim 15 or the polynucleotide as described in claim 14 is integrated into its genome. A test plate, characterized in that the test plate comprises: a substrate and a test strip, wherein the test strip contains the antibody as described in claim 1, or the recombinant protein as described in claim 7, or a combination thereof.