Non-fucosylated Anti-FGFR2b monoclonal antibody

Abstract

The present invention provides a non-fucosylated anti-FGFR2-IIIb antibody. Specifically, the present invention provides a non-fucosylated anti-FGFR2-IIIb antibody, a method for expressing the non-fucosylated FGFR2-IIIb antibody by using FUT8 knockout cell lines, and an application of the antibody in the treatment of diseases. Compared with conventionally expressed antibodies, the non-fucosylated antibody of the present invention exhibits enhanced FcyRllla binding affinity. The antibody of the present invention can enhance ADCC effect, and has potential for applications in the field of oncotherapy.

Description

Fucosylated anti-FGFR2b monoclonal antibody

[0001] This application claims priority to Chinese Patent Application No. 202411516256.8, filed on October 28, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine. Specifically, this invention relates to a fucosylated anti-FGFR2IIIb monoclonal antibody. Background Technology

[0003] Fibroblast growth factor receptor (FGFR) belongs to a subfamily of the tyrosine kinase receptor superfamily. There are four FGFR genes in the human genome (FGFR1-4).

[0004] FGFR-mediated signaling pathways are essential for normal cell growth and differentiation, participating in physiological processes such as angiogenesis, cell proliferation and migration, regulation of organ development, and wound healing. However, mutations or overexpression of FGFR can lead to excessive activation of the FGFR signaling pathway, further inducing the transformation of normal cells into cancerous cells. Specifically, excessive activation of RAS-RAF-MAPK can stimulate cell proliferation and differentiation; excessive activation of PI3K-AKT inhibits apoptosis; SATA is closely related to promoting tumor invasion and metastasis and enhancing tumor immune escape capabilities; and the PLCγ signaling pathway is an important pathway for regulating tumor cell metastasis.

[0005] FGFRs contain three extracellular immunoglobulin-like domains (D1-D3), a hydrophobic single-transmembrane helical structure, and an intracellular tyrosine kinase domain, with an 8-residue acid cassette between D1 and D2. In FGFRs 1-3, ligand binding specificity largely depends on alternative splicing at the C-terminus of the D3 domain. There are two alternative splices at the C-terminus of the D3 domain, encoded by exon 8 or 9 to generate FGFRb or FGFRc isoforms. These b and c isoforms are typically confined to epithelial and mesenchymal tissues, respectively. In this way, selective splicing of the receptor allows the ligand to activate the receptor in adjacent mesenchymal or epithelial tissues without activating autocrine signals.

[0006] The primary ligands for the FGFR2IIIb (or FGFR2b) form of FGFR2 are FGF1, FGF7, FGF10, and FGF22. FGFR2b is highly expressed in tumors through FGFR2 gene amplification or transcriptional upregulation of FGFR2b subtypes, with FGFR2 amplification being the most common FGFR2 gene aberration. In immunohistochemical section analysis of gastric cancer patients, FGFR2b expression was detected in approximately 60% of tumor samples, with 2%–9% of these samples showing FGFR2b overexpression at levels ranging from 31% to 61%. These gastric cancer samples often exhibit diffuse overexpression and are associated with aggressive characteristics of gastric cancer, including higher-grade T stages, more frequent lymph node metastasis, and lower overall survival. Further research has established that overexpression of the FGFR2b receptor or FGFR2 gene amplification is directly related to poor prognosis in gastric cancer patients. Besides gastric cancer, aberrant activation of the FGF / FGFR2 signaling pathway has also been observed in other cancers, including but not limited to esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, lung cancer (e.g., non-small cell lung cancer), and cholangiocarcinoma. Therefore, inhibiting FGFR2 signaling may be an effective mechanism for treating various cancers.

[0007] Modifying the Fc domain of antibodies can enhance antibody-dependent cell-mediated cytotoxicity (ADCC), thereby increasing antitumor activity. Previous studies have found that mutations in the Fc domain can enhance the binding affinity of antibodies to Fc receptors, thus strengthening ADCC activity; for example, the S239D / A330L / I332E mutant can enhance cytotoxicity. Furthermore, alterations in the glycosylation of the Fc domain can also affect its effector function. It has been reported that a reduction in core fucosylation in antibodies alters the function of Fc effectors, particularly Fcγ receptor binding and ADCC activity.

[0008] Therefore, there is a need in this field to develop an anti-FGFR2IIIb monoclonal antibody with a stronger ADCC effect. Summary of the Invention

[0009] The purpose of this invention is to provide an anti-FGFR2IIIb monoclonal antibody with a strong ADCC effect.

[0010] In a first aspect of the invention, an antibody against FGFR2Ⅲb is provided, the antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementarity-determining region (CDR) selected from the group consisting of:

[0011] (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 CDR is defined according to the Kabat rule;

[0012] (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 rules;

[0013] (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 rules;

[0014] (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, wherein the CDRs are defined according to the Kabat rules;

[0015] (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, wherein the CDRs are defined according to the Kabat rules; and

[0016] (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, wherein the CDR is defined according to the IMGT rule;

[0017] Furthermore, the variable region of the light chain has a complementary determinant region (CDR) selected from the following group:

[0018] (1) 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 CDR is defined according to the Kabat rule;

[0019] (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, wherein the CDR is defined according to the Kabat rule;

[0020] (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, wherein the CDRs are defined according to the Kabat rules; and

[0021] (4) VL-CDR1 shown in SEQ ID 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;

[0022] Furthermore, any amino acid sequence in the above-mentioned CDR sequence also includes a derivative sequence which optionally involves the addition, deletion, modification and / or substitution of 1-2 amino acids, such that the derivative antibody composed of the heavy chain and light chain containing the derived CDR sequence can retain the binding affinity of FGFR2Ⅲb or its derivative protein.

[0023] The fucosylation ratio of the antibody is ≤10%, preferably ≤5%, ≤3%, ≤2%, or ≤1%.

[0024] In another preferred embodiment, the fucosylation rate of the antibody is 0%-0.1%.

[0025] In another preferred embodiment, the antibody does not have fucosylation.

[0026] In a first aspect of the invention, an antibody against FGFR2Ⅲb is provided, the antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementarity-determining region (CDR) selected from the group consisting of:

[0027] (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 CDR is defined according to the Kabat rule;

[0028] (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 rules;

[0029] (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 rules;

[0030] (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, wherein the CDRs are defined according to the Kabat rules;

[0031] (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, wherein the CDRs are defined according to the Kabat rules; and

[0032] (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, wherein the CDR is defined according to the IMGT rule;

[0033] Furthermore, the variable region of the light chain has a complementary determinant region (CDR) selected from the following group:

[0034] (1) 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 CDR is defined according to the Kabat rule;

[0035] (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, wherein the CDR is defined according to the Kabat rule;

[0036] (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, wherein the CDRs are defined according to the Kabat rules; and

[0037] (4) VL-CDR1 shown in SEQ ID 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;

[0038] The fucosylation ratio of the antibody is ≤10%, preferably ≤5%, ≤3%, ≤2%, or ≤1%.

[0039] In another preferred embodiment, the fucosylation rate of the antibody is 0%-0.1%.

[0040] In another preferred embodiment, the antibody does not have fucosylation.

[0041] 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 group consisting of:

[0042] (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 rules;

[0043] (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:14, wherein the CDRs are defined according to the Kabat rules;

[0044] (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, wherein the CDRs are defined according to the Kabat rules;

[0045] (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, wherein the CDRs are defined according to the Kabat rules;

[0046] (5) 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, wherein the CDRs are defined according to the Kabat rules; and

[0047] (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, wherein the CDRs are defined according to the Kabat rules; and

[0048] (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-CDR1 shown in SEQ ID 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;

[0049] The fucosylation ratio of the antibody is ≤10%, preferably ≤5%, ≤3%, ≤2%, or ≤1%.

[0050] In another preferred embodiment, the fucosylation rate of the antibody is 0%-0.1%.

[0051] In another preferred embodiment, the antibody does not have fucosylation.

[0052] In another preferred embodiment, the antibody specifically binds to FGFR2Ⅲb or a derivative thereof.

[0053] In another preferred embodiment, the antibody is capable of specifically binding to FGFR2Ⅲb derived from humans, mice, and cynomolgus monkeys.

[0054] In another preferred embodiment, the FGFR2Ⅲb is cell surface FGFR2Ⅲb or soluble FGFR2Ⅲb.

[0055] In another preferred embodiment, the NCBI accession number of the FGFR2Ⅲb is NP_075259.

[0056] In another preferred embodiment, the FGFR2Ⅲb-derived protein is an FGFR2Ⅲb S252W mutant.

[0057] In another preferred embodiment, the affinity KD value of the antibody binding to FGFR2Ⅲb is ≤1×10⁻⁶. -7 M, preferably ≤1×10 -8 M, preferably ≤5×10 -9 M.

[0058] In another preferred embodiment, the antibody blocks the binding of FGFR2Ⅲb or its derivative protein to FGF1, FGF7 or FGF10.

[0059] In another preferred embodiment, the blocking finger reduces the binding rate of the FGF1, FGF7 or FGF10 to FGFR2Ⅲb by 50%, preferably by 70%, and more preferably by 90%.

[0060] In another preferred embodiment, the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO:24, 26, 28, 30 or 31.

[0061] In another preferred embodiment, the heavy chain further includes a heavy chain constant region.

[0062] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0063] In another preferred embodiment, the variable region of the light chain has the amino acid sequence shown in SEQ ID NO:25, 27 or 29.

[0064] In another preferred embodiment, the light chain further includes a light chain constant region.

[0065] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0066] In another preferred embodiment, 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.

[0067] In another preferred embodiment, 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 sequences shown in SEQ ID NO: 24, 26, 28, 30, 31, or 4 in the sequence listing.

[0068] 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.

[0069] In another preferred embodiment, the antibody has:

[0070] (1) The heavy chain variable region as shown in SEQ ID NO:24 and the light chain variable region as shown in SEQ ID NO:25;

[0071] (2) The heavy chain variable region as shown in SEQ ID NO:26 and the light chain variable region as shown in SEQ ID NO:27;

[0072] (3) The heavy chain variable region as shown in SEQ ID NO:28 and the light chain variable region as shown in SEQ ID NO:29;

[0073] (4) The heavy chain variable region as shown in SEQ ID NO:30 and the light chain variable region as shown in SEQ ID NO:29;

[0074] (5) The heavy chain variable region as shown in SEQ ID NO:31 and the light chain variable region as shown in SEQ ID NO:29;

[0075] (6) the heavy chain variable region as shown in SEQ ID NO:26 and the light chain variable region as shown in SEQ ID NO:29; or

[0076] (7) The heavy chain variable region as shown in SEQ ID NO:4 and the light chain variable region as shown in SEQ ID NO:5.

[0077] In another preferred embodiment, the heavy chain variable region of the antibody further includes a human-derived frame region, and / or the light chain variable region of the antibody further includes a human-derived frame region.

[0078] In another preferred embodiment, the heavy chain variable region of the antibody further includes a mouse-derived frame region, and / or the light chain variable region of the antibody further includes a mouse-derived frame region.

[0079] In another preferred embodiment, the antibody is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or combinations thereof.

[0080] 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 sequences shown in SEQ ID NO: 32, 34, 36, 38, 39, or 22.

[0081] 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.

[0082] In another preferred embodiment, the antibody has:

[0083] (1) The heavy chain as shown in SEQ ID NO:32 and the light chain as shown in SEQ ID NO:33;

[0084] (2) The heavy chain as shown in SEQ ID NO:34 and the light chain as shown in SEQ ID NO:35;

[0085] (3) Heavy chains as shown in SEQ ID NO:36 and light chains as shown in SEQ ID NO:37;

[0086] (4) Heavy chains as shown in SEQ ID NO:38 and light chains as shown in SEQ ID NO:37;

[0087] (5) Heavy chains as shown in SEQ ID NO:39 and light chains as shown in SEQ ID NO:37;

[0088] (6) The heavy chain as shown in SEQ ID NO:34 and the light chain as shown in SEQ ID NO:37; or

[0089] (7) The heavy chain as shown in SEQ ID NO:22 and the light chain as shown in SEQ ID NO:23.

[0090] In another preferred embodiment, the fucosylation rate of the antibody is ≤0.5% or ≤0.1%.

[0091] In another preferred embodiment, the fucosylation ratio of the antibody is 0%-0.1%.

[0092] In another preferred embodiment, the antibody is generated in host cells with protein fucosylation defects.

[0093] In another preferred embodiment, the host cell is α-1,6-fucosyltransferase (FUT8) deficient.

[0094] In another preferred embodiment, the defects include gene knockout, reduced expression, and / or reduced activity.

[0095] In another preferred embodiment, the antibody is generated under conditions in which protein fucosylation is inhibited.

[0096] In another preferred embodiment, the condition inhibits the expression and / or activity of FUT8.

[0097] In another preferred embodiment, the conditions include the addition of an inhibitor of FUT8.

[0098] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0099] In another preferred embodiment, the host cell is Escherichia coli.

[0100] In another preferred embodiment, the cell is a mammalian cell.

[0101] In another preferred embodiment, the host cell is a CHO cell.

[0102] In another preferred embodiment, the host cell is a CHOK1 cell or a CHOS cell.

[0103] In a second aspect of the invention, a composition is provided comprising the antibody described in the first aspect of the invention, characterized in that at least 95% of the anti-FGFR2Ⅲb antibody in the composition is unfucosylated.

[0104] In a third aspect of the invention, a recombinant protein is provided, said recombinant protein comprising:

[0105] (i) the antibody as described in the first aspect of the invention; and

[0106] (ii) Optional tag sequences to assist in expression and / or purification.

[0107] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0108] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0109] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0110] In another preferred embodiment, the recombinant protein specifically targets FGFR2Ⅲb.

[0111] In another preferred embodiment, the recombinant protein is a fusion protein.

[0112] In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against FGFR2Ⅲb), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0113] In another preferred embodiment, the bispecific or multispecific antibody not only targets 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).

[0114] In a fourth aspect of the invention, a host cell is provided, wherein the host cell genome integrates a polynucleotide encoding an antibody as described in the first aspect of the invention or a recombinant protein as described in the second aspect of the invention, or the host cell contains a vector comprising the polynucleotide.

[0115] The host cells are engineered to lose or weaken the fucosylation modification function of intracellular proteins.

[0116] In another preferred embodiment, the host cell is FUT8 deficient.

[0117] In another preferred embodiment, the defects include gene knockout, reduced expression, and / or reduced activity.

[0118] In another preferred embodiment, the host cell is a CHO cell.

[0119] In another preferred embodiment, the host cell is a CHOK1 cell or a CHOS cell.

[0120] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, and mammalian cell viruses.

[0121] In another preferred embodiment, the vector is an adenovirus, a retrovirus, or another vector.

[0122] In a fifth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: antibodies as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, host cells as described in the third aspect of the invention, or combinations thereof, wherein the active ingredient is used for:

[0123] (a) Preparation of diagnostic reagents, test plates or kits; and / or

[0124] (b) To prepare medicines for the prevention and / or treatment of diseases associated with abnormal expression or function of FGFR2Ⅲb or its derivative proteins.

[0125] In another preferred embodiment, the reagent is used to detect FGFR2Ⅲb or its derivative protein.

[0126] In another preferred embodiment, the reagent, detection plate, or kit is used to detect diseases associated with abnormal expression or function of FGFR2Ⅲb or its derivative proteins.

[0127] In another preferred embodiment, the reagent, test plate, or kit is used to predict the risk of tumors or cancer.

[0128] In another preferred embodiment, the agent is used to prevent and / or treat tumors or cancer.

[0129] In another preferred embodiment, the tumor includes solid tumors and hematologic malignancies.

[0130] In another preferred embodiment, the tumor is a tumor that highly expresses FGFR2Ⅲb or its S252W mutant.

[0131] In another preferred embodiment, the tumor is selected from: breast cancer, gastric cancer, esophageal cancer, colorectal cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0132] In a sixth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0133] (i) an active ingredient selected from the group consisting of: antibodies as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, host cells or combinations thereof as described in the third aspect of the invention; and

[0134] (ii) Pharmaceutically acceptable carriers.

[0135] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0136] In another preferred embodiment, the pharmaceutical composition is an injection.

[0137] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of an antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a host cell as described in the third aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0138] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of tumors or cancer.

[0139] In a seventh aspect of the invention, a method for detecting FGFR2Ⅲb or a derivative protein thereof in a sample is provided, the method comprising the steps of:

[0140] (1) Contact the sample with the antibody as described in the first aspect of the present invention;

[0141] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of FGFR2Ⅲb or its derivative protein in the sample.

[0142] In another preferred embodiment, the detection is for in vitro, non-therapeutic, and non-diagnostic purposes.

[0143] In an eighth aspect of the invention, a composition for in vitro detection of FGFR2Ⅲb or its derivative protein in a sample is provided, comprising an antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, and immune cells or combinations thereof as active ingredients as described in the third aspect of the invention.

[0144] In a ninth aspect of the invention, a detection plate is provided, the detection plate comprising: a substrate (support plate) and a test strip, the test strip containing an antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, or a combination thereof.

[0145] In a tenth aspect of the invention, a kit is provided, the kit comprising:

[0146] (1) A first container containing an antibody as described in the first aspect of the invention; and / or

[0147] (2) A second container containing a secondary antibody against the antibody as described in the first aspect of the present invention;

[0148] or,

[0149] The kit contains a detection plate as described in the ninth aspect of the present invention.

[0150] In an eleventh aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising the steps of:

[0151] (a) Culture the host cells as described in the third aspect of the present invention under suitable expression conditions;

[0152] (b) Isolating a recombinant polypeptide from a culture, said recombinant polypeptide being an antibody as described in the first aspect of the invention or a recombinant protein as described in the second aspect of the invention.

[0153] In another preferred embodiment, in step (a), the host cells are cultured under conditions in which protein fucosylation is inhibited.

[0154] In another preferred embodiment, the condition inhibits the expression and / or activity of FUT8.

[0155] In another preferred embodiment, the conditions include the addition of an inhibitor of FUT8.

[0156] In a twelfth aspect of the invention, the use of an antibody as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or a host cell as described in the third aspect of the invention, and / or a pharmaceutical composition as described in the fifth aspect of the invention, in the preparation of a medicament for treating diseases associated with abnormal expression or function of FGFR2Ⅲb is provided.

[0157] In another preferred embodiment, the FGFR2Ⅲb expression abnormality refers to FGFR2Ⅲb overexpression.

[0158] In another preferred embodiment, the overexpression refers to the ratio of the expression level (F1) of FGFR2Ⅲb to the expression level (F0) under physiological conditions (i.e., F1 / F0) being ≥1.5, preferably ≥2, and more preferably ≥2.5.

[0159] In another preferred embodiment, the drug is used for the prevention and / or treatment of tumors.

[0160] In another preferred embodiment, the drug is used to prevent and / or treat tumor occurrence, growth and / or metastasis.

[0161] In another preferred embodiment, the tumor includes solid tumors and hematologic malignancies.

[0162] In another preferred embodiment, the tumor is a tumor that highly expresses FGFR2Ⅲb or its S252W mutant.

[0163] In another preferred embodiment, the tumor is selected from: breast cancer, gastric cancer, esophageal cancer, colorectal cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0164] In a thirteenth aspect of the invention, a method for treating a disease associated with abnormal expression or function of FGFR2Ⅲb or a derivative thereof is provided, comprising administering to a subject in need an effective amount of an antibody as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or a pharmaceutical composition as described in the fifth aspect of the invention, or a combination thereof.

[0165] 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, breast cancer, or a combination thereof.

[0166] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0167] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0168] Figure 1 shows the binding activity of 27E9A11 with FGFR2Ⅲb-his.

[0169] Figure 2 shows the inhibitory activity of 27E9A11 on the binding of FGF7 / FGFR2Ⅲb.

[0170] Figure 3 shows the inhibitory activity of 27E9A11 on the binding of FGF10 / FGFR2Ⅲb.

[0171] Figure 4 shows the binding activity of III-0 to human FGFR2Ⅲb protein.

[0172] Figure 5 shows the activity of III-0 in blocking the binding of FGF1 / FGFR2Ⅲb, the activity in blocking the binding of FGF7 / FGFR2Ⅲb, and the activity in blocking the binding of FGF10 / FGFR2Ⅲb.

[0173] Figure 6 shows the binding activity of III-0 to SNU-16 cells.

[0174] Figure 7 shows the binding activity of III-0 to CHO-FGFR2Ⅲb cells.

[0175] 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.

[0176] 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.

[0177] Figure 10 shows the activities of III-0 and III-10 in blocking the binding of FGF1 / FGFR2Ⅲb, the activities in blocking the binding of FGF7 / FGFR2Ⅲb, and the activities in blocking the binding of FGF10 / FGFR2Ⅲb.

[0178] Figure 11 shows the activities of III-11, III-12, III-13, III-14, and III-15 in blocking the binding of FGF1 / FGFR2Ⅲb, and in blocking the binding of FGF10 / FGFR2Ⅲb.

[0179] Figure 12 shows the binding activity of III-0, III-10, III-11, III-14, and III-15 to CHO-FGFR2Ⅲb.

[0180] Figure 13 shows the binding activity of III-0, III-10, III-11, III-14, III-15 with SNU-16.

[0181] Figure 14 shows the ADCC activity of III-10, III-11, III-12, III-13, III-14, and III-15.

[0182] Figure 15 shows the activity of FGFR2Ⅲb in III-10 binding mice and cynomolgus monkeys.

[0183] Figure 16 shows the ability of III-10 to inhibit the growth of SNU16 tumors.

[0184] Figure 17 shows the results of antibody glycoform analysis.

[0185] Figure 18 shows the ADCC activity of the fucosylated antibody C121.

[0186] Figure 19 shows the expression level of FGFR2IIIb protein in 4T1 cells.

[0187] Figure 20 shows a comparison of FGFR2IIIb protein expression levels between 4T1 cells and SNU-16 cells.

[0188] Figure 21 shows the ability of the fucosylated antibody C121 to inhibit 4T1 tumor growth in vivo. Figure 22 shows that C121 treatment does not affect the body weight of mice. Detailed Implementation

[0189] Through extensive and in-depth research, the inventors have obtained a class of fucosylated anti-FGFR2Ⅲb antibodies. This invention utilizes Fut8 knockout cells to produce fucosylated FGFR2Ⅲb antibodies. Testing showed that, compared to conventionally expressed antibodies, the fucosylated antibodies of this invention exhibit significantly enhanced FcγRIIIa (F176 / V176) binding affinity. Furthermore, the fucosylated antibodies of this invention can enhance ADCC effects and unexpectedly inhibit the growth of 4T1 breast cancer tumors with low FGFR2Ⅲb expression.

[0190] the term

[0191] To facilitate understanding of the invention, 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 one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0192] 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 this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0193] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0194] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, comprising the antibody against FGFR2Ⅲb of the present invention and compositions thereof, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount that effectively relieves the symptoms of one or more diseases (therapeutic effective amount).

[0195] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0196] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0197] FGFR2Ⅲb

[0198] The terms “FGFR2IIIb” or “FGFR2b” are used interchangeably in this application. FGFR2Ⅲb is a transmembrane protein encoded by the FGFR2 gene (Fibroblast Growth Factor Receptor 2), NCBI number NP_075259. This 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, while the D3 domain regulates the binding activity of the receptor protein and ligand. The FGFR2 gene exhibits alternative splicing in different tissues, with two different splice isoforms existing in the D3 domain. When epithelial cells express D3, exon 7 and exon 8 are selected to form the FGFR2IIIb protein; when mesenchymal cells express D3, exon 7 and exon 9 are selected to form the FGFR2IIIc protein.

[0199] The FGFR2 gene exists in multiple pathogenic mutations in somatic and cancer cells. The S252W mutation (the change from serine to tryptophan at amino acid position 252) is the most common and has been extensively studied. Located in exon 7, between the D2 and D3 domains, the S252 site alters the affinity between the ligand and receptor, promoting ligand-independent FGFR2 dimerization and constitutive activation of FGFR2 kinase, thus inducing tumors or other diseases. Endometrioid adenocarcinoma is the most frequently detected tumor type with the FGFR2 S252W mutation, accounting for 12.52% of patients in this category, while the FGFR2 S252W mutation accounts for 4.71% of all endometrioid adenocarcinoma patients. Apert syndrome is one of the most severe types of cranial syndromes in humans, an autosomal dominant inherited disease, with two-thirds of patients exhibiting the FGFR2 S252W mutation.

[0200] Antibody

[0201] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 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 the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0202] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions 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)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0203] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0204] As used herein, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a largely homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which typically contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized through hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the antibody's properties and that it is obtained from a largely homogeneous population of antibodies; this should not be interpreted as requiring any special methods to produce the antibody.

[0205] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. 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 regions.

[0206] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on the lower chain region; (iii) Fd fragments consisting of VH and CH1 domains; and (iv) Fv fragments consisting of the VH and VL domains of a single arm of the antibody. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.

[0207] This invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments or fusion proteins formed by antibodies and other sequences, such as Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0208] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation. An epitope can be a discontinuous three-dimensional spatial site on the antigen, recognized by the antibody or antigen-binding fragment of the present invention.

[0209] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... - 7 M, for example, approximately less than 10 -8 M, 10 -9 M or l0 -10M or lower affinity (KD) binding.

[0210] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (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 recombination techniques well known in the art.

[0211] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0212] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0213] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[0214] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0215] The term "CDR" refers to the hypervariable region within the variable domain of an antibody that primarily facilitates antigen binding. One of the most commonly used definitions of CDR is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. Furthermore, IMGT (Lefranc, 2003) and Chothia (Al-Lazikani, 1997) have provided CDR definition rules, which are well known to those skilled in the art.

[0216] In a preferred embodiment of the present invention, the light chain of the antibody includes the aforementioned light chain variable region and light chain constant region, wherein the light chain constant region may be mouse-derived or human-derived.

[0217] As used herein, the term "antibody Fc region" or "human immunoglobulin Fc region" encompasses the constant region polypeptide of an antibody other than the heavy chain constant region 1 (CH1), such as the two constant region domains CH2 and CH3 at the carboxyl terminus of the heavy chain constant region of human immunoglobulin IgA, IgD, and IgG, and the three constant region domains CH2, CH3, and CH4 at the carboxyl terminus of the heavy chain constant region of human immunoglobulin IgE and IgM, and also includes the flexible hinge region at the amino terminus of these domains. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as containing residues from A231 to its terminal residues.

[0218] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0219] Table A

[0220] As used in this article, the terms “ADCC” or “antibody-dependent cell-mediated cytotoxicity” include cell-mediated reactions in which nonspecific cytotoxic cells expressing FcγR recognize antibodies bound to target cells, leading to target cell lysis. In various contexts, enhanced ADCC effector function can refer to enhanced titer or enhanced efficacy. In the experimental context, “titer” refers to the effect of observing specific therapeutic efficacy in EC. 50 The concentration of the antibody at that time (half-maximum effective concentration). In the experimental context, "efficacy" refers to the maximum possible effector function of the antibody at saturation level.

[0221] Anti-FGFR2Ⅲb antibody

[0222] 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 derived protein (S252W mutant) as described in the first aspect of the present invention.

[0223] The function of the antibody of the present invention is determined by the specific gene sequences of the variable regions of the antibody's light and heavy chains. The antibody of the present invention can specifically bind to FGFR2Ⅲb, exhibiting high affinity and efficiently blocking the binding of FGFR2Ⅲb to its ligands FGF1, FGF7, and FGF10. Using the variable region gene or complementarity-determining region (CDR) gene of the antibody of the present invention, different forms of genetically engineered antibodies can be modified and produced in any expression system utilizing prokaryotic and eukaryotic cells.

[0224] This invention provides an antibody against FGFR2Ⅲb, the antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementarity-determining region (CDR) selected from the group consisting of:

[0225] (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 CDR is defined according to the Kabat rule;

[0226] (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 rules;

[0227] (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 rules;

[0228] (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, wherein the CDRs are defined according to the Kabat rules;

[0229] (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, wherein the CDRs are defined according to the Kabat rules; and

[0230] (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, wherein the CDR is defined according to the IMGT rule;

[0231] Furthermore, the variable region of the light chain has a complementary determinant region (CDR) selected from the following group:

[0232] (1) 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 CDR is defined according to the Kabat rule;

[0233] (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, wherein the CDR is defined according to the Kabat rule;

[0234] (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, wherein the CDRs are defined according to the Kabat rules; and

[0235] (4) VL-CDR1 shown in SEQ ID 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.

[0236] Furthermore, the amino acid sequence also includes a sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence, preferably with at least 80% homology or sequence identity, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% amino acid sequence.

[0237] Methods for determining sequence homology or identity known to those skilled in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, 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., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG 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 for identity determination.

[0238] Preferably, the antibody described herein is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0239] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising 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 fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0240] The antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0241] The antibody derivatives described in this 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.

[0242] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0243] The animal is preferably a mammal, such as a mouse.

[0244] The antibodies of this invention can be chimeric antibodies, humanized antibodies, CDR-grafted and / or modified antibodies that target FGFR2Ⅲb (e.g., human FGFR2Ⅲb, mouse FGFR2Ⅲb or cynomolgus monkey FGFR2Ⅲb).

[0245] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0246] In the above-described content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0247] Fucosylation-free antibodies

[0248] As used herein, the terms "fucosylated antibody" or "antibody lacking fucosylation" refer to an antibody with a low level of fucosylation or no fucosylation in its constant region glycosylation. Methods for measuring fucosylation include any methods known in the art, including those described in this application. The fucosylation level can be obtained by measuring the proportion of fucosylated glycosylated structures in the antibody relative to all glycosylated structures. The antibodies of this invention contain glycosylation modifications in the antibody's Fc region, such as the glycosylation modification located at Asn297 (according to EU numbering rules) in the antibody's Fc region.

[0249] The antibodies or antigen-binding fragments of the present invention have reduced fucosylation levels, thereby contributing to enhanced binding of the Fc region to FcγRIIIa, and consequently improving the ADCC effect of the antibody. In some embodiments, the antibodies or antigen-binding fragments of the present invention have little or no fucosylation. For a single antibody, "no fucosylation" means that the proportion of fucosylated glycosylation structures in the antibody is ≤10% of all glycosylated glycosylation structures; "no fucosylation" means that the proportion of fucosylated glycosylation structures in the antibody is 0% or below a detectable level. For example, the proportion of fucosylated glycosylation structures in the glycosylation structures contained in the antibodies of the present invention may be ≤10%. In some embodiments, the proportion of fucosylated glycosylation structures may be ≤5%, for example ≤3%, ≤2%, ≤1%, ≤0.5%, or ≤0.1%. In some embodiments, the proportion of fucosylated glycosylation structures is 0%-1%, for example 0%-0.5%, 0%-0.3%, or 0%-0.1%. In other embodiments, the proportion of fucose in the glycosyl structure of the antibody of the present invention is 0% or less than a detectable level. For compositions containing the antibody of the present invention, "fucosylation-free" means that the proportion of the antibody with fucosylation modification in the composition is ≤5%, for example ≤3%, ≤2%, ≤1%, ≤0.5%, or ≤0.1% of the total antibody.

[0250] The antibodies or antigen-binding fragments of the present invention can be produced using techniques known to those skilled in the art for producing fucosylated antibodies. For example, the antibodies of the present invention can be produced using cells engineered to lose their protein fucosylation modification function. In some embodiments, the cells used to produce the antibodies of the present invention may be cells with the Fut8 gene knocked out. In some embodiments, the cells are Chinese hamster ovary (CHO) cells, such as CHOK1 cells, CHOS cells, or other CHO-derived cells. In some embodiments, the cells are Fut8 gene knocked-out CHOK1 cells.

[0251] Compared to control antibodies with the same sequence (e.g., antibodies produced in cells conventionally capable of protein fucosylation modification), the fucosylation-free antibodies of the present invention exhibit increased FcγRIIIa binding activity. In some embodiments, the affinity KD value of the fucosylation-free antibodies of the present invention for FcγRIIIa (F176 or V176) is 1 / 2 or less, 1 / 5 or less, or 1 / 10 or less of that of the control antibodies. Compared to control antibodies with the same sequence (e.g., antibodies produced in cells conventionally capable of protein fucosylation modification), the fucosylation-free antibodies of the present invention exhibit increased ADCC activity.

[0252] Recombinant protein

[0253] 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.

[0254] Preferably, the recombinant protein further includes 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-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and more preferably a human-derived antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat-derived antibody light chain constant region or a human-derived antibody light chain constant region, and more preferably a human-derived antibody light chain constant region.

[0255] In another preferred embodiment, the recombinant protein comprises the antibody of the present invention.

[0256] The recombinant protein is a conventional protein in the art, preferably one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single chain antibody fragment (scFv), single domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies.

[0257] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0258] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, comprising an Fd segment of a light chain variable region, a light chain constant region, and a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0259] 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.

[0260] The single-region antibody described is a conventional single-region antibody in the art, which includes only the heavy chain variable region.

[0261] The recombinant protein is prepared using conventional methods in the art. Preferably, the preparation method involves isolating the protein from an expression transformant or obtaining it through artificial synthesis of the protein sequence. The method for isolating the protein from the expression transformant is preferably as follows: cloning a polynucleotide molecule encoding the protein and carrying a point mutation into a recombinant vector; transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant; and culturing the obtained recombinant expression transformant to isolate and purify the recombinant protein.

[0262] Antibody preparation

[0263] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0264] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0265] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0266] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0267] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0268] The host cell can be a prokaryotic cell; a lower eukaryotic cell; or a higher eukaryotic cell, such as a mammalian cell.

[0269] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody is then purified using conventional separation and purification methods well-known to those skilled in the art.

[0270] In some embodiments, the host cells may be engineered to lose or attenuate the intracellular protein fucosylation modification function. The host cells may be FUT8-deficient, including FUT8 gene knockout, reduced expression, and / or reduced activity. In other embodiments, the host cells are cultured under conditions that inhibit protein fucosylation. These conditions inhibit FUT8 expression and / or activity. For example, the conditions may include the addition of a FUT8 inhibitor.

[0271] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of 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 monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0272] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art.

[0273] application

[0274] The present invention also provides the use of the antibodies, recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for the preparation of diagnostic agents or pharmaceuticals.

[0275] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal FGFR2Ⅲb expression or function.

[0276] In this invention, the diseases associated with abnormal FGFR2Ⅲb expression or function are conventional diseases associated with abnormal FGFR2Ⅲb expression or function in the art. Preferably, the diseases associated with abnormal FGFR2Ⅲb expression or function are cancer.

[0277] In this invention, the cancer is a conventional cancer in the art, preferably gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, bile duct cancer, lung cancer, or non-small cell lung cancer.

[0278] Detection uses and kits

[0279] The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0280] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include, for example, resected tumor samples, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0281] The samples used in this invention include fixed or preserved cell or tissue samples.

[0282] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0283] Pharmaceutical Composition

[0284] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or its active fragment or its fusion protein or corresponding immune cell, and a pharmaceutically acceptable carrier.

[0285] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy.

[0286] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal expression or function of FGFR2Ⅲb or its derivative proteins.

[0287] The pharmaceutical compositions of the present invention contain a safe and effective amount of the monoclonal antibody described above, as well as a pharmaceutically acceptable carrier or excipient. The pharmaceutical formulation should be matched to the route of administration. The dosage of the active ingredient is a therapeutically effective amount. Furthermore, the peptides of the present invention can be used in conjunction with other therapeutic agents.

[0288] 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.

[0289] Preferably, the pharmaceutical composition of this invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents.

[0290] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0291] This invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases 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 or breast cancer.

[0292] Methods and compositions for detecting FGFR2Ⅲb in samples

[0293] The present invention also provides a method for detecting FGFR2Ⅲb in a sample (e.g., detecting cells overexpressing FGFR2Ⅲb), comprising the following steps: contacting the antibody with the sample to be tested in vitro, and detecting whether the antibody and the sample to be tested bind to form an antigen-antibody complex.

[0294] The term "overexpression" is conventional 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 changes in protein degradation), as well as local overexpression and enhanced functional activity due to changes in protein transport patterns (increased cell membrane localization) (e.g., in the case of increased enzymatic hydrolysis of the substrate).

[0295] In this invention, the detection method for whether or not an antigen-antibody complex is formed is a conventional detection method in the art, and preferably a flow cytometry (FACS) assay.

[0296] This invention provides a composition for detecting FGFR2Ⅲb in a sample, comprising the above-described antibody, recombinant protein, immune cells, or a combination thereof as active ingredients. Preferably, it further comprises a compound composed of functional fragments of the above-described antibody as an active ingredient.

[0297] The main advantages of this invention include:

[0298] 1) The anti-FGFR2Ⅲb antibody of the present invention can bind to FGFR2Ⅲb, which has high affinity and can efficiently block the binding of FGFR2Ⅲb to its ligands FGF1, FGF7 and FGF10.

[0299] 2) The antibody of the present invention has excellent binding activity against FGFR2Ⅲb from multiple species, and also has excellent specificity, and basically does not bind to FGFR2Ⅲc; the antibody of the present invention has better selectivity for FGFR2Ⅲb compared to FGFR2Ⅲc.

[0300] 3) The antibody of the present invention can mediate the specific killing of FGFR2IIIb-overexpressing cancer cells by immune cells through ADCC.

[0301] 4) The antibody of the present invention has a significant antitumor effect in tumor-bearing mouse models, and the antibody of the present invention has a significant antitumor effect even at low doses.

[0302] 5) This invention provides a fucosylated-free anti-FGFR2Ⅲb antibody. Compared to conventionally expressed antibodies, the fucosylated-free antibody of this invention significantly enhances the binding affinity of FcγRIIIa (F176 / V176) and exhibits a further enhanced ADCC effect.

[0303] 6) The fucosylated antibody of the present invention unexpectedly inhibits the growth of 4T1 breast cancer tumors with low expression of FGFR2Ⅲb.

[0304] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0305] Example 1: Obtaining anti-human FGFR2Ⅲb antibody

[0306] The inventors immunized 6-8 week old female Balb / c mice with FGFR2Ⅲb(D2+D3) (SEQ ID NO:1) as the antigen. Spleen cells from the immunized mice were fused with SP2 / 0-AG14 cells to form hybridoma cells, and an appropriate amount of the fused cells was seeded into 96-well plates. On days 7-10 post-fusion, the supernatant from each well was collected, and the binding activity of mouse antibodies secreted by the hybridoma cells to human FGFR2Ⅲb-his(ECD) (SEQ ID NO:2) and FGFR2Ⅲc-his(ECD) (SEQ ID NO:3), as well as their inhibitory activity against the binding of FGF7 / FGFR2Ⅲb and FGF10 / FGFR2Ⅲb (FGF7 and FGF10 were purchased from Nearshore Protein, catalog numbers: CM88 and CR11, respectively) was detected by ELISA. Finally, several hybridoma cell lines specifically binding to FGFR2Ⅲb-his(ECD) but not to FGFR2Ⅲc-his(ECD) were obtained. The hybridoma cell line with the best inhibition of FGF7 / FGFR2Ⅲb and FGF10 / FGFR2Ⅲb binding activity was 27E9A11. Sequencing revealed its secreted antibody, which was named 27E9A11 antibody. The cDNA sequences of the heavy chain variable region and light chain variable region corresponding to the 27E9A11 antibody are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively. The antigen complementarity determinant (CDR) sequence of the 27E9A11 antibody is shown in Table 1.

[0307] Table 1. Antigen complementarity determinants (CDRs) of mouse antibody 27E9A11

[0308] Detection of the binding activity of 27E9A11 antibody to human FGFR2Ⅲb protein

[0309] A 1 μg / mL human FGFR2Ⅲb-his protein solution was coated into 100 μL / well of a 96-well high-affinity plate and incubated overnight at 4°C with shaking. The next day, the plates were washed three times with 300 μL PBST (Tween 20: 0.5‰), then blocked with 100 μL / well of 5% BSA / PBS for 2 hours with shaking at room temperature. The plates were then washed three times with 300 μL PBST. A serial dilution solution of the 27E9A11 antibody sample was prepared using PBS. 100 μL of this solution was added to each well of a 96-well plate and incubated for 1 hour with shaking at room temperature. The plates were then washed three times with 300 μL PBST. A secondary antibody solution of goat anti-mouse IgG HRP (Thermo Fisher, catalog number A16090) was prepared and added to each well of a 96-well plate with 100 μL of this solution. The plates were incubated for 30 minutes with shaking at room temperature. The plates were then washed four times with 300 μL PBST. Add 100 μL / well of TMB (tetramethylbenzidine) and develop color for 3 minutes. Add 100 μL / well of 0.6 N H₂SO₄ to stop the color development and measure the OD. 450 nm.

[0310] Detection of the binding of human FGF factors (FGF7 or FGF10) to human FGFR2Ⅲb protein blocked by 27E9A11 antibody.

[0311] A 1 μg / mL solution of human FGF factor and a 5 μg / mL solution of heparin sodium (Sinopharm, catalog number 63007131, hereinafter the same) was used to coat 96-well high-affinity plates with 100 μL / well. The plates were incubated overnight at 4°C with shaking. The next day, the plates were washed three times with 300 μL of PBST (Tween 20: 0.5‰), then blocked with 100 μL / well of 5% BSA / PBS for 2 hours with shaking at room temperature. The plates were washed three times with 300 μL of PBST. A serial dilution solution of the 27E9A11 antibody sample was prepared using PBS, and 1 μg / mL of FGFR2Ⅲb-huFc was added to each solution at a 1:1 ratio and premixed. 100 μL of each solution was added to a 96-well plate and incubated for 2 hours with shaking at room temperature. The plates were washed three times with 300 μL of PBST. Prepare secondary antibody (goat anti-mouse IgG HRP solution, Abcam, catalog number ab6858, hereinafter the same) and add 100 μL / well to a 96-well plate, incubate at room temperature with shaking for 1 hour. Wash four times with 300 μL PBST. Add 100 μL / well of TMB (tetramethylbenzidine) and incubate for 5 min. Add 100 μL / well of 0.6N H2SO4 to stop the color development and measure OD. 450 nm.

[0312] Results: The binding activity of the 27E9A11 antibody to FGFR2Ⅲb-his is shown in Figure 1. EC 50 See Table 2; the inhibitory activity of the 27E9A11 antibody against FGF7 / FGFR2Ⅲb binding is shown in Figure 2, IC50. 50See Table 2; the inhibitory activity of the 27E9A11 antibody against FGF10 / FGFR2Ⅲb binding is shown in Figure 3, IC50. 50 See Table 2; the results show that the 7E9A11 antibody has strong binding activity to FGFR2Ⅲb-his, EC 50 The concentration was 0.050 μg / mL; and the 27E9A11 antibody exhibited strong inhibitory activity against the binding of human FGF factors (FGF7 and FGF10) to human FGFR2Ⅲb protein, with an IC50 concentration of 0.050 μg / mL. 50 The concentrations were 0.417 μg / mL and 0.545 μg / mL, respectively.

[0313] Table 2. Binding and blocking abilities of antibody 27E9A11

[0314] Example 2: Binding of chimeric antibody III-0 to human FGFR2Ⅲb protein

[0315] The variable regions of the heavy chain and light chain of mouse antibody 27E9A11 were linked to the constant regions of the heavy chain and κ chain of human IgG1, respectively, to obtain human-mouse chimeric antibody III-0, the heavy chain sequence of which is shown in SEQ ID NO:22 and the light chain sequence of which is shown in SEQ ID NO:23.

[0316] The binding activity of III-0 to human FGFR2Ⅲb protein was investigated, and the detection method is described in Example 1 (the secondary antibody was changed to goat anti-human IgG HRP). The results are shown in Figure 4. III-0 effectively binds to human FGFR2Ⅲb protein, EC50... 50 The concentration was 40.42 ng / mL.

[0317] Example 3: III-0 blocks the binding of human FGF factors (FGF1 / FGF7 / FGF10) to human FGFR2Ⅲb protein.

[0318] Coat 96-well high-affinity plates with 100 μL / well of 1 μg / mL human FGF factor and 5 μg / mL heparin sodium solution, and incubate overnight at 4°C with shaking. The next day, wash three times with 300 μL PBST (Tween 20: 0.5‰), then block with 100 μL / well of 5% BSA / PBS for 2 hours with shaking at room temperature. Wash three times with 300 μL PBST. Prepare serial dilutions of antibody samples with PBS, adding 1 μg / mL FGFR2Ⅲb-mFc at a 1:1 ratio and premixing. Add 100 μL / well of each solution to a 96-well plate and incubate for 2 hours with shaking at room temperature. Wash three times with 300 μL PBST. Prepare secondary antibody (goat anti-mouse IgG HRP) solution, add 100 μL / well of each solution to a 96-well plate, and incubate for 1 hour with shaking at room temperature. Wash four times with 300 μL PBST. Add 100 μL / well of TMB (tetramethylbenzidine) and develop color for 5 min. Add 100 μL / well of 0.6 N H₂SO₄ to stop the color development and measure the OD. 450 nm.

[0319] The results are shown in Figure 5. III-0 can effectively block the binding of FGF1 / 7 / 10 to FGFR2Ⅲb, and its IC 50 See Table 3.

[0320] Table 3. Blocking ability of antibody III-0

[0321] Example 4: Binding of III-0 to FGFR2Ⅲb protein on SNU-16 cells

[0322] SNU-16 cells are derived from human gastric cancer and naturally overexpress human FGFR2Ⅲb protein. SNU-16 cells were digested and centrifuged, then rinsed with PBS at 2x10⁻⁶ ppm. 6 Resuspend the cells at a density of cells / mL, mix well, and transfer 100 μL to a centrifuge tube. Centrifuge and discard the supernatant. Prepare serial dilutions of the antibody samples using PBS. Add 100 μL to each centrifuge tube and incubate at 4°C with shaking for 1 hour. Centrifuge the cells, discard the supernatant, and mix with 400 μL of PBS. Repeat 3 times. Prepare the secondary antibody (Thermo Fisher, catalog number A21091) for goat anti-human IgG (H+L) flow cytometry detection. Add 100 μL / well to a centrifuge tube and incubate at 4°C with shaking for 30 minutes. Centrifuge the cells at 2000 rpm for 3 minutes, discard the supernatant, and mix with 400 μL of PBS. Repeat twice. Transfer to a flow cytometer (Beckman, Cytoflex) for detection. The results are shown in Figure 6. III-0 can effectively recognize and bind to SNU-16 cells, and the bound EC... 50 See Table 4.

[0323] Example 5: Binding of chimeric antibody to FGFR2Ⅲb protein on CHO-FGFR2Ⅲb cells

[0324] The inventors constructed a CHO-K1 cell line overexpressing FGFR2Ⅲb protein. The CHO-FGFR2Ⅲb cells were digested and centrifuged, then rinsed with PBS at 2 x 10⁻⁶ ppm. 6 Resuspend the cells at a density of cells / mL, mix well, and transfer 100 μL to a centrifuge tube. Centrifuge and discard the supernatant. Prepare serial dilutions of the antibody sample using PBS. Add 100 μL to each centrifuge tube and incubate at 4°C with shaking for 1 hour. Centrifuge the cells, discard the supernatant, and mix with 400 μL of PBS. Repeat 3 times. Prepare the secondary antibody (goat anti-human IgG (H+L) for flow cytometry detection). Add 100 μL / well to a centrifuge tube and incubate at 4°C with shaking for 30 minutes. Centrifuge the cells at 2000 rpm for 3 minutes, discard the supernatant, and mix with 400 μL of PBS. Repeat twice. Transfer to a flow cytometer (Beckman, Cytoflex) for detection. The results are shown in Figure 7. III-0 can effectively recognize and bind to CHO-FGFR2Ⅲb cells, and bind EC... 50 See Table 4.

[0325] Table 4. Binding ability of antibody III-0 to different cells.

[0326] Example 6: Binding of humanized antibodies to human FGFR2Ⅲb and FGFR2Ⅲb (S252W) proteins

[0327] Humanized antibody III-10 was obtained by humanizing chimeric antibody III-0 using the CDR transplantation method. Further affinity maturation modification of III-10 was then performed. Specifically, single-point saturation mutations were performed on each amino acid site of the CDR region of antibody III-10. Mutation hotspots with antigen-specific binding ability were screened using ELISA, and these hotspots were then combined to obtain candidate antibody mutation sequences. The affinity between the candidate antibodies and the antigen was detected using the SPR method, ultimately yielding five antibodies: III-11, III-12, III-13, III-14, and III-15. The sequences of these antibodies are shown in the table below.

[0328] Table 5. Variable region sequences of affinity-modified antibodies.

[0329] (CDR sequence defined by Kabat rules)

[0330] S252W is a relatively common FGFR2Ⅲb mutation in tumors. The binding activity of a series of humanized antibodies against III-0 to FGFR2Ⅲb, and to FGFR2Ⅲb (S252W), was tested. The detection method is described in Example 2. The results are shown in Figure 8. The binding activity of the humanized antibodies against III-0 to human FGFR2Ⅲb and FGFR2Ⅲb (S252W) proteins is comparable to that of III-0. The EC50 values ​​of each antibody... 50 See Table 6.

[0331] Table 6. Binding ability of affinity-modified antibodies to FGFR2IIIb / FGFR2IIIb (S252W)

[0332] Example 7: Binding of anti-human FGFR2Ⅲb antibody to human FGFR2Ⅲc protein

[0333] A 1 μg / mL human FGFR2Ⅲc protein solution was coated onto 96-well high-affinity plates at 100 μL / well and incubated overnight at 4°C with shaking. The next day, the plates were washed three times with 300 μL PBST (Tween 20: 0.5‰), then blocked with 100 μL / well of 5% BSA / PBS for 2 hours with shaking at room temperature. The plates were washed three times with 300 μL PBST. A serial dilution solution of antibody samples was prepared using PBS. 100 μL of this solution was added to each well of the 96-well plate and incubated for 1 hour with shaking at room temperature. The plates were washed three times with 300 μL PBST. A secondary antibody solution of goat anti-human IgG HRP was prepared and added to each well at 100 μL / well. The plates were incubated for 30 minutes with shaking at room temperature. The plates were washed four times with 300 μL PBST. 100 μL / well of TMB (tetramethylbenzidine) was added, and the plates were developed for 3 minutes. 100 μL / well of 0.6N H2SO4 was added to stop the development, and the OD was measured. 450 nm.

[0334] The test results are shown in Figure 9. Antibodies III-0, III-10, and III-11 showed weak or no binding to human FGFR2Ⅲc protein. Antibodies III-12, III-13, III-14, and III-15 exhibited relatively weak binding activity to human FGFR2Ⅲc protein. These results confirm that the antibodies of this invention possess high specificity, and in clinical applications, they can reduce unnecessary binding, decrease potential side effects, and improve drug safety.

[0335] Example 8: Humanized antibody blocks the binding of human FGF factors (FGF1 / FGF7 / FGF10) to human FGFR2Ⅲb protein.

[0336] The specific detection method is described in Example 3. The detection results are shown in Figure 10. Both III-10 and III-0 can block the binding of FGF1 / FGFR2Ⅲb. The activity of III-10 in blocking the binding of FGF10 / FGFR2Ⅲb is comparable to that of III-0. 50 See Table 7.

[0337] Table 7. Blocking ability of antibodies III-0 and III-10

[0338] Example 9: Humanized antibody (affinity maturation) blocks the binding of human FGF factors (FGF1 / FGF10) to human FGFR2Ⅲb protein.

[0339] The specific detection method is described 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 their blocking abilities are comparable, as shown in Table 8.

[0340] Table 8. Blocking ability of affinity maturation antibodies

[0341] Example 10: Binding of humanized antibody to FGFR2Ⅲb protein on CHO-FGFR2Ⅲb cells

[0342] The specific detection method is described in Example 5. The results are shown in Figure 12. The binding activity of III-10, III-11, III-14, and III-15 to CHO-FGFR2Ⅲb was comparable to that of III-0. The EC50 of each antibody... 50 See Table 9.

[0343] Example 11: Binding of humanized antibody to FGFR2Ⅲb protein on SNU-16 cells

[0344] The specific detection method is described in Example 4. The results are shown in Figure 13. The binding activity of III-10, III-11, III-14, and III-15 to SNU-16 was comparable to that of III-0. The EC50 of each antibody... 50 See Table 9.

[0345] Table 9. Binding ability of humanized antibodies to different cells.

[0346] Example 12: ADCC effect of humanized antibodies

[0347] The inventors constructed a stable Jurkat-NFAT-Luc-CD16A cell line expressing the CD16 receptor and the NFAT (Nuclear Factor of Activated T-cells) reactive element. Using CHO-FGFR2Ⅲb cells as target cells, the cells were digested, 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. Serial dilutions of the antibody samples were prepared using complete culture medium. The supernatant was discarded from the 384-well plates, and 15 μL of antibody solution was added to each well. Pre-incubation was performed at 37°C for 1 hour. Jurkat-NFAT-Luc-CD16A cells were resuspended in complete culture medium, with 15 μL of cell suspension added to each well, and incubated at 37°C for 4 hours. ONE-Glo TM Luciferase assay solution (Promega, catalog number E6110) was added at 30 μL / well to a 384-well plate and reacted for 1–3 minutes. The plates were then transferred to a multi-plate reader (Tecan Spark 20M) for detection. The results are shown in Figure 14. The EC50 values ​​of the antibodies were roughly equivalent. Regarding activation efficacy, all antibodies were able to activate immune cells, with III-14 exhibiting the strongest activation effect.

[0348] Table 10 ADCC effects of humanized antibodies

[0349] Example 13: Binding of humanized antibody to mouse / cynomolgus monkey FGFR2Ⅲb protein

[0350] A 1 μg / mL mouse / cynomolgus monkey FGFR2Ⅲb protein solution was coated into 100 μL / well of a 96-well high-affinity plate and incubated overnight at 4°C with shaking. The next day, the plates were washed three times with 300 μL PBST (Tween 20: 0.5‰), then blocked for 2 hours with 100 μL / well of 5% BSA / PBS and shaken at room temperature. The plates were washed three times with 300 μL PBST. A serial dilution of the antibody samples was prepared using PBS. 100 μL / well of the solution was added to each well of the 96-well plate and incubated for 1 hour with shaking at room temperature. The plates were washed three times with 300 μL PBST. A secondary antibody solution of goat anti-human IgG HRP was prepared and added to each well of the 96-well plate with 100 μL / well and incubated for 30 minutes with shaking at room temperature. The plates were washed four times with 300 μL PBST. 100 μL / well of TMB (tetramethylbenzidine) was added, and the plates were developed for 3 minutes. Add 100 μL / well of 0.6 N H2SO4 to stop color development, then measure the OD. 450 nm.

[0351] The results are shown in Figure 15. III-10 can bind to FGFR2Ⅲb and EC in mice and cynomolgus monkeys. 50 See Table 11.

[0352] Table 11 Binding ability of humanized antibodies to mouse / cynomolgus monkey FGFR2IIIb

[0353] Example 14: Detection of antibody affinity

[0354] Surface plasmon resonance (SPR) was used to detect antigen-antibody affinity. Using FGFR2Ⅲb-his as the antigen, a specific concentration of antibody was incubated with a protein A sensor chip for antibody capture. During the antigen binding phase, serially diluted FGFR2Ⅲb-his protein was used as the mobile phase to bind with the captured antibody on the sensor chip. During the dissociation phase, continuous elution was performed with HBS-EP buffer. The binding of the antibody to FGFR2Ⅲb-his on the sensor chip was quantitatively detected using a Biacore 8k (GE Healthcare). The results are shown in Table 12. The affinity of antibodies III-11, III-12, III-13, III-14, and III-15 for FGFR2Ⅲb-his was significantly higher than that of III-0 and III-10.

[0355] Table 12 Affinity of anti-human FGFR2Ⅲb antibody to FGFR2Ⅲb-his

[0356] Example 15: Efficacy study of III-10 in a mouse SNU-16 tumor model

[0357] For the SNU16 tumor model, SNU16 gastric cancer cells (5.0 × 10⁻⁶) in serum-free culture medium were... 6 (Number) were injected subcutaneously into the upper right abdomen of female SCID mice. When the tumor volume reached 170 mm²... 3 On day 5 post-inoculation, mice were randomly divided into groups of 8. Intraperitoneal injections of III-10 antibody and human albumin (control) were administered twice weekly for 3 weeks. The concentration of III-10 antibody and human albumin control was 5 mg / kg. Tumor volume and mouse weight were measured and recorded every 2-3 days during this period.

[0358] As shown in Figure 16, III-10 exhibited a significant ability to inhibit SNU16 tumor growth. Further experimental results showed that the antibody of this invention could also significantly inhibit SNU16 tumors at low doses.

[0359] Example 16: Preparation of Fucosylated Antibody

[0360] The expression sequences (SEQ ID NOs: 32 and 33) of the heavy and light chains of antibody III-10 from Example 6 were constructed on the pGenHT1.0-DGV expression vector (purchased from Nanjing Pengbo Biotechnology Co., Ltd.) to obtain the recombinant plasmid pGenHT1.0-DGV-L3 (Light chain) (Heavy chain). Subsequently, it was transfected into CHOK1-ADCC+ cells (Nanjing Pengbo Biotechnology Co., Ltd., catalog number DMF: 036229) and CHOK1SV GS-KO cells (purchased from Lonza). The Fut8 gene was knocked out in CHOK1-ADCC+ cells, resulting in the loss of intracellular protein fucosylation modification function. Therefore, it can express afucosylated antibody, thereby enhancing the binding force of the antibody to the Fc receptor FcγRIIIa (CD16a) and increasing antibody-dependent cytotoxicity (ADCC) activity. CHOK1SV GS-KO cells were normal cells.

[0361] After transfection, methionine sulfoximine (MSX) was used to screen for cells that stably expressed non-fucosylated antibodies (VA583-FUT8) and those expressing conventional antibodies (VA583-GS). The VA583-FUT8 cell pool was plated using a single-clone imaging and plating system (VIPS). Each clone was expanded and cultured, and yield was evaluated. Single-clone cell lines with clear clonal origin and high yield were selected and named C121. Their yields are shown in Table 13. The antibodies produced by VA583-GS and C121 were named VA583-GS and C121, respectively.

[0362] Table 13 Antibody Yield

[0363] Example 17: Sugar Form Analysis

[0364] Ultra-high performance liquid chromatography (UPLC) was used to analyze the glycoforms of VA583-GS and C121 antibodies, identifying eight major glycoforms, the structures of which are shown in Figure 17. Only three glycoforms—G0F, G1Fa, and G1Fb—contained fucose. The proportions of each glycoform relative to the total number of glycoforms were calculated, and the results are shown in Table 14. The fucose proportion, i.e., the fucosylation proportion, is the sum of the proportions of the G0F, G1Fa, and G1Fb glycoforms. The analysis results showed that the C121 antibody had a 0% fucose modification proportion, indicating that it lacked fucosylation modification.

[0365] Table 14 Sugar Form Analysis

[0366] Example 18: Affinity Detection

[0367] The affinity between antibody molecules and FcγRIIIa (F176) and FcγRIIIa (V176) receptors was detected using surface plasmon resonance (SPR) technology. The results are shown in Tables 15 and 16. The KD values ​​of the unfucosylated C121 antibody molecule with the two receptor proteins were significantly lower than those of the ordinary antibody VA583-GS, indicating that the binding ability of C121 antibody with the two receptor proteins is significantly stronger than that of ordinary antibody VA583-GS. Correspondingly, the ADCC activity activated by C121 antibody is also stronger than that of VA583-GS.

[0368] Table 15. Affinity test results of antibodies with human FcγRIIIa (F176) protein

[0369] Table 161 Affinity test results of antibody with human FcγRIIIa (V176) protein

[0370] Example 19: ADCC Activity Detection

[0371] Using CHO-FGFR2Ⅲb cells as the target cells, they were digested, centrifuged, resuspended in culture medium, and seeded into 96-well plates at a density of 20,000 cells / well, and cultured overnight. Serial dilutions of the antibody samples were prepared using complete culture medium, and Jurkat-FcγRIIIa-F158 cells were diluted to 3 × 10⁻⁶ cells / well with complete culture medium. 6 Cells / mL, then discard the culture supernatant in the 96-well plate, add 25 μL of fresh culture medium + 25 μL of antibody diluent + 25 μL of Jurkat-NFAT-Luc-CD16A cell suspension to each well, mix well and incubate at 37°C for 6 hours. Prepare ONE-Glo TM Luciferase assay solution (Promega, catalog number E6110) was added to 96-well plates at a rate of 70 μL / well and reacted for 1–3 minutes. The plates were then transferred to a SpectraMax iD5 microplate reader for detection. The results are shown in Figure 18 and Table 17. The ADCC activity of the fucosylated antibody C121 was significantly stronger than that of the conventional antibody VA583-GS.

[0372] Table 17 ADCC Activity

[0373] Example 20: In vivo antitumor drug efficacy

[0374] The efficacy of the drug was evaluated using a 4T1 mouse orthotopic breast cancer model. Flow cytometry was used to detect the proportion of 4T1 cells expressing FGFR2IIIb protein, and the results are shown in Figure 19. The vast majority of cells expressed FGFR2IIIb protein. Further Western blotting experiments were used to compare the intracellular FGFR2IIIb protein expression levels in 4T1 cells and SNU-16 cells, which highly express FGFR2IIIb. The results are shown in Figure 20. The FGFR2IIIb expression level in 4T1 cells was significantly lower than that in SNU-16 cells. Therefore, using the 4T1 model for efficacy evaluation is more convincing.

[0375] 50,000 4T1 cells in serum-free medium were subcutaneously inoculated into the third mammary gland on the right side of female C57 mice. When the tumor volume reached approximately 70 mm³ (10 days post-inoculation), the mice were randomly divided into groups of 8. Intraperitoneal injections of C121 antibody, VA583-GS antibody, and human albumin (as a control) were administered at a concentration of 1 mg / kg, twice weekly for two weeks. Tumor volume and mouse weight were measured and recorded every 2-3 days. Drug efficacy was assessed by tumor volume, while mouse weight data was used to ensure that the overall health of the animals was not affected by the test drug.

[0376] The tumor volume results are shown in Figure 21. The fucose-free antibody C121 showed a significant ability to inhibit 4T1 tumor growth, while the ordinary antibody VA583-GS did not show anti-4T1 activity. Mouse body weight data are shown in Figure 22. There were no significant changes in body weight among the three groups of mice, indicating that the tested drug had no effect on the overall health of the mice.

[0377] The sequence of the present invention is shown in Table 18 below.

[0378] Table 18 Sequence List

[0379] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An antibody against FGFR2Ⅲb, said antibody comprising a heavy chain and a light chain, wherein, The variable region of the heavy chain has a complementarity-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 CDR is 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 rules; (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 rules; (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, wherein the CDRs are defined according to the Kabat rules; (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, wherein the CDRs are defined according to the Kabat rules; 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, wherein the CDR is defined according to the IMGT rule; Furthermore, the variable region of the light chain has a complementary determinant region (CDR) selected from the following group: (1) 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 CDR is 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, wherein the CDR is 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, wherein 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, wherein the CDRs are defined according to the IMGT rules; Furthermore, any amino acid sequence in the above-mentioned CDR sequence also includes a derivative sequence which optionally involves the addition, deletion, modification and / or substitution of 1-2 amino acids, such that the derivative antibody composed of the heavy chain and light chain containing the derived CDR sequence can retain the binding affinity of FGFR2Ⅲb or its derivative protein. Wherein, the fucosylation ratio of the antibody is ≤10%, preferably, the fucosylation ratio of the antibody is ≤5%, ≤3%, ≤2%, or ≤1%, and more preferably, the antibody is not fucosylated.

2. The antibody as described in claim 1, 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 group consisting of: (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 rules; (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:14, wherein the CDRs are defined according to the Kabat rules; (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, wherein the CDRs are defined according to the Kabat rules; (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, wherein the CDRs are defined according to the Kabat rules; (5) 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, wherein the CDRs are defined according to the Kabat rules; (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, wherein the CDRs are defined according to the Kabat rules; 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-CDR1 shown in SEQ ID 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.

3. The antibody as described in claim 1, 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 with it; 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 with it.

4. The antibody as described in claim 1, characterized in that, The antibody has the following characteristics: (1) The heavy chain variable region as shown in SEQ ID NO:24 and the light chain variable region as shown in SEQ ID NO:25; (2) The heavy chain variable region as shown in SEQ ID NO:26 and the light chain variable region as shown in SEQ ID NO:27; (3) The heavy chain variable region as shown in SEQ ID NO:28 and the light chain variable region as shown in SEQ ID NO:29; (4) The heavy chain variable region as shown in SEQ ID NO:30 and the light chain variable region as shown in SEQ ID NO:29; (5) The heavy chain variable region as shown in SEQ ID NO:31 and the light chain variable region as shown in SEQ ID NO:29; (6) the heavy chain variable region as shown in SEQ ID NO:26 and the light chain variable region as shown in SEQ ID NO:29; or (7) The heavy chain variable region as shown in SEQ ID NO:4 and the light chain variable region as shown in SEQ ID NO:

5.

5. The antibody as described in claim 1, 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 with it; 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 with it.

6. The antibody as described in claim 1, characterized in that, The antibody has the following characteristics: (1) The heavy chain as shown in SEQ ID NO:32 and the light chain as shown in SEQ ID NO:33; (2) The heavy chain as shown in SEQ ID NO:34 and the light chain as shown in SEQ ID NO:35; (3) Heavy chains as shown in SEQ ID NO:36 and light chains as shown in SEQ ID NO:37; (4) Heavy chains as shown in SEQ ID NO:38 and light chains as shown in SEQ ID NO:37; (5) Heavy chains as shown in SEQ ID NO:39 and light chains as shown in SEQ ID NO:37; (6) The heavy chain as shown in SEQ ID NO:34 and the light chain as shown in SEQ ID NO:37; or (7) The heavy chain as shown in SEQ ID NO:22 and the light chain as shown in SEQ ID NO:

23.

7. A composition comprising the antibody as described in any one of claims 1-6, characterized in that, At least 95% of the anti-FGFR2Ⅲb antibody in the composition is unfucosylated.

8. A recombinant protein, said recombinant protein comprising: (i) the antibody as described in claim 1; as well as (ii) Optional tag sequences to assist in expression and / or purification.

9. A host cell, characterized in that, The host cell genome integrates a polynucleotide encoding the antibody as described in claim 1 or the recombinant protein as described in claim 8, or the host cell contains a vector comprising the polynucleotide. The host cells are engineered to lose their protein fucosylation modification function.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 8, the host cell of claim 9, or a combination thereof; and (ii) Pharmaceutically acceptable carriers.

11. Use of the antibody of claim 1, the recombinant protein of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 10 in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal expression or function of FGFR2Ⅲb.

12. The use as described in claim 12, characterized in that, The disease described is cancer.

13. The use as described in claim 12, characterized in that, The cancers mentioned are selected from: breast cancer, stomach cancer, esophageal cancer, colorectal cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, bile duct cancer, lung cancer, and non-small cell lung cancer.

14. A detection plate, characterized in that, The detection 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 8, or a combination thereof.

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