CN118852431A - Anti-FGFR2b mAb - Google Patents

Abstract

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

Description

Anti-FGFR2b mAb

Technical Field

The present invention relates to the field of biomedicine and in particular to an anti-FGFR2IIIb monoclonal antibody.

Background Art

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

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

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

The main ligands of the FGFR2IIIb (or FGFR2b) form of FGFR2 are FGF1, FGF7, FGF10, and FGF22. FGFR2b can be highly expressed in tumors through amplification of the FGFR2 gene or transcriptional upregulation of the FGFR2b subtype, of which FGFR2 amplification is the most common FGFR2 gene aberration. In the immunohistochemical section analysis of gastric cancer patients, FGFR2b expression can be detected in approximately 60% of tumor samples, of which 2%-9% of samples can detect FGFR2b overexpression, and the level of overexpression ranges from 31% to 61%. These gastric cancer samples often show diffuse type and are associated with the aggressive characteristics of gastric cancer, including higher-level T stage, more frequent lymph node metastasis, and lower overall survival rate. With the deepening of research, it has been determined that the overexpression of the FGFR2b receptor or the amplification of the FGFR2 gene is directly related to the poor prognosis of gastric cancer patients. In addition to gastric cancer, abnormal 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 (such as non-small cell lung cancer), bile duct cancer, etc. Therefore, inhibiting FGFR2 signaling may be an effective mechanism for treating a variety of cancers.

Summary of the invention

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

In the first aspect of the present invention, an anti-FGFR2IIIb antibody is provided, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementary determining region (CDR) selected from the following group:

(1) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:7, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(2) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(3) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(4) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, 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 rule; and

(6) VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11, wherein the CDRs are defined according to IMGT rules;

Furthermore, the variable region of the light chain has a complementarity determining region (CDR) selected from the group consisting of:

(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 CDRs are defined according to the Kabat rule;

(2) VL-CDR1 shown in SEQ ID NO: 17, VL-CDR2 shown in SEQ ID NO: 18, and VL-CDR3 shown in SEQ ID NO: 14, wherein the CDRs are defined according to the Kabat rule;

(3) VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rule; and

(4) VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to IMGT rules;

Furthermore, any one of the amino acid sequences in the above-mentioned CDR sequences also includes a derivative sequence that is optionally added, deleted, modified and/or substituted with 1-2 amino acids, and enables the derivative antibody composed of the heavy chain and light chain containing the derived CDR sequence to retain the binding affinity for FGFR2Ⅲb or its derivative protein.

In the first aspect of the present invention, an anti-FGFR2IIIb antibody is provided, wherein the antibody comprises a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementary determining region (CDR) selected from the following group:

(1) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:7, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(2) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(3) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(4) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, 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 rule; and

(6) VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11, wherein the CDRs are defined according to IMGT rules;

Furthermore, the variable region of the light chain has a complementarity determining region (CDR) selected from the group consisting of:

(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 CDRs are defined according to the Kabat rule;

(2) VL-CDR1 shown in SEQ ID NO: 17, VL-CDR2 shown in SEQ ID NO: 18, and VL-CDR3 shown in SEQ ID NO: 14, wherein the CDRs are defined according to the Kabat rule;

(3) VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rule; and

(4) VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to IMGT rules.

In another preferred embodiment, the antibody has a heavy chain variable region CDR (VH-CDR) and a light chain variable region CDR (VL-CDR) selected from the following groups:

(1) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:7, VH-CDR3 shown in SEQ ID NO:8, VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:13, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to the Kabat 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; and

(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 IMGT rules.

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

In another preferred example, the antibody is capable of specifically binding to FGFR2IIIb from humans, mice and cynomolgus monkeys.

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

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

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

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

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

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

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

In another preferred embodiment, the heavy chain also includes a heavy chain constant region.

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

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

In another preferred embodiment, the light chain also includes a light chain constant region.

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

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

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

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

In another preferred embodiment, the antibody has:

(1) a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO: 25;

(2) a heavy chain variable region as shown in SEQ ID NO:26 and a light chain variable region as shown in SEQ ID NO:27;

(3) a heavy chain variable region as shown in SEQ ID NO:28 and a light chain variable region as shown in SEQ ID NO:29;

(4) a heavy chain variable region as shown in SEQ ID NO:30 and a light chain variable region as shown in SEQ ID NO:29;

(5) a heavy chain variable region as shown in SEQ ID NO:31 and a light chain variable region as shown in SEQ ID NO:29;

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

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

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

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

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

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

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

In another preferred embodiment, the antibody has:

(1) a heavy chain as shown in SEQ ID NO:32 and a light chain as shown in SEQ ID NO:33;

(2) a heavy chain as shown in SEQ ID NO:34 and a light chain as shown in SEQ ID NO:35;

(3) a heavy chain as shown in SEQ ID NO:36 and a light chain as shown in SEQ ID NO:37;

(4) a heavy chain as shown in SEQ ID NO:38 and a light chain as shown in SEQ ID NO:37;

(5) a heavy chain as shown in SEQ ID NO:39 and a light chain as shown in SEQ ID NO:37;

(6) a heavy chain as shown in SEQ ID NO:34 and a light chain as shown in SEQ ID NO:37; or

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

In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein comprises:

(i) the antibody according to the first aspect of the present invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

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

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

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

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

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

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

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

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

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

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

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

In a fifth aspect of the present invention, a use of an active ingredient is provided, wherein the active ingredient is selected from the group consisting of the antibody according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the immune cell according to the fourth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for:

(a) preparing diagnostic reagents, test panels or test kits; and/or

(b) preparing drugs for preventing and/or treating diseases associated with abnormal expression or function of FGFR2IIIb or its derivative proteins.

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

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

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

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

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

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

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

In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising:

(i) an active ingredient, wherein the active ingredient is selected from the group consisting of the antibody according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the immune cell according to the fourth aspect of the present invention, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

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

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

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

In another preferred embodiment, the pharmaceutical composition is used to prevent and/or treat tumors or cancers.

In the seventh aspect of the present invention, a polynucleotide is provided, which encodes a polypeptide selected from the following group:

(1) The antibody according to the first aspect of the present invention;

(2) the recombinant protein as described in the second aspect of the present invention; or

(3) The chimeric antigen receptor (CAR) construct as described in the third aspect of the present invention.

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

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

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

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

In the tenth aspect of the present invention, a method for detecting FGFR2IIIb or a protein derived therefrom in a sample is provided, the method comprising the steps of:

(1) contacting the sample with the antibody according to the first aspect of the present invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of FGFR2IIIb or its derivative protein in the sample.

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

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

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

In the thirteenth aspect of the present invention, a kit is provided, comprising:

(1) a first container, wherein the first container contains the antibody according to the first aspect of the present invention; and/or

(2) a second container, wherein the second container contains a secondary antibody against the antibody described in the first aspect of the present invention;

or,

The kit contains the detection plate as described in the twelfth aspect of the present invention.

In a fourteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

(a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention.

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

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

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

In another preferred embodiment, the drug is used to prevent and/or treat tumors.

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

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

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

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

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

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 shows the binding activity of 27E9A11 to FGFR2IIIb-his.

FIG2 shows the inhibitory activity of 27E9A11 on FGF7/FGFR2IIIb binding.

FIG3 shows the inhibitory activity of 27E9A11 on FGF10/FGFR2IIIb binding.

FIG4 shows the binding activity of III-0 to human FGFR2IIIb protein.

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

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

FIG. 7 shows the binding activity of III-0 to CHO-FGFR2IIIb cells.

FIG8 shows the binding activity of III-0, III-10, III-11, III-14, and III-15 to human FGFR2IIIb and FGFR2IIIb(S252W) proteins.

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

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

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

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

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

FIG14 shows the ADCC activities of III-10, III-11, III-12, III-13, III-14, and III-15.

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

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

DETAILED DESCRIPTION

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

the term

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

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

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

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

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not occur.

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

FGFR2IIIb

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

There are multiple pathogenic mutations in the FGFR2 gene in somatic and cancer cells, among which S252W (amino acid 252 mutates from serine to tryptophan) is the most common and well-studied mutation site. The S252 site is located in exon 7, between the D2 domain and the D3 domain. When it mutates to tryptophan, it changes the affinity between the ligand and the receptor, promotes ligand-independent FGFR2 dimerization and constitutive activation of FGFR2 kinase, and induces tumors or other diseases. Endometrial adenocarcinoma is the most common tumor type with FGFR2 S252W mutations. 12.52% of patients in this group have FGFR2 mutations, and FGFR2 S252W mutations account for 4.71% of all endometrial adenocarcinoma patients. Apert syndrome is one of the most severe types of human skull syndromes. It is an autosomal dominant hereditary disease, and 2/3 of patients have FGFR2 S252W mutations.

Antibody

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

As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

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

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

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

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

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

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

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

In the present invention, antibodies include murine, 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 parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, such as a chimeric antibody having a variable region from a monoclonal antibody from a mouse, and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

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

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

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

In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

Table A

Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

As used herein, the term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" includes a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibodies on target cells, causing target cell lysis. In various aspects, enhancing ADCC effector function can mean enhanced titer or enhanced efficacy. "Titer" as used in an experimental context means the concentration of the antibody when a specific therapeutic efficacy EC ₅₀ is observed (half-maximal effective concentration). "Efficacy" as used in an experimental context means the maximum possible effector function of an antibody at a saturation level.

Anti-FGFR2IIIb antibody

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 antibodies against FGFR2IIIb and its derivative protein (S252W mutant) described in the first aspect of the present invention.

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

The present invention provides an anti-FGFR2IIIb antibody, the antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain has a complementary determining region (CDR) selected from the following group:

(1) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:7, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(2) VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(3) VH-CDR1 shown in SEQ ID NO:6, VH-CDR2 shown in SEQ ID NO:19, and VH-CDR3 shown in SEQ ID NO:8, wherein the CDRs are defined according to the Kabat rule;

(4) 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 rule; and

(6) VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11, wherein the CDRs are defined according to IMGT rules;

Furthermore, the variable region of the light chain has a complementarity determining region (CDR) selected from the group consisting of:

(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 CDRs are defined according to the Kabat rule;

(2) VL-CDR1 shown in SEQ ID NO: 17, VL-CDR2 shown in SEQ ID NO: 18, and VL-CDR3 shown in SEQ ID NO: 14, wherein the CDRs are defined according to the Kabat rule;

(3) VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:18, and VL-CDR3 shown in SEQ ID NO:20, wherein the CDRs are defined according to the Kabat rule; and

(4) VL-CDR1 shown in SEQ ID NO:40, VL-CDR2 shown in SAS, and VL-CDR3 shown in SEQ ID NO:14, wherein the CDRs are defined according to IMGT rules.

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

Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Biology, 1996. Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Preferably, the antibody described herein is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single-chain antibody (scFv), a single-domain antibody (sdAb), and a single-domain antibody (Signle-domain antibody), as well as a monoclonal antibody or a polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be prepared by a variety of approaches and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.

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

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

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

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

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

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

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

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

Recombinant protein

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

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

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

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

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

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

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

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

Wherein, the preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: obtaining by separation from an expression transformant that recombinantly expresses the protein or obtaining by artificially synthesizing a protein sequence. The separation from the expression transformant that recombinantly expresses the protein is preferably the following method: cloning a polynucleotide molecule encoding the protein and having a point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant, obtaining a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the recombinant protein.

Polynucleotide

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

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

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

Carrier

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

The recombinant expression vector can be obtained by conventional methods in the art, that is, the polynucleotide molecule of the present invention is connected to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned polynucleotide molecule. The vector preferably includes various plasmids, cosmids, phages or virus vectors, etc.

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

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

Antibody preparation

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

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

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

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

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

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

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

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

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

application

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

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

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

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

Test Uses and Kits

The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

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

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

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 comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

Pharmaceutical composition

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

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

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

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

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

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

In the present invention, preferably, the amount of the pharmaceutical composition administered is an effective amount, which is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using experiments that do not exceed routine.

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

Method and composition for detecting FGFR2Ⅲb in samples

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

The meaning of overexpression is conventional in the art, referring 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 increased functional activity (such as in the case of increased enzymatic hydrolysis of the substrate) due to changes in protein transport patterns (increased cell membrane localization).

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

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

The main advantages of the present invention include:

1) The anti-FGFR2IIIb antibody of the present invention can bind to FGFR2IIIb with high affinity and can effectively block the binding of FGFR2IIIb to its ligands FGF1, FGF7 and FGF10.

2) The antibodies of the present invention have excellent binding activity to FGFR2Ⅲb from various species, and at the same time have excellent specificity, and basically do not bind to FGFR2Ⅲc; the antibodies of the present invention have good selectivity for FGFR2Ⅲb relative to FGFR2Ⅲc.

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

4) The antibody of the present invention has a significant tumor-suppressing effect in a tumor-bearing mouse model, and the antibody of the present invention has a significant tumor-suppressing effect even at a low dose.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

Example 1: Acquisition of anti-human FGFR2IIIb antibody

The inventors used FGFR2Ⅲb (D2+D3) (SEQ ID NO: 1) as an antigen to immunize female Balb/c mice aged 6-8 weeks. The spleen cells of the immunized mice were taken for hybridoma cell fusion with SP2/0-AG14 cells, and an appropriate amount of the fused cells were plated on a 96-well plate. The supernatant of each well was taken on the 7th to 10th day after fusion, and the binding activity of the mouse antibodies secreted by the hybridoma cells with human FGFR2Ⅲb-his (ECD) (SEQ ID NO: 2) and FGFR2Ⅲc-his (ECD) (SEQ ID NO: 3) was detected by ELISA, and the inhibitory activity of the mouse antibodies secreted by the hybridoma cells with FGF7/FGFR2Ⅲb, FGF10/FGFR2Ⅲb binding (FGF7 and FGF10 were purchased from Jinan Protein, with the item numbers: CM88 and CR11, respectively). Finally, multiple hybridoma cells that specifically bind to FGFR2Ⅲb-his (ECD) and do not bind to FGFR2Ⅲc-his (ECD) were obtained. Among them, the hybridoma cell with the best inhibition of FGF7/FGFR2Ⅲb and FGF10/FGFR2Ⅲb binding activity was 27E9A11, and its secretory antibody was obtained by sequencing, and the antibody was named 27E9A11 antibody. The heavy chain variable region cDNA sequence and light chain variable region cDNA sequence corresponding to the 27E9A11 antibody, the heavy chain variable region sequence encoded by it is shown in SEQ ID NO: 4; the light chain variable region sequence encoded by it is shown in SEQ ID NO: 5. The antigen complementary determinant cluster (CDR) sequence of the 27E9A11 antibody is shown in Table 1.

Table 1 Antigenic complementarity determining clusters (CDRs) of mouse antibody 27E9A11

Binding activity detection of 27E9A11 antibody to human FGFR2Ⅲb protein

Coat a 96-well high affinity plate with 100 μL/well (100 microliters per well) of a 1 μg/mL human FGFR2Ⅲb-his protein solution at 4°C overnight with shaking. The next day, wash three times with 300 μL PBST (Tween20: 0.5‰), then block with 100 μL/well of 5% BSA/PBS for 2 hours and shake at room temperature. Wash three times with 300 μL PBST. Prepare a gradient dilution solution of the 27E9A11 antibody sample with PBS. Add the solution to a 96-well plate at 100 μL/well and shake at room temperature for 1 hour. Wash three times with 300 μL PBST. Prepare a secondary antibody goat anti-mouse IgG HRP solution (Thermo Fisher, catalog number A16090, the same below), add it to a 96-well plate at 100 μL/well, and shake at room temperature for 30 minutes. Wash four times with 300 μL PBST. 100 μL/well TMB (tetramethylbenzidine) was added to develop color for 3 minutes. 100 μL/well 0.6 NH ₂ SO ₄ was added to stop the color development and the OD ₄₅₀ nm was detected.

Detection of 27E9A11 antibody blocking the binding of human FGF factor (FGF7 or FGF10) to human FGFR2Ⅲb protein

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

Results: The binding activity of 27E9A11 antibody to FGFR2Ⅲb-his is shown in Figure 1, and _EC50 is shown in Table 2; the inhibitory activity of 27E9A11 antibody on FGF7/FGFR2Ⅲb binding is shown in Figure 2, and _IC50 is shown in Table 2; the inhibitory activity of 27E9A11 antibody on FGF10/FGFR2Ⅲb binding is shown in Figure 3, and _IC50 is shown in Table 2.

Table 2 Binding and blocking abilities of antibody 27E9A11

Antibody _EC50 (FGFR2b) IC ₅₀ (FGF7/FGFR2b) IC ₅₀ (FGF10/FGFR2b) 27E9A11 0.050μg/mL 0.417 μg/mL 0.545 μg/mL

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

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

The binding activity of III-0 to human FGFR2IIIb protein was studied, and the detection method was as described in Example 1 (the secondary antibody was changed to goat anti-human IgG HRP). The results are shown in Figure 4, and III-0 can effectively bind to human FGFR2IIIb protein with an EC ₅₀ of 40.42 ng/mL.

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

Coat a 96-well high affinity plate with 1 μg/mL human FGF factor and 5 μg/mL heparin sodium solution at 100 μL/well and shake overnight at 4°C. Wash three times with 300 μL PBST (Tween20: 0.5‰) the next day, then block with 100 μL/well 5% BSA/PBS for 2 hours and shake at room temperature. Wash three times with 300 μL PBST. Prepare gradient dilution solutions of antibody samples with PBS, and add 1 μg/mL FGFR2Ⅲb-mFc at 1:1 to premix. Add 100 μL/well to 96-well plate and shake at room temperature for 2 hours. Wash three times with 300 μL PBST. Prepare secondary goat anti-mouse IgG HRP solution, add 100 μL/well to 96-well plate and shake at room temperature for 1 hour. Wash four times with 300 μL PBST. 100 μL/well TMB (tetramethylbenzidine) was added and color development was carried out for 5 min. 100 μL/well 0.6 NH ₂ SO ₄ was added to stop color development and OD ₄₅₀ nm was detected.

The results are shown in FIG5 . III-0 can effectively block the binding of FGF1/7/10 to FGFR2Ⅲb. Its IC ₅₀ is shown in Table 3 .

Table 3 Blocking ability of antibody III-0

Antibody IC ₅₀ (FGF1/FGFR2b) IC ₅₀ (FGF7/FGFR2b) IC ₅₀ (FGF10/FGFR2b) III-0 0.904 μg/mL 0.694 μg/mL 0.840 μg/mL

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

SNU-16 is a cell derived from human gastric cancer, which naturally overexpresses human FGFR2Ⅲb protein. SNU-16 cells were digested and centrifuged, and resuspended in PBS solution at a density of 2x10 ⁶ cells/mL. After mixing, 100 μL was transferred to a centrifuge tube and centrifuged to discard the supernatant. PBS was used to prepare gradient dilution solutions of antibody samples. 100 μL was added to the centrifuge tube respectively and oscillated at 4°C for 1 hour. The cells were centrifuged, the supernatant was discarded, and 400 μL PBS was used to mix, and repeated 3 times. The secondary antibody goat anti-human IgG (H+L) flow detection antibody (Thermo Fisher, catalog number A21091, the same below) was prepared, and 100 μL/well was added to the centrifuge tube and oscillated at 4°C for 30 minutes. The cells were centrifuged at 2000 rpm for 3 minutes, the supernatant was discarded, and 400 μL PBS was used to mix, and repeated 2 times. Transfer to flow cytometry detection (Beckman, Cytoflex). The results are shown in FIG6 . III-0 can effectively recognize and bind to SNU-16 cells. The binding EC ₅₀ is shown in Table 4 .

Example 5: Binding of chimeric antibodies to FGFR2IIIb protein on CHO-FGFR2IIIb cells

The inventors constructed a CHO-K1 cell line that overexpresses FGFR2Ⅲb protein. CHO-FGFR2Ⅲb cells were digested and centrifuged, and resuspended with PBS solution at a density of 2x10 ⁶ cells/mL. After mixing, 100 μL was transferred to a centrifuge tube, and the supernatant was discarded by centrifugation. A gradient dilution solution of the antibody sample was prepared with PBS. 100 μL was added to the centrifuge tube respectively, and oscillated at 4°C for 1 hour. The cells were centrifuged, the supernatant was discarded, and 400 μL PBS was used to mix, and repeated 3 times. The secondary antibody goat anti-human IgG (H+L) flow detection antibody was prepared, added to the centrifuge tube at 100 μL/well, and oscillated at 4°C for 30 minutes. The cells were centrifuged at 2000 rpm for 3 minutes, the supernatant was discarded, and 400 μL PBS was used to mix, and repeated 2 times. Transfer to a flow cytometer for detection (Beckman, Cytoflex). The results are shown in FIG7 . III-0 can effectively recognize and bind to CHO-FGFR2Ⅲb cells. The binding EC ₅₀ is shown in Table 4 .

Table 4 Binding ability of antibody III-0 to different cells

Antibody EC ₅₀ (SNU-16) _EC50 (CHO-FGFR2b) III-0 1.641 μg/mL 1.010μg/mL

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

The chimeric antibody III-0 was humanized by CDR transplantation to obtain the humanized antibody III-10. Then, III-10 was subjected to affinity maturation. Specifically, single-point saturation mutation was performed on each amino acid site in the CDR region of the III-10 antibody, and the mutation hotspots with antigen-specific binding ability were screened by ELISA, and then these hotspots were combined to obtain candidate antibody mutation sequences. The affinity of the candidate antibodies to the antigen was detected by the SPR method, and finally 5 antibodies were obtained, namely III-11, III-12, III-13, III-14, and III-15. The sequences of these antibodies are shown in the following table.

Table 5 Affinity-modified antibody variable region sequences

(CDR sequence defined by Kabat rules)

S252W is a common mutation of FGFR2Ⅲb on tumors. The binding activity of a series of humanized antibodies of III-0 to FGFR2Ⅲb and FGFR2Ⅲb(S252W) was tested. The detection method is shown in Example 2. The test results are shown in Figure 8. The binding activity of the humanized antibody of III-0 to human FGFR2Ⅲb and FGFR2Ⅲb(S252W) protein is comparable to that of III-0, and the EC ₅₀ of each antibody is shown in Table 6.

Table 6 Affinity-modified antibodies binding to FGFR2IIIb/FGFR2IIIb(S252W)

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

Coat a 96-well high affinity plate with 100 μL/well of a 1 μg/mL human FGFR2Ⅲc protein solution at 4°C overnight with shaking. The next day, wash three times with 300 μL PBST (Tween20: 0.5‰), then block with 100 μL/well of 5% BSA/PBS for 2 hours and shake at room temperature. Wash three times with 300 μL PBST. Prepare a gradient dilution solution of the antibody sample with PBS. Add the solution to a 96-well plate at 100 μL/well and shake at room temperature for 1 hour. Wash three times with 300 μL PBST. Prepare a secondary goat anti-human IgG HRP solution, add it to a 96-well plate at 100 μL/well and shake at room temperature for 30 minutes. Wash four times with 300 μL PBST. Add 100 μL/well TMB (tetramethylbenzidine) and color for 3 minutes. 100 μL/well 0.6 NH ₂ SO ₄ was added to stop color development, and OD ₄₅₀ nm was detected.

The test results are shown in Figure 9. III-0, III-10, and III-11 bind very weakly or not at all to human FGFR2 IIIc protein. III-12, III-13, III-14, and III-15 have weak binding activity to human FGFR2 IIIc protein. The results confirm that the antibodies of the present invention have high specificity, can reduce unnecessary binding in clinical applications, reduce potential side effects, and improve drug safety.

Example 8: Humanized antibodies block the binding of human FGF factors (FGF1/FGF7/FGF10) to human FGFR2IIIb protein

The specific detection method is shown in Example 3. The detection results are shown in Figure 10. Both III-10 and III-0 can block FGF1/FGFR2IIIb binding. The activity of III-10 in blocking FGF10/FGFR2IIIb binding is equivalent to that of III-0. The IC ₅₀ is shown in Table 7.

Table 7 Blocking ability of antibodies III-0 and III-10

Antibody IC ₅₀ (FGF1/FGFR2b) IC ₅₀ (FGF7/FGFR2b) IC ₅₀ (FGF10/FGFR2b) III-0 0.692 μg/mL 0.603 μg/mL 0.745 μg/mL III-10 0.581 μg/mL 0.565 μg/mL 0.724 μg/mL

Example 9: Humanized antibody (affinity maturation) blocks the binding of human FGF factor (FGF1/FGF10) to human FGFR2IIIb protein

The specific detection method is shown in Example 3. The detection results are shown in Figure 11. III-11, III-12, III-13, III-14, and III-15 can all block the binding of FGF1/10 to FGFR2IIIb, and the blocking ability is comparable, as shown in Table 8.

Table 8 Blocking ability of affinity matured antibodies

Antibody IC ₅₀ (FGF1/FGFR2b) IC ₅₀ (FGF10/FGFR2b) III-11 1.179 μg/mL 0.976 μg/mL III-12 1.083μg/mL 0.999μg/mL III-13 0.966μg/mL 0.905 μg/mL III-14 0.974 μg/mL 0.937 μg/mL III-15 0.941 μg/mL 0.805 μg/mL

Example 10: Binding of humanized antibodies to FGFR2IIIb protein on CHO-FGFR2IIIb cells

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

Example 11: Binding of humanized antibodies to FGFR2IIIb protein on SNU-16 cells

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

Table 9 Binding ability of humanized antibodies to different cells

Antibody _EC50 (CHO-FGFR2b) EC ₅₀ (SNU-16) III-0 0.342 μg/mL 0.740 μg/mL III-10 0.257 μg/mL 0.471 μg/mL III-11 0.190 μg/mL 0.319 μg/mL III-14 0.263 μg/mL 0.526 μg/mL III-15 0.260 μg/mL 0.378 μg/mL

Example 12: ADCC effect of humanized antibodies

The inventors constructed a Jurkat-NFAT-Luc-CD16A stable cell line expressing CD16 receptor and NFAT (Nuclear Factor of Activated T-cells) reaction element. CHO-FGFR2Ⅲb was used as the target cell, and the CHO-FGFR2Ⅲb cells were digested and centrifuged, and resuspended in culture medium at a density of 1.3E+06 cells/mL. After mixing, 60 μL was transferred to a 384-well plate and cultured overnight. The gradient dilution solution of the antibody sample was prepared with complete culture medium. The supernatant was discarded from the 384-well plate, and 15 μL of the antibody solution was added to the plate, and pre-incubated at 37°C for 1 hour. Jurkat-NFAT-Luc-CD16A cells were resuspended in complete culture medium, 15 μL of cell suspension was added to each well, and placed in a 37°C incubator for 4 hours. Prepare ONE-Glo ^™ Luciferase detection solution (Promega, catalog number E6110), add 30 μL/well to 384-well plate, react for 1-3 minutes. Transfer to multifunctional microplate reader (Tecan Spark 20M) for detection. The results are shown in Figure 14. The EC50 of each antibody is roughly the same. In terms of activation effect, each antibody can activate immune cells, among which III-14 has the strongest activation effect.

Table 10 ADCC effect of humanized antibodies

Antibody _EC50 (μg/mL) E _max (RFU) III-10 0.383 1170 III-11 0.419 1082 III-12 0.396 1007 III-13 0.404 983 III-14 0.352 1433 III-15 0.388 749

Example 13: Binding of humanized antibodies to mouse/cynomolgus monkey FGFR2IIIb protein

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

The results are shown in Figure 15. III-10 can bind to mouse and cynomolgus monkey FGFR2Ⅲb, and the EC ₅₀ is shown in Table 11.

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

Antibody EC ₅₀ (mouse FGFR2b) EC ₅₀ (Cynomolgus FGFR2b) III-10 0.127 μg/mL 0.128 μg/mL

Example 14: Detection of Antibody Affinity

Surface plasmon resonance (SPR) was used to detect antigen-antibody affinity. Using FGFR2Ⅲb-his as the antigen, a certain concentration of antibody was incubated with a protein A sensor chip for antibody capture. In the antigen binding stage, gradiently diluted FGFR2Ⅲb-his protein was used as the mobile phase to bind to the antibody captured on the sensor chip. In the dissociation stage, HBS-EP buffer was used for continuous elution. The binding of the antibody to FGFR2Ⅲb-his on the sensor chip was quantitatively detected using Biacore 8k (GE Healthcare). The test results are shown in Table 12. The affinity of III-11, III-12, III-13, III-14, and III-15 to FGFR2Ⅲb-his is significantly improved compared to III-0 and III-10.

Table 12 Affinity of anti-human FGFR2Ⅲb antibodies to FGFR2Ⅲb-his

Example 15: Efficacy study of III-10 in the mouse SNU-16 tumor model

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

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

The sequences of the present invention are shown in Table 13 below.

Table 13 Sequence Listing

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

Claims (17)

1. An antibody against FGFR2 iiib, said antibody comprising a heavy chain and a light chain, wherein the variable region of said heavy chain has Complementarity Determining Regions (CDRs) selected from the group consisting of:

(1) A VH-CDR1 as shown in SEQ ID NO. 6, a VH-CDR2 as shown in SEQ ID NO. 7, and a VH-CDR3 as shown in SEQ ID NO. 8, said CDRs being defined according to the Kabat rules;

(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, said CDRs being defined according to the Kabat rules;

(3) A VH-CDR1 as shown in SEQ ID NO. 6, a VH-CDR2 as shown in SEQ ID NO. 19, and a VH-CDR3 as shown in SEQ ID NO. 8, said CDRs being defined according to the Kabat rules;

(4) VH-CDR1 as shown in SEQ ID No. 15, VH-CDR2 as shown in SEQ ID No.19, and VH-CDR3 as shown in SEQ ID No. 8, said CDRs being 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, said CDRs being 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, said CDRs being defined according to IMGT rules;

and, the variable region of the light chain has a Complementarity Determining Region (CDR) selected from the group consisting of:

(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, said CDRs being defined according to the Kabat rules;

(2) VL-CDR1 shown in SEQ ID NO. 17, VL-CDR2 shown in SEQ ID NO. 18, and VL-CDR3 shown in SEQ ID NO. 14, said CDRs being defined according to the Kabat rules;

(3) VL-CDR1 shown in SEQ ID NO. 12, VL-CDR2 shown in SEQ ID NO. 18, and VL-CDR3 shown in SEQ ID NO. 20, said CDRs being 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, said CDRs being defined according to the IMGT rules;

and, any one of the above-mentioned CDR sequences further includes a derivative sequence which is optionally added, deleted, modified and/or substituted for 1 to 2 amino acids and which enables a derivative antibody composed of a heavy chain and a light chain containing the derivative CDR sequence to retain FGFR2 iiib or its derivative protein binding affinity.

2. The antibody of claim 1, wherein said 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) A VH-CDR1 shown in SEQ ID No. 6, a VH-CDR2 shown in SEQ ID No. 7, a VH-CDR3 shown in SEQ ID No. 8, a VL-CDR1 shown in SEQ ID No. 12, a VL-CDR2 shown in SEQ ID No. 13, and a VL-CDR3 shown in SEQ ID No. 14, said CDRs being 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, said CDRs being defined according to the Kabat rules;

(3) A VH-CDR1 as shown in SEQ ID No. 6, a VH-CDR2 as shown in SEQ ID No. 19, a VH-CDR3 as shown in SEQ ID No. 8, a VL-CDR1 as shown in SEQ ID No. 12, a VL-CDR2 as shown in SEQ ID No. 18, and a VL-CDR3 as shown in SEQ ID No. 20, said CDRs being 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, said CDRs being 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, said CDRs being 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, said CDRs being 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, said CDRs being defined according to the IMGT rules.

3. The antibody of claim 1, wherein the heavy chain variable region of the antibody comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID NOs 24, 26, 28, 30, 31 or 4; and/or the light chain variable region of the antibody comprises or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence shown in SEQ ID No. 25, 27, 29 or 5.

4. The antibody of claim 1, wherein the antibody has:

(1) A heavy chain variable region as shown in SEQ ID NO. 24 and a light chain variable region as shown in SEQ ID NO. 25;

(2) A heavy chain variable region as shown in SEQ ID NO. 26 and a light chain variable region as shown in SEQ ID NO. 27;

(3) A heavy chain variable region as set forth in SEQ ID NO. 28 and a light chain variable region as set forth in SEQ ID NO. 29;

(4) A heavy chain variable region as shown in SEQ ID NO. 30 and a light chain variable region as shown in SEQ ID NO. 29;

(5) A heavy chain variable region as shown in SEQ ID NO. 31 and a light chain variable region as shown in SEQ ID NO. 29;

(6) A heavy chain variable region as set forth in SEQ ID NO. 26 and a light chain variable region as set forth in SEQ ID NO. 29; or (b)

(7) A heavy chain variable region as shown in SEQ ID NO. 4 and a light chain variable region as shown in SEQ ID NO. 5.

5. The antibody of claim 1, wherein the heavy chain has an amino acid sequence as shown in or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to SEQ ID No. 32, 34, 36, 38, 39, or 22; and/or the light chain has an amino acid sequence as shown in or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to SEQ ID No. 33, 35, 37 or 23.

6. The antibody of claim 1, wherein the antibody has:

(1) A heavy chain as shown in SEQ ID NO. 32 and a light chain as shown in SEQ ID NO. 33;

(2) A heavy chain as shown in SEQ ID NO. 34 and a light chain as shown in SEQ ID NO. 35;

(3) A heavy chain as shown in SEQ ID NO. 36 and a light chain as shown in SEQ ID NO. 37;

(4) A heavy chain as shown in SEQ ID NO. 38 and a light chain as shown in SEQ ID NO. 37;

(5) A heavy chain as shown in SEQ ID NO. 39 and a light chain as shown in SEQ ID NO. 37;

(6) A heavy chain as shown in SEQ ID NO. 34 and a light chain as shown in SEQ ID NO. 37; or (b)

(7) A heavy chain as shown in SEQ ID NO. 22 and a light chain as shown in SEQ ID NO. 23.

7. A recombinant protein, said recombinant protein comprising:

(i) The antibody of claim 1; and

(Ii) Optionally a tag sequence to assist expression and/or purification.

8. A Chimeric Antigen Receptor (CAR) construct, the antigen binding region of which comprises a single chain variable fragment (scFv) that specifically binds to FGFR2 iiib or a derivative protein thereof, and which scFv has the heavy chain variable region and the light chain variable region of the antibody of claim 1.

9. A recombinant immune cell that expresses the CAR construct of claim 8 exogenously.

10. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises:

(i) An active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 7, the immune cell of claim 9, or a combination thereof; and

(Ii) A pharmaceutically acceptable carrier.

11. Use of an antibody according to claim 1, a recombinant protein according to claim 7, an immune cell according to claim 9, or a pharmaceutical composition according to claim 10 for the manufacture of a medicament for the prevention and/or treatment of a disease.

12. The use according to claim 12, wherein the disease is cancer.

13. The use of claim 12, wherein the cancer is selected from the group consisting of: gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

14. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The antibody of claim 1;

(2) The recombinant protein of claim 7; or (b)

(3) The Chimeric Antigen Receptor (CAR) construct of claim 8.

15. A carrier, characterized in that, the vector contains the polynucleotide of claim 14.

16. An engineered host cell comprising the vector of claim 15 or the polynucleotide of claim 14 integrated into the genome.

17. A test plate, said test plate comprising: a substrate and a test strip comprising the antibody of claim 1, or the recombinant protein of claim 7, or a combination thereof.