WO2025130860A1 - Anti-fgfr2b antibody and use thereof - Google Patents

Abstract

Provided in the present invention are a human FGFR2 antibody and the use thereof. Specifically, disclosed in the present invention is an antibody specifically binding to human FGFR2. The antibody has relatively high selective binding to subtype FGFR2b, has relatively weak binding to other subtypes such as FGFR2c, and has relatively strong killing activity toward tumor cells endogenously expressing FGFR2b such as KATOIII and SNU-16, and thus can be used in the development of drugs and diagnostic reagents for treating FGFR2b positive tumors.

Description

Anti-FGFR2b antibodies and their applications

This disclosure claims the priority of the Chinese patent application filed with the China Patent Office on December 18, 2023, with application number 202311753054.0 and invention name “Anti-FGFR2b antibodies and their applications”, the entire contents of which are incorporated by reference in this disclosure.

Technical Field

The present invention relates to the field of antibodies, and in particular to FGFR2b antibodies and applications thereof.

Background Art

The fibroblast growth factor (FGF) family consists of 22 known FGFs, which are divided into 7 subfamilies based on activity and sequence similarity. Currently, only 4 fibroblast growth factor receptors (FGFRs) 1-4 and their subtypes are known to bind to FGF. When FGFR binds to ligands and heparin, it will induce FGFR to form dimers, and the tyrosine kinase domain in the cell will be autophosphorylated, activating multiple signal transduction pathways in the cell, such as proliferation (STATs, RAS/p38/JNKs, and RAS/MAPK/ERK), survival (STATs and PI3K/AKT) and cytoskeleton regulation (PLC/Ca ²⁺ ), etc., promoting cell proliferation, survival, differentiation, and participating in the regulation of physiological processes in the body, such as embryonic development, organ development, wound healing and angiogenesis. Different subtypes of FGFR2 have different ligands, and FGFR2b is a high-affinity receptor of the FGF7 subfamily. Different subtypes are expressed in different tissues. FGFR2b is mainly expressed in epithelial tissues (such as epithelial cells on the surface of the colorectal lumen, epithelial cells from the base to the lower third of the esophagus, epithelial wall cells on the surface of the gastric cavity, squamous epithelium on the surface of the uterus, vascular smooth muscle cells, etc.), and FGFR2c is mainly expressed in interstitial tissues.

When FGFR is overexpressed and mutated, it will cause abnormalities in the FGFR signaling pathway, promoting the occurrence and development of tumors by promoting cell proliferation, survival, migration and angiogenesis. FGFR2 is overexpressed and mutated in a variety of solid tumors, among which the FGFR2b subtype is expressed in gastric cancer, non-small cell lung cancer squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer. Gastric cancer is a common and highly prevalent malignant tumor in the world, with an annual incidence of more than one million patients and a mortality rate second only to lung cancer and breast cancer. However, the current treatment options for gastric cancer are very limited, especially for the relatively high proportion of HER2-negative patients, there is a lack of effective treatment options. About 30% of patients with HER2-negative advanced gastric cancer and gastroesophageal junction cancer are FGFR2b positive. Immunohistochemical staining, sequencing and other methods have determined that FGFR2b subtypes are specifically highly expressed in gastric cancer tissues rather than FGFR2c subtypes, while FGFR2b expression is not detected in adjacent tissues and normal organs (except skin). FGFR2b can be used as a therapeutic target for these gastric cancer patients. In addition, FGFR2b is highly expressed (2+/3+IHC) in 31% of non-small cell lung cancer squamous cell carcinoma, 13% of triple-negative breast cancer, 40% of ovarian cancer, 4% of pancreatic cancer, and 22% of intrahepatic bile duct cancer. Therefore, the development of treatments targeting FGFR2b is of great significance for the treatment of various cancers.

Summary of the invention

In a first aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 2 (FGFR2), wherein the antibody or the antigen-binding fragment thereof has one or more of the following properties:

(1) Inhibit the binding of FGF7 to FGFR2;

(2) Inhibit FGF7-induced tumor cell proliferation;

(3) binds to FGFR2 with an affinity of no greater than 5×10 ⁻⁸ M; and

(4) Binds to FGFR2b but not to FGFR2c.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region in combination with the following LCDRs and HCDRs:

(1) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 37, 38 and 39, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 34, 35 and 36;

(2) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 43, 44 and 45, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 40, 41 and 42;

(3) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 49, 50 and 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 46, 47 and 48;

(4) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 54, 50 and 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 52, 53 and 48;

(5) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 58, 59 and 60, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 55, 56 and 57;

(6) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 63, 59 and 64, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 46, 61 and 62;

(7) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 68, 69 and 70, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 65, 66 and 67;

(8) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 74, 75 and 39, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 71, 72 and 73;

(9) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 79, 80 and 81, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 76, 77 and 78;

(10) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 85, 86 and 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 82, 83 and 84;

(11) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 90, 86 and 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 87, 88 and 89;

(12) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 94, 86 and 60, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 91, 92 and 93;

(13) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 98, 86 and 64, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 95, 96 and 97;

(14) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 102, 103 and 70, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 99, 100 and 101;

(15) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 43, 44 and 45, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 40, 127 and 42;

(16) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 43, 44 and 45, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 40, 128 and 42;

(17) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 43, 44 and 45, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 40, 129 and 42;

(18) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 49, 50 and 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 46, 135 and 48;

(19) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.54, 50, 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.52, 140, 48;

(20) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 54, 50, 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 52, 141, 48;

(21) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs. 58, 59 and 60, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs. 55, 146 and 57;

(22) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NO.63, 59, 64, and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NO.46, 152, 62; or

(23) A sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the six CDRs described in any one of (1) to (22), or a sequence of six CDRs having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity.

In some embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region and a heavy chain variable region combination as shown below:

The light chain variable region comprises a sequence as shown in SEQ ID NO.19, 21, 23, 25, 27, 29, 31, 118, 119, 122, 123, 130, 131, 132, 136, 142, 143, 144, 147, 148, 153, 154 or 155, and the heavy chain variable region comprises a sequence as shown in SEQ ID NO.18, 20, 22, 24, 26, 28, 30, 120, 121, 124, 125, 126, 133, 134, 137, 138, 139, 145, 149, 150, 151, 156 or 157, or

The light chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region shown in any of the preceding items, and the heavy chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable region shown in any of the preceding items.

In a second aspect, the present disclosure provides a multispecific antigen-binding molecule, which comprises the aforementioned FGFR2 antibody or antigen-binding fragment thereof, and an antigen-binding molecule that binds to an antigen other than FGFR2, or an antigen-binding molecule that binds to a FGFR2 epitope different from the FGFR2 epitope bound by the aforementioned antibody or antigen-binding fragment thereof.

In a third aspect, the present disclosure provides an immunoconjugate comprising the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned multispecific antigen-binding molecule.

In a fourth aspect, the present disclosure provides a chimeric antigen receptor (CAR), which comprises at least a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the aforementioned FGFR2 antibody or its antigen-binding fragment, or the aforementioned multispecific antigen-binding molecule.

In a fifth aspect, the present disclosure provides an immune effector cell, which expresses the aforementioned chimeric antigen receptor, or comprises a nucleic acid fragment encoding the aforementioned chimeric antigen receptor.

In a sixth aspect, the present disclosure provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule or chimeric antigen receptor.

In a seventh aspect, the present disclosure provides a vector comprising the aforementioned isolated nucleic acid fragment.

In an eighth aspect, the present disclosure provides a host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (such as Escherichia coli), fungi (such as yeast), insect cells or mammalian cells (such as CHO cell line or 293T cell line).

In a ninth aspect, the present disclosure provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule.

In a tenth aspect, the present disclosure provides a method for preparing the aforementioned immune effector cells, comprising introducing a nucleic acid fragment encoding CAR into the immune effector cells, and starting the immune effector cells to express the aforementioned CAR.

In an eleventh aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared according to the aforementioned method, immune effector cell prepared according to the aforementioned method; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.

In a twelfth aspect, the present disclosure provides a method for treating a tumor or cancer, comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared according to the aforementioned method, immune effector cell prepared according to the aforementioned method, or the aforementioned pharmaceutical composition; the tumor or cancer is a tumor or cancer expressing FGFR2.

In a thirteenth aspect, the present disclosure provides an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared according to the aforementioned method, immune effector cell prepared according to the aforementioned method, or the aforementioned pharmaceutical composition for use in preparing a drug for treating tumors or cancer; the tumor or cancer is a tumor or cancer expressing FGFR2.

In a fourteenth aspect, the present disclosure provides an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared according to the aforementioned method, immune effector cell prepared according to the aforementioned method, or the aforementioned pharmaceutical composition, for use in treating tumors or cancers; the tumors or cancers are tumors or cancers expressing FGFR2.

In a fifteenth aspect, the present disclosure provides a kit comprising an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared according to the aforementioned method, immune effector cell prepared according to the aforementioned method, or the aforementioned pharmaceutical composition.

In a sixteenth aspect, the present disclosure provides a method for detecting the expression of FGFR2b in a biological sample using the aforementioned FGFR2 antibody or an antigen-binding fragment thereof, or a multispecific antibody.

In a seventeenth aspect, the present disclosure provides a use of the aforementioned FGFR2 antibody or its antigen-binding fragment, or multispecific antibody in the preparation of an FGFR2b detection reagent.

Beneficial effects:

1. The FGFR2 antibody provided by the present disclosure has high selectivity for subtypes, has strong binding activity to FGFR2b, and weak binding to FGFR2c, and has enhanced tumor killing specificity and reduced nonspecific killing of normal organs;

2. The FGFR2 antibody provided by the present disclosure has a higher affinity for FGFR2b and can exert a stronger tumor killing effect at a lower dose.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Binding reaction of chimeric antibody to human FGFR2(β)b protein;

Figure 2. Binding reaction of chimeric antibody to SNU-16 cells;

Figure 3. Chimeric antibodies inhibit cell proliferation induced by the ligand FGF7;

Figure 4. In vitro killing activity of chimeric antibodies;

Figure 5. Binding reaction of humanized antibodies to human FGFR2(β)b protein;

Figure 6. Binding reaction of humanized antibody to KATOIII membrane protein;

Figure 7. Binding reaction of humanized antibodies to SNU-16 cells;

Figure 8. Humanized antibodies inhibit cell proliferation induced by ligand FGF7;

Figure 9. In vitro killing activity of humanized antibodies;

Figure 10. Chimeric antibody regulates SNU-16-#232 tumor growth and weight change curve of tumor-bearing mice;

Figure 11. Humanized antibody regulates KATOIII-#729 tumor growth and weight change curve of tumor-bearing mice;

FIG. 12 . Chimeric antibodies regulate SNU-16-#232 tumor growth and weight change curve of tumor-bearing mice.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Definitions and explanations of terms

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings understood by those of ordinary skill in the art. Furthermore, unless otherwise indicated herein, terms in the singular shall include the plural, and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless expressly indicated otherwise.

The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

The term "and/or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

The term FGFR (Fibroblast Growth Factor Receptors) in this article refers to fibroblast growth factor receptors, which generally include four types: FGFR1-FGFR4 (also known as CD331-334). FGFR is a branch of the tyrosinase receptor family and is a type I transmembrane protein. The extracellular region contains a ligand binding site composed of 2 or 3 immunoglobulin-like domains (IgI to IgIII); a single-pass transmembrane region; and an intracellular region, which contains a tyrosine kinase domain. FGFR usually functions as a dimer. Among them, the extracellular domain contains three Ig-like regions D1 (IgI), D2 (IgII), and D3 (IgIII), of which D1 and Acidic box form an autoinhibitory region; D2 and D3 are responsible for binding to ligands (D2 binds to heparan sulfate on the cell surface, and D3 has two forms, IIIb and IIIc, due to alternative splicing. Only one subtype, IIIc, has been found in FGFR4). According to the number of Ig-like domains, FGFR can be divided into two forms, one is α type, which contains three regions of IgI, IgII, and IgIII; the other is β type, which contains only IgII and IgIII. Exemplarily, FGFR2b in the present disclosure includes "FGFR2(α)b" and "FGFR2(β)b".

Human FGF is classified into 22 types of FGF (FGF1 to FGF14 and FGF16 to FGF23). Except for FGF19, FGF21, and FGF23, which are endocrine, the rest are produced by paracrine, among which FGF7 can only bind to FGFR2 of type IIIb. The binding of FGFR to FGF mediates the activation and transmission of signaling pathways such as RAS-RAF-MAPK, PI3K-AKT, JAK-STAT, and PLCγ. FGFR gene mutations are commonly found in solid tumors such as lung cancer, liver cancer, intrahepatic bile duct cancer, breast cancer, gastric cancer, uterine cancer, and bladder cancer, and the types and frequencies of FGFR mutations in different cancer types also vary.

FGFR has the following activities: (1) binds to FGF; (2) this binding causes FGFR dimerization; (3) this dimerization causes FGFR phosphorylation at specific tyrosine residues in FGFR; (4) this phosphorylation promotes the recruitment of adaptor proteins such as FGFR substrate 2α (FRS2α); and (5) this transduces the signal generated by FGF stimulation to cells or tissues expressing FGFR or activates signal transduction.

The terms "FGFR2b" and "FGFR2IIIb" can be used interchangeably to refer to fibroblast growth factor receptor 2IIIb splicing forms. An exemplary human FGFR2IIIb is shown in GenBank accession number NP_075259.4 (date: July 7, 2013). FGFR2IIIb protein is usually combined with one or two or more FGFs selected from FGF1, FGF3, FGF7 (KGF), FGF10, FGF22 and FGF23. FGFR2IIIb protein can be combined with other FGFs, and may not be combined with mutant forms of the FGFs included in the above group.

The terms "FGFR2c" and "FGFR2IIIc" can be used interchangeably to refer to fibroblast growth factor receptor 2IIIc splicing forms. An exemplary human FGFR2IIIc is shown in GenBank accession number NP_000132.3 (date: July 7, 2013). FGFR2IIIc protein is generally in conjunction with one or two or more FGFs selected from FGF1, FGF2, FGF4, FGF6, FGF9, FGF17, FGF18, FGF21 and FGF23. FGFR2IIIc protein can be in conjunction with other FGFs, and may not be in conjunction with mutant forms of the FGFs included in the above group.

In some embodiments, the antibodies disclosed herein bind to FGFR2b with high affinity and bind to FGFR2c with low affinity or do not bind to FGFR2c. Typically, the antibodies or antigen-binding fragments thereof disclosed herein bind to FGFR2b with an affinity or binding activity 5, 10, 100, 1000 or 10,000 times higher than that of FGFR2c, and the activity can be indicated by detecting the binding strength of the antibody to the protein by methods such as ELISA or FACS.

The term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, sugar, or lipid) that is bound by an antigen binding molecule (e.g., an antibody, antibody fragment, or a scaffold protein comprising an antibody binding region). Epitopes are often composed of chemically active clusters of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural characteristics and specific charge properties. An epitope can be composed of adjacent or juxtaposed non-adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes composed of adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes may include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some instances, if two antibodies show competitive binding to an antigen, they may bind to the same epitope within the antigen.

A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids and/or carbohydrates within an antigenic protein that are bound by an antibody specific for that epitope.

A "linear epitope" comprises contiguous polypeptides, amino acids and/or carbohydrates within an antigenic protein that are bound by an antibody specific for that epitope.

The term "antibody" is used in the broadest sense herein and refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of the heavy chain of an immunoglobulin and/or sufficient sequence from the variable region of the light chain of an immunoglobulin, so as to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds including, for example, biocompatible polymers. Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used to transplant CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

The term "antibody" herein includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Due to the different amino acid compositions and arrangement sequences of the constant regions of the heavy chains of immunoglobulins, their antigenicity is also different. Based on this, the "immunoglobulins" herein can be divided into five categories, or isotypes of immunoglobulins, namely, IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chains, δ chains, γ chains, α chains, and ε chains, respectively. The same class of Ig can be divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the disulfide bonds of the heavy chain, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into κ chains or λ chains by the differences in the constant regions. Each of the five classes of Ig can have κ chains or λ chains.

The term "antibody" in this article also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNARs) found in cartilaginous fish such as sharks.

The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

The term "heavy chain antibody" herein refers to an antibody lacking the light chain of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

In this article, the terms "nanobody" and "single domain antibody" (sdAb) have the same meaning and can be used interchangeably. They refer to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, a heavy chain antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

The term "multi-specificity" herein refers to the ability of an antibody or its antigen-binding fragment to bind, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific", "trispecific", "tetraspecific" and the like refer to the number of different epitopes that an antibody can bind. For example, conventional monospecific IgG type antibodies have two identical antigen binding sites (paratopes), so they can only bind to the same epitope (rather than binding to different epitopes). In contrast, multispecific antibodies have at least two different types of paratopes/binding sites, so they can bind to at least two different epitopes. As described herein, "complementarity determining region" refers to the antigen binding site of an antibody. In addition, a single "specificity" can refer to one, two, three or more than three identical complementary determining regions in a single antibody (the actual number of complementary determining regions/binding sites in a single antibody molecule is referred to as "valence"). For example, a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes. Accordingly, a multispecific antibody comprises at least two (different) complementary determining regions/binding sites. Therefore, the term "multispecific antibody" refers to an antibody having more than one paratope and having the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but also generally includes proteins, e.g. antibodies that specifically bind three or more different epitopes, scaffolds, ie antibodies with three or more paratopes/binding sites.

The term "valent" herein refers to the presence of a specified number of binding sites in an antibody/antigen binding molecule. Thus, the terms "monovalent", "divalent", "tetravalent" and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively.

"Full length antibody" and "intact antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody.

"Antigen-binding fragment" and "antibody fragment" are used interchangeably herein, and they do not have the entire structure of a complete antibody, but only contain a part or a partial variant of a complete antibody, and the part or a partial variant has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, F(ab') ₂ , Fab', Fab'-SH, Fd, Fv, scFv, diabody, and single domain antibody.

The term "chimeric antibody" herein refers to an antibody having variable sequences of an immunoglobulin derived from one source organism (such as rat, mouse, rabbit or alpaca) and constant regions of an immunoglobulin derived from a different organism (such as human). Methods for producing chimeric antibodies are known in the art.

The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and/or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.

The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

The term "variable region" herein refers to the region of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen, and "heavy chain variable region" is used interchangeably with "VH" and "HCVR", and "light chain variable region" is used interchangeably with "VL" and "LCVR". The variable domains of the heavy and light chains of natural antibodies generally have similar structures, each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "complementarity determining region" is used interchangeably with "CDR" herein, and generally refers to the hypervariable region (HVR) found in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). As understood in the art, the amino acid position representing the hypervariable region of an antibody can vary according to the context and various definitions known in the art. Some positions within the variable domain can be considered as hybrid hypervariable positions, because these positions can be considered to be within the hypervariable region under a set of standards (such as IMGT or KABAT), and are considered to be outside the hypervariable region under different sets of standards (such as KABAT or IMGT). One or more of these positions can also be found in an extended hypervariable region. The present application includes antibodies comprising modifications in these hybrid hypervariable positions. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region can be abbreviated as LCDR. The variable domains of the native heavy and light chains each include four framework regions that mainly adopt a sheet configuration, which are connected by three CDRs (CDR1, CDR2 and CDR3), which form a loop connecting the sheet structure, and in some cases form a part of the sheet structure. The CDRs in each chain are held together in close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with the CDRs from other antibody chains contribute to the formation of the antibody antigen binding site.

"CDR" herein can be annotated and defined by methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http://cao.labshare.cn/AbRSA/cdrs.php), the abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and the IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results). CDR herein includes overlaps and subsets of amino acid residues defined in different ways.

The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat. The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions. The term "IMGT numbering system" herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al.

The term "heavy chain constant region" herein refers to the carboxyl terminal portion of the heavy chain of an antibody, which is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interactions with Fc receptors, which have a more conservative amino acid sequence relative to the variable domains of the antibody. The "heavy chain constant region" is selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment", the former having a structure substantially similar to that of a natural antibody constant region, while the latter only includes "a portion of the full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" is selected from an Fc or CH3 domain.

The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to an antigen, and the light chain constant region is selected from a constant kappa domain or a constant lambda domain.

The term "Fc region" herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Exemplarily, the human IgG heavy chain Fc region may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, removing one or more, particularly one or two amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447) may be present or absent. Typically, an IgG Fc region comprises an IgG CH2 and an IgG CH3 domain, and optionally, may further comprise a complete or partial hinge region, but does not comprise a CH1 domain. The "CH2 domain" of a human IgG Fc region generally extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the residues at the C-terminus of the CH2 domain in the Fc region (i.e., from an amino acid residue at about position 341 of IgG to an amino acid residue at about position 447). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced in one of its chains and a "cavity" or "hole" introduced correspondingly in the other of its chains; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). As described herein, such variant CH3 domains may be used to promote heterodimerization of two different antibody heavy chains. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index.

The term "Fc variant" herein refers to changes in Fc structure or function caused by one or more amino acid substitutions, insertions or deletions at appropriate sites on Fc. "Interactions between Fc variants" refers to the formation of space filling effects, electrostatic guidance, hydrogen bonding, hydrophobic interactions, etc. between Fc variants designed by mutation. Interactions between Fc variants help to form stable heterodimeric proteins. The preferred mutation design is a "Knob-into-Hole" form of mutation design.

The mutation design technology of Fc variants has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion protein forms. Representative ones include the "Knob-into-Hole" form proposed by Cater et al.; the heterodimeric form containing Fc formed by electrostatic steering (Electrostatic Steering) by Amgen technicians (US20100286374A1); the heterodimeric form (SEEDbodies) formed by IgG/Ig chain exchange proposed by Jonathan H.Davis et al.; the bispecific molecules formed by DuoBody platform technology of Genmab; the heterodimeric protein form formed by Xencor technicians through comprehensive structural calculation and Fc amino acid mutation, and different modes of action; the heterodimeric protein form obtained by the charge network-based Fc modification method (CN201110459100.7) of Suzhou Alphamab; and other genetic engineering methods based on Fc amino acid changes or functional modification methods to achieve the formation of heterodimeric functional proteins. The Knob/Hole structure on the Fc variant fragment described in the present application refers to two Fc fragments that are mutated separately, and after the mutation, they can be combined in the form of "Knob-into-Hole". It is preferred to use the "knob-into-hole" model of Cater et al. to perform site mutation transformation on the Fc region, so that the first Fc variant and the second Fc variant obtained can be combined together in the form of "knob-into-hole" to form a heterodimer. It is within the scope of those skilled in the art to select a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass. The Fc region of human antibodies IgG1, IgG2, IgG3, and IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred. Randomly select one of the first Fc variant or the second Fc variant to make a knob mutation and the other to make a hole mutation. (The above content is incorporated herein by reference).

An antibody with "altered" FcR binding affinity or ADCC activity is one that has enhanced or diminished FcR binding affinity and/or ADCC activity compared to a parent antibody, wherein the antibody or parent antibody differs in at least one structural aspect. An antibody that "exhibits increased binding" to an FcR binds to at least one FcR with a better affinity than the parent antibody. An antibody that "exhibits decreased binding" to an FcR binds to at least one FcR with a lower affinity than the parent antibody. An antibody that exhibits decreased binding to an FcR may have little or no detectable binding to an FcR, e.g., 0-20% binding to an FcR compared to a native sequence IgG Fc region.

"Enhanced affinity for FcγRIIIA" refers to an antibody having a higher affinity for FcγRIIIA (also referred to as CD16a in some cases) than a parent antibody, wherein the antibody or parent antibody differs in at least one structural aspect. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated and the parent antibody is fucosylated. Any suitable method for determining affinity for FcγRIIIA can be used. In some embodiments, affinity for FcγRIIIA is determined by the methods described herein. In some embodiments, the antibody with enhanced affinity for FcγRIIIA has enhanced ADCC activity. In some embodiments, the antibody with enhanced affinity for FcγRIIIA has enhanced affinity for FcγRIIIA(V158). In some embodiments, the antibody with enhanced affinity for FcγRIIIA has enhanced affinity for FcγRIIIA(F158).

The term "Affibody" refers to a small, non-immunoglobulin affinity protein that has a triple-stranded β-sheet structure. Its microstructure is a compact spherical structure and it can bind to specific target molecules with high affinity and low immunogenicity.

The term "conservative amino acid" herein generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Exemplarily, the amino acids within each of the following groups are conservative amino acid residues of each other, and the replacement of the amino acid residues within the group is a replacement of conservative amino acids:

1) Alanine (A), serine (S), threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), Lysine (K), Histidine (H);

5) isoleucine (I), leucine (L), methionine (M), valine (V); and

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percent identity between the two sequences varies with the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced for optimal comparison of the two sequences and the length of each space.

Mathematical algorithms can be used to compare sequences between two sequences and calculate the percent identity. For example, using the Needlema and Wunsch algorithm (available at www.gcg.com) that has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6, the percent identity between two amino acid sequences is determined. For another example, using the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6, the percent identity between two nucleotide sequences is determined. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossum62 scoring matrix using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present application can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present application. In order to obtain gapped alignment results for comparison purposes, gapped BLAST can be used. When using BLAST and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, which comprises at least (1) an extracellular antigen binding domain, such as a heavy chain variable region and/or a light chain variable region of an antibody, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain.

The term "signal peptide" is used interchangeably and refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of the polypeptide from mammalian cells. The leader sequence may be cleaved during export of the polypeptide from mammalian cells to form the mature protein. Leader sequences may be natural or artificial, and they may be heterologous or homologous to the protein to which they are attached. Exemplarily, the signal sequence may comprise MGWSWILLFLLSVTAGVHS (SEQ ID NO: 158).

The term "nucleic acid" herein includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone bonding or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for direct expression of the antibodies of the present application in vitro and/or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA carrier and/or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo.

An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

The term "vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "host cell" herein refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initially transformed cell are included herein.

The term "pharmaceutical composition" herein refers to a preparation which is in a form which permits the biological activity of the active ingredient contained therein to be effective, and which contains no additional ingredients which are unacceptably toxic to a subject to which the pharmaceutical composition is administered.

The term "pharmaceutically acceptable carrier" herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. and combinations thereof, which are known to those skilled in the art. Except for situations where they are incompatible with the active ingredient, any conventional carrier is considered for use in treatment or pharmaceutical compositions.

The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include those who already have a condition or disease, as well as those who are susceptible to a condition or disease or those for whom a condition or disease is to be prevented. When referring to terms such as slow down, alleviate, weaken, alleviate, and relieve, their meanings also include elimination, disappearance, non-occurrence, and the like.

The term "subject" herein refers to an organism that is treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, a primate (e.g., a monkey), or a non-primate mammal that is treated for a disease or condition.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent that is effective in preventing or alleviating a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. "Effective amount" also refers to an amount of a compound sufficient to relieve symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevent, or alleviate these conditions. When an active ingredient is administered alone to an individual, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

The term "cancer" herein refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. The term "tumor" or "neoplasm" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

Non-limiting example cancers include gastric cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer and esophageal cancer. In some embodiments, cancer comprises FGFR2 gene amplification. In some embodiments, FGFR2 amplification includes>3 FGFR2:CEN10 (chromosome 10 centromere) ratio. In some embodiments, the cancer comprising FGFR2 gene overexpresses FGFR2IIIb. In some embodiments, the degree of overexpression of FGFR2IIIb in cancer comprising FGFR2 overexpression is higher than FGFR2IIIc. In some embodiments, the normalized level of FGFR2IIIb expressed by cancer comprising FGFR2 amplification exceeds 2 times, 3 times, 5 times or 10 times of the normalized level expressed by FGFR2IIIc. In some embodiments, the expression level is normalized to GUSB. In some embodiments, cancer overexpresses FGFR2IIIb but does not include FGFR2 gene amplification. In some embodiments, gastric cancer comprises FGFR2 gene amplification. In some embodiments, gastric cancer comprising FGFR2 gene amplification overexpresses FGFR2IIIb. In some embodiments, gastric cancer comprising FGFR2 gene amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, gastric cancer comprising FGFR2 gene amplification expresses FGFR2IIIb at a normalized level that is more than 2 times, 3 times, 5 times or 10 times the normalized level expressed by FGFR2IIIc. In some embodiments, the expression level is normalized to GUSB. In some embodiments, gastric cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification. In some embodiments, overexpression is mRNA overexpression. In some embodiments, overexpression is protein overexpression.

The term "EC50" herein refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the antibody concentration at which 50% of its maximal effect is observed, and can be measured by methods known in the art.

DETAILED DESCRIPTION OF THE INVENTION

Antibodies that bind to human FGFR2

In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 2 (FGFR2), wherein the antibody or the antigen-binding fragment thereof comprises one or more of the following properties:

(1) Inhibit the binding of FGF7 to FGFR2;

(2) Inhibit FGF7-induced tumor cell proliferation;

(3) binds to FGFR2 with an affinity of no greater than 5×10 ⁻⁸ M; and

(4) Binds to FGFR2b but not to FGFR2c.

In some embodiments, the antigen-binding fragment is selected from one or more of F(ab) ₂ , Fab', Fab, Fv fragment, scFv, nanobody or affibody.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and/or a heavy chain variable region.

In some embodiments, the antibody or antigen-binding fragment thereof binds to human, mouse or monkey FGFR2.

In some embodiments, the antibody or antigen-binding fragment thereof is chimeric, humanized, or fully human.

In some embodiments, the antibody or antigen-binding fragment thereof is defucosylated.

CDR

The CDR of an antibody is a part of the variable region and is a key part in determining the specificity of an antibody. The CDR of an antibody can be determined by a variety of coding systems, generally including CCG, Kabat, CHothia, IMGT, AbM, etc. In the present disclosure, the CDR generally covers the CDR sequence obtained according to any CDR division method, and also covers its variants, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; and also covers its homologs, wherein the homologs have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity sequences with the amino acid sequence of the CDR. In certain embodiments, the isolated antibodies or antigen-binding proteins described in the present disclosure are defined by the Kabat or IMGT numbering system.

In the present disclosure, the antibody or antigen-binding fragment thereof comprises three LCDRs of the light chain variable region: LCDR1, LCDR2 and LCDR3.

In some embodiments, the LCDRs may comprise LCDRs in a light chain variable region sequence as shown in SEQ ID NO. 19, 21, 23, 25, 27, 29, 31, 118, 119, 122, 123, 130, 131, 132, 136, 142, 143, 144, 147, 148, 153, 154 or 155.

In some embodiments, the LCDRs may include LCDR1, LCDR2 and LCDR3 of the following sequence:

(1) LCDR1 as shown in SEQ ID NO.37, LCDR2 as shown in SEQ ID NO.38, and LCDR3 as shown in SEQ ID NO.39;

(2) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44, and LCDR3 as shown in SEQ ID NO.45;

(3) LCDR1 as shown in SEQ ID NO.49, LCDR2 as shown in SEQ ID NO.50, and LCDR3 as shown in SEQ ID NO.51;

(4) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50, and LCDR3 as shown in SEQ ID NO.51;

(5) LCDR1 as shown in SEQ ID NO.58, LCDR2 as shown in SEQ ID NO.59, and LCDR3 as shown in SEQ ID NO.60;

(6) LCDR1 as shown in SEQ ID NO.63, LCDR2 as shown in SEQ ID NO.59, and LCDR3 as shown in SEQ ID NO.64;

(7) LCDR1 as shown in SEQ ID NO.68, LCDR2 as shown in SEQ ID NO.69, and LCDR3 as shown in SEQ ID NO.70;

(8) LCDR1 as shown in SEQ ID NO.74, LCDR2 as shown in SEQ ID NO.75, and LCDR3 as shown in SEQ ID NO.39;

(9) LCDR1 as shown in SEQ ID NO.79, LCDR2 as shown in SEQ ID NO.80, and LCDR3 as shown in SEQ ID NO.81;

(10) LCDR1 as shown in SEQ ID NO.85, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.51;

(11) LCDR1 as shown in SEQ ID NO.90, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.51;

(12) LCDR1 as shown in SEQ ID NO.94, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.60;

(13) LCDR1 as shown in SEQ ID NO.98, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.64;

(14) LCDR1 as shown in SEQ ID NO.102, LCDR2 as shown in SEQ ID NO.103 and LCDR3 as shown in SEQ ID NO.70; or

(15) LCDRs whose sequences have 1, 2, 3 amino acid substitutions, deletions and/or insertions or have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any of the groups of LCDRs in (1) to (14).

In some embodiments, the antibody or antigen-binding fragment thereof comprises three HCDRs of the heavy chain variable region: HCDR1, HCDR2, and HCDR3.

In some embodiments, the HCDRs may comprise HCDRs in a heavy chain variable region sequence as shown in SEQ ID NO. 18, 20, 22, 24, 26, 28, 30, 120, 121, 124, 125, 126, 133, 134, 137, 138, 139, 145, 149, 150, 151, 156 or 157.

In some embodiments, the HCDRs may comprise HCDR1, HCDR2 and HCDR3 with the following sequences:

(1) HCDR1 as shown in SEQ ID NO.34, HCDR2 as shown in SEQ ID NO.35, and HCDR3 as shown in SEQ ID NO.36;

(2) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.41, and HCDR3 as shown in SEQ ID NO.42;

(3) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.47, and HCDR3 as shown in SEQ ID NO.48;

(4) HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.53 and HCDR3 as shown in SEQ ID NO.48;

(5) HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.56 and HCDR3 as shown in SEQ ID NO.57;

(6) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.61, and HCDR3 as shown in SEQ ID NO.62;

(7) HCDR1 as shown in SEQ ID NO.65, HCDR2 as shown in SEQ ID NO.66, and HCDR3 as shown in SEQ ID NO.67;

(8) HCDR1 as shown in SEQ ID NO.71, HCDR2 as shown in SEQ ID NO.72, and HCDR3 as shown in SEQ ID NO.73;

(9) HCDR1 as shown in SEQ ID NO.76, HCDR2 as shown in SEQ ID NO.77, and HCDR3 as shown in SEQ ID NO.78;

(10) HCDR1 as shown in SEQ ID NO.82, HCDR2 as shown in SEQ ID NO.83, and HCDR3 as shown in SEQ ID NO.84;

(11) HCDR1 as shown in SEQ ID NO.87, HCDR2 as shown in SEQ ID NO.88 and HCDR3 as shown in SEQ ID NO.89;

(12) HCDR1 as shown in SEQ ID NO.91, HCDR2 as shown in SEQ ID NO.92, and HCDR3 as shown in SEQ ID NO.93;

(13) HCDR1 as shown in SEQ ID NO.95, HCDR2 as shown in SEQ ID NO.96 and HCDR3 as shown in SEQ ID NO.97;

(14) HCDR1 as shown in SEQ ID NO.99, HCDR2 as shown in SEQ ID NO.100, and HCDR3 as shown in SEQ ID NO.101;

(15) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.127, and HCDR3 as shown in SEQ ID NO.42;

(16) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.128 and HCDR3 as shown in SEQ ID NO.42;

(17) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.129 and HCDR3 as shown in SEQ ID NO.42;

(18) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.135, and HCDR3 as shown in SEQ ID NO.48;

(19) HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.140 and HCDR3 as shown in SEQ ID NO.48;

(20) HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.141, and HCDR3 as shown in SEQ ID NO.48;

(21) HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.146, and HCDR3 as shown in SEQ ID NO.57;

(22) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.152 and HCDR3 as shown in SEQ ID NO.62; or

(23) HCDRs whose sequences have 1, 2, 3 amino acid substitutions, deletions and/or insertions or are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any of the HCDRs in (1) to (22).

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region in combination with the following LCDRs and HCDRs:

(1) LCDR1 as shown in SEQ ID NO.37, LCDR2 as shown in SEQ ID NO.38 and LCDR3 as shown in SEQ ID NO.39, and HCDR1 as shown in SEQ ID NO.34, HCDR2 as shown in SEQ ID NO.35 and HCDR3 as shown in SEQ ID NO.36;

(2) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.41 and HCDR3 as shown in SEQ ID NO.42;

(3) LCDR1 as shown in SEQ ID NO.49, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.47 and HCDR3 as shown in SEQ ID NO.48;

(4) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.53 and HCDR3 as shown in SEQ ID NO.48;

(5) LCDR1 as shown in SEQ ID NO.58, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.60, and HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.56 and HCDR3 as shown in SEQ ID NO.57;

(6) LCDR1 as shown in SEQ ID NO.63, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.64, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.61 and HCDR3 as shown in SEQ ID NO.62;

(7) LCDR1 as shown in SEQ ID NO.68, LCDR2 as shown in SEQ ID NO.69 and LCDR3 as shown in SEQ ID NO.70, and HCDR1 as shown in SEQ ID NO.65, HCDR2 as shown in SEQ ID NO.66 and HCDR3 as shown in SEQ ID NO.67;

(8) LCDR1 as shown in SEQ ID NO.74, LCDR2 as shown in SEQ ID NO.75 and LCDR3 as shown in SEQ ID NO.39, and HCDR1 as shown in SEQ ID NO.71, HCDR2 as shown in SEQ ID NO.72 and HCDR3 as shown in SEQ ID NO.73;

(9) LCDR1 as shown in SEQ ID NO.79, LCDR2 as shown in SEQ ID NO.80 and LCDR3 as shown in SEQ ID NO.81, and HCDR1 as shown in SEQ ID NO.76, HCDR2 as shown in SEQ ID NO.77 and HCDR3 as shown in SEQ ID NO.78;

(10) LCDR1 as shown in SEQ ID NO.85, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.82, HCDR2 as shown in SEQ ID NO.83 and HCDR3 as shown in SEQ ID NO.84;

(11) LCDR1 as shown in SEQ ID NO.90, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.87, HCDR2 as shown in SEQ ID NO.88 and HCDR3 as shown in SEQ ID NO.89;

(12) LCDR1 as shown in SEQ ID NO.94, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.60, and HCDR1 as shown in SEQ ID NO.91, HCDR2 as shown in SEQ ID NO.92 and HCDR3 as shown in SEQ ID NO.93;

(13) LCDR1 as shown in SEQ ID NO.98, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.64, and HCDR1 as shown in SEQ ID NO.95, HCDR2 as shown in SEQ ID NO.96 and HCDR3 as shown in SEQ ID NO.97;

(14) LCDR1 as shown in SEQ ID NO.102, LCDR2 as shown in SEQ ID NO.103 and LCDR3 as shown in SEQ ID NO.70, and HCDR1 as shown in SEQ ID NO.99, HCDR2 as shown in SEQ ID NO.100 and HCDR3 as shown in SEQ ID NO.101;

(15) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.127 and HCDR3 as shown in SEQ ID NO.42;

(16) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.128 and HCDR3 as shown in SEQ ID NO.42;

(17) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.129 and HCDR3 as shown in SEQ ID NO.42;

(18) LCDR1 as shown in SEQ ID NO.49, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.135 and HCDR3 as shown in SEQ ID NO.48;

(19) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.140 and HCDR3 as shown in SEQ ID NO.48;

(20) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.141 and HCDR3 as shown in SEQ ID NO.48;

(21) LCDR1 as shown in SEQ ID NO.58, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.60, and HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.146 and HCDR3 as shown in SEQ ID NO.57;

(22) LCDR1 as shown in SEQ ID NO.63, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.64, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.152 and HCDR3 as shown in SEQ ID NO.62; or,

(23) A sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the six CDRs described in any one of (1) to (22), or a sequence of six CDRs having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity.

Antibody variable region

In the present disclosure, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH).

In some embodiments, the light chain variable region comprises a sequence as shown in SEQ ID NO. 19, 21, 23, 25, 27, 29, 31, 118, 119, 122, 123, 130, 131, 132, 136, 142, 143, 144, 147, 148, 153, 154 or 155, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the shown sequence.

In some embodiments, the heavy chain variable region comprises a sequence as shown in SEQ ID NO. 18, 20, 22, 24, 26, 28, 30, 120, 121, 124, 125, 126, 133, 134, 137, 138, 139, 145, 149, 150, 151, 156 or 157, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the shown sequence.

In some embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region and a heavy chain variable region combination as shown below:

(1) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.19 and SEQ ID NO.18, respectively;

(2) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.21 and SEQ ID NO.20, respectively;

(3) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.23 and SEQ ID NO.22, respectively;

(4) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.25 and SEQ ID NO.24, respectively;

(5) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.27 and SEQ ID NO.26, respectively;

(6) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.29 and SEQ ID NO.28, respectively;

(7) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.31 and SEQ ID NO.30, respectively;

(8) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.118 and SEQ ID NO.120, respectively;

(9) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.118 and SEQ ID NO.121, respectively;

(10) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.119 and SEQ ID NO.120, respectively;

(11) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.122 and SEQ ID NO.126, respectively;

(12) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.123 and SEQ ID NO.124, respectively;

(13) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.123 and SEQ ID NO.125, respectively;

(14) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.130 and SEQ ID NO.133, respectively;

(15) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.131 and SEQ ID NO.134, respectively;

(16) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.132 and SEQ ID NO.134, respectively;

(17) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.136 and SEQ ID NO.137, respectively;

(18) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.136 and SEQ ID NO.138, respectively;

(19) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.136 and SEQ ID NO.139, respectively;

(20) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.142 and SEQ ID NO.145, respectively;

(21) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.143 and SEQ ID NO.145, respectively;

(22) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.144 and SEQ ID NO.145, respectively;

(23) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.147 and SEQ ID NO.149, respectively;

(24) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.148 and SEQ ID NO.150, respectively;

(25) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.147 and SEQ ID NO.151, respectively;

(26) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.153 and SEQ ID NO.156, respectively;

(27) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.154 and SEQ ID NO.157, respectively;

(28) The light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO.155 and SEQ ID NO.156; or

(29) The light chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region shown in any one of (1) to (28) above, and the heavy chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable region shown in any one of (1) to (28) above.

Antibody constant region

In the present disclosure, the antibody or antigen-binding fragment thereof may comprise a light chain and/or heavy chain constant region sequence.

In some embodiments, the heavy chain constant region may comprise the heavy chain constant region sequence of human or murine antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

In some embodiments, the heavy chain constant region comprises the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4.

In some embodiments, the heavy chain constant region comprises a heavy chain constant region derived from a human IgG1 antibody.

In some embodiments, the light chain constant region comprises the constant region of a human or murine λ or κ chain.

Multispecific Antibodies

In another aspect, the present disclosure provides a multispecific antigen-binding molecule comprising the aforementioned FGFR2 antibody or antigen-binding fragment thereof, and an antigen-binding molecule that binds to an antigen other than FGFR2, or binds to a different FGFR2 epitope than the aforementioned antibody or antigen-binding fragment thereof.

In some embodiments, other antigens besides FGFR2 may include: CD3 (preferably CD3ε), CD16, NKG2D, NKp46, NKp30, CD137, CD258, PD-1, PD-L1, 4-1BB, CD40, CD64, EGFR, VEGF, HER2, HER1, HER3, IGF-1R, phosphatidylserine (PS), C-Met, HSA, MSLN, blood-brain barrier receptor, GPC3, PSMA, CD33, GD2, ROR1, ROR2, FRα or Gucy2C.

In some embodiments, the antigen-binding molecule for the other antigen is an antibody or an antigen-binding fragment thereof.

In some embodiments, the multispecific antigen-binding molecule can be bispecific, trispecific, or tetraspecific.

In some embodiments, the multispecific antigen-binding molecule can be bivalent, trivalent, tetravalent, pentavalent, or hexavalent.

Immunoconjugates

In another aspect, the present disclosure provides an immunoconjugate comprising the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned multispecific antigen-binding molecule.

In some embodiments, the immunoconjugate further comprises a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent and a photosensitizer.

Immune effector cells

On the other hand, the present disclosure provides a chimeric antigen receptor (CAR), which comprises at least a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the aforementioned FGFR2 antibody or its antigen-binding fragment, or the aforementioned multispecific antigen-binding molecule.

In another aspect, the present disclosure provides an immune effector cell, which expresses the aforementioned chimeric antigen receptor, or comprises a nucleic acid fragment encoding the aforementioned chimeric antigen receptor.

In some embodiments, the immune effector cells are selected from T cells, NK cells, NKT cells, DNT cells, monocytes, macrophages, dendritic cells or mast cells.

In some embodiments, the T cell is selected from a cytotoxic T cell (CTL), a regulatory T cell (Treg), or a helper T cell (Th).

In some embodiments, the immune effector cells are autologous immune effector cells or allogeneic immune effector cells.

On the other hand, the present disclosure provides a method for preparing the aforementioned immune effector cell, comprising introducing a nucleic acid fragment encoding CAR into the immune effector cell, and starting the immune effector cell to express the aforementioned CAR.

Nucleic acid molecules

In another aspect, the present disclosure provides one or more isolated nucleic acid molecules, which may be nucleotides, deoxynucleotides and/or ribonucleotides of any length in isolated form, encoding the aforementioned antibodies or antigen-binding fragments thereof, multispecific antigen-binding molecules or chimeric antigen receptors.

Carrier

On the other hand, the disclosure provides a vector comprising the aforementioned isolated nucleic acid fragment. The vector can transform, transduce or transfect a host cell so that the genetic material elements it carries are expressed in the host cell. For example, the vector may include a promoter, a transcriptor, an enhancer, a replicon, a selection element and a reporter gene, etc. For example, the vector may include a component that assists in entering the cell. In order to replicate the nucleic acid molecule in the vector, the 5' end and the 3' end of the nucleic acid molecule may also include a long terminal repeat sequence (LTR).

Host cells

In another aspect, the present disclosure provides a host cell comprising the aforementioned isolated nucleic acid molecule or isolated vector.

In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterium (eg, E. coli), a fungus (eg, yeast), an insect cell, or a mammalian cell (eg, a CHO cell line or a 293T cell line).

The cell may include progeny of a single cell. Progeny may not necessarily be completely identical (either in the morphology of total DNA complement or in genome) to the original parent cell due to natural, accidental or deliberate mutation.

In some embodiments, the host cell is genetically modified such that the expression of fucosyltransferase is reduced or absent.

In some embodiments, the fucosyltransferase comprises an α-1,6-fucosyltransferase.

In some embodiments, the genetic modification comprises knocking out the FUT8 gene of the host cell.

Pharmaceutical composition

In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, the aforementioned isolated nucleic acid molecule, immune effector cells, and optionally a pharmaceutically acceptable adjuvant.

In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, stabilizers, excipients, solubilizers, surfactants, emulsifiers and/or preservatives.

In some embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

In some embodiments, the pharmaceutical composition can be administered parenterally, percutaneously, intracavitary, intraarterially, intrathecally and/or intranasally or injected directly into a tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection.

Combination or co-administration of drugs

On the other hand, the present disclosure provides a combination of drugs, which refers to a product produced by mixing or combining multiple active ingredients, and can be divided into fixed and non-fixed combinations.

The term "fixed combination" refers to an active ingredient, such as one or more of the antibodies or antigen-binding fragments thereof, multispecific antibodies or immune effector cells described in the present disclosure, combined with one or more other active ingredients, in the form of a single entity (such as a mixed injection) or fixed specifications and dosages, which are simultaneously applied to patients or subjects.

The term "non-fixed combination" means that one or more active ingredients, such as the antibodies or antigen-binding fragments thereof, multispecific antibodies or immune effector cells described in the present disclosure, are administered to a patient or subject simultaneously, concurrently or sequentially (without specific time limits) as separate entities with other one or more active ingredients, and such administration provides therapeutically effective levels of the two or more active ingredients in the patient or subject.

In some embodiments, the one or more other active ingredients comprise an anti-tumor agent.

In some embodiments, the pharmaceutical composition can be administered by various routes, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

Preparation method

In another aspect, the present disclosure provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule, the method comprising culturing the cell under conditions such that the antigen-binding protein is expressed.

use

On the other hand, the present disclosure provides a method for treating tumors or cancer, comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, a product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition.

On the other hand, the present disclosure provides a use of an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule, immune effector cell prepared according to the aforementioned method, or the aforementioned pharmaceutical composition in the preparation of a drug for treating tumors or cancer.

On the other hand, the present disclosure provides an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition for treating tumors or cancer.

In some embodiments, the tumor or cancer is a tumor or cancer expressing FGFR2, such as gastric cancer, non-small cell lung cancer, squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, colorectal cancer.

In another aspect, the present disclosure provides a method for blocking the interaction between FGFR2 and its ligand (such as FGF7), comprising administering the antibody or antigen-binding fragment thereof, multispecific antibody or pharmaceutical composition to a subject in need thereof.

Detection Kits

In another aspect, the present disclosure provides a kit comprising an effective amount of the aforementioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, immune effector cell, a product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition.

In another aspect, the present disclosure provides a method for detecting the expression of FGFR2b in a biological sample using the aforementioned FGFR2 antibody or antigen-binding fragment thereof, or multispecific antibody.

In another aspect, the present disclosure provides a use of the aforementioned FGFR2 antibody or antigen-binding fragment thereof, or multispecific antibody in the preparation of a FGFR2b detection reagent.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

Example

Example 1: Preparation of FGFR2-related antigens, control antibodies and construction of stable cell lines

1.1 Preparation of FGFR2 antigen

Human FGFR2(α)b protein (Uniprot:P21802-3), human FGFR2(β)b protein (NCBI:NP_001138391.1), human FGFR2(β)c protein (NCBI:NP_001138387.1), cynomolgus monkey FGFR2(β)b protein (Uniprot:A0A2K5TLA3), mouse FGFR2(α)b protein (GenBank:ABL89196.1), and mouse FGFR2(β)b protein (Uniprot:P21803-2) were used as FGFR2 templates, and the amino acid sequences of the extracellular domain (ECD) of FGFR2 of different species and subtypes were connected to human IgG1Fc (hFc) (as shown in SEQ ID NO.1-6 in Table 1) or different His tags to obtain immune antigens or detection proteins. At the same time, commercial antigens human FGFR2(β)b-his (purchased from Acro, catalog number: FGB-H5223), cynomolgus monkey FGFR2(β)b-his (purchased from Acro, catalog number: FGB-C52H6), mouse FGFR2(β)b-his (purchased from Acro, catalog number: FGB-M52H5) and human FGFR2(β)c (purchased from Acro, catalog number: FGC-H5225) were purchased for identification and screening of antibodies.

Table 1 FGFR2 extracellular region and hFc amino acid sequence

The corresponding nucleotide sequences were cloned into pTT5 vector (purchased from UBO Biotechnology, catalog number: VT2202), and plasmids were prepared according to established standard molecular biology methods.

The expression vector and transfection reagent PEI (purchased from Polysciences, catalog number: 24765-1) were added to OPTI-MEM (purchased from Gibco, catalog number: 11058021) and mixed well, then allowed to stand for 15 minutes, added to Expi293F cells (purchased from Thermofisher, catalog number: A14527), and cultured in a shaking incubator at 5% CO ₂ , 120 rpm, and 37° C. On the second day of transfection, OPM-293ProFeed (purchased from Shanghai Aopumai, catalog number: F081918-001) and 6 g/L glucose (purchased from Sigma-Aldrich, catalog number: G8270-5KG) were added. On the sixth day of transfection, the cell culture supernatant was collected.

Proteins containing His tags were purified from cell culture supernatants using Ni columns (purchased from Cytiva, catalog number 17371206). First, equilibrate with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4) (purchased from Bio-Tech, catalog number B548117-0500), and then load the clarified culture supernatant onto the Ni column, controlling the flow rate at 5 mL/min. After loading, wash the Ni column with equilibration buffer, the volume of which is 3-5 times the volume of the Ni column bed. Elute the protein bound to Ni with an eluent containing 250 mM imidazole. After the sample is appropriately concentrated, it is further purified using PBS-equilibrated gel chromatography Superdex200 (purchased from Cytiva, product number 28990946) to remove aggregates, collect the monomer peak, and then sterile filter it using a 0.22 μm filter (purchased from Millipore, product number SLGVR13SL). The concentration is determined using Nanodrop (purchased from Thermofisher), the antibody purity is determined using HPLC-SEC, and the protein endotoxin content is detected using an endotoxin detection kit (purchased from Andus). After passing the test, the samples are divided into separate sets for use and stored at -80°C.

Proteins containing Fc tags were purified from cell culture supernatants using a Protein A column (purchased from Cytiva, catalog number 17549802). The Protein A column was first equilibrated with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4), and then the clarified culture supernatant was loaded onto the Protein A column, with the flow rate controlled at 10 mL/min. After loading, the Protein A column was washed with equilibration buffer, and the volume of the equilibration buffer was 3-5 times the volume of the Protein A column bed. The proteins bound to the Protein A column were eluted with an eluent (50 mM NaAc-HAc, pH 3.5). The eluted proteins were collected and Tris buffer (purchased from Sinopharm, catalog number 30188336) was added to neutral pH. After the sample is appropriately concentrated, it is further purified using gel chromatography Superdex200 equilibrated with PBS to remove aggregates, collect the monomer peak, and then sterile filter it with a 0.22μm filter. The concentration is determined using Nanodrop, the antibody purity is determined using HPLC-SEC, and the protein endotoxin content is detected using an endotoxin detection kit. After passing the test, it is divided into separate sets for use and stored at -80°C.

1.2 Preparation of control antibodies

At present, the only antibody drug targeting FGFR2b in clinical stage is Amgen's Bemarituzumab, which is a first-in-class targeting antibody FPA144 developed by Five Prime Therapeutics. The antibody sequence of the anti-FGFR2b positive control antibody Bemarituzumab (FPA144) is the HuGAL-FR21 sequence in patent US8603987B2. The sequence is obtained according to the patent, and the variable region of the antibody is connected to the human antibody hIgG1 (as shown in Table 2, the heavy chain of SEQ ID NO.8 and the light chain of SEQ ID NO.9) or the mouse antibody IgG2a constant region sequence (as shown in Table 2, the heavy chain of SEQ ID NO.10 and the light chain of SEQ ID NO.11), and used for the positive control antibodies (named FPA144-hIgG1 and FPA144-mIgG2a) in the screening.

The negative control antibody is an antibody against fluorescein isothiocyanate (FITC) that does not bind to FGFR2 (the sequence is shown in Table 2, named hIgG1 or mIgG2a).

The nucleotide sequences corresponding to the heavy and light chains of the antibody were cloned into the pTT5 vector to obtain plasmids expressing the heavy and light chains of the antibody, and expressed in Expi293F cells according to the method in Section 1.1 above, and purified according to the purification method of proteins containing Fc tags.

Table 2 Heavy and light chain sequences of control antibodies


Note: Antibody constant regions are in italics.

1.3 Construction of human FGFR2b stable transgenic cell line

The nucleotide sequences encoding the full-length amino acid sequence of human FGFR2(α)b (Uniprot: P21802-3, as shown in Table 3 SEQ ID NO.16) and the full-length amino acid sequence of human FGFR2(β)b (NCBI: NP_001138391.1, as shown in Table 3 SEQ ID NO.17) were cloned into the pLVX vector (purchased from Uniprot Biotechnology, catalog number VT1465) and viral particles were prepared in HEK293T cells (purchased from the Chinese Academy of Sciences).

Table 3 Full-length amino acid sequences of different subtypes of FGFR2b in different species


Note: Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

1.3.1 Construction of human FGFR2(α)b-HEK293T and human FGFR2(β)b-HEK293T stable cell lines

HEK293T cells (purchased from the Chinese Academy of Sciences) were infected with human FGFR2 (α) b and human FGFR2 (β) b lentiviruses, respectively, and then selectively cultured for 2 weeks in DMEM medium (purchased from Gibco, catalog number: 11995-065) containing 10% (v/v) fetal bovine serum (purchased from Excell Bio, catalog number: FSP500) containing 5 μg/mL puromycin (purchased from Gibco, catalog number: A11138-03), monoclonal cells were plated on 96-well plates, and cultured in a 37°C, 5% CO ₂ incubator. After about 2 weeks, some monoclonal clones were selected for expansion. The amplified clones were labeled with positive control antibody FPA144-hIgG1 and secondary antibodies, and flow cytometry analysis was performed to select monoclonal cell lines with good growth and high fluorescence intensity for continued expansion and cryopreservation in liquid nitrogen.

1.3.2 Construction of human FGFR2(α)b-Ba/F3 stable transgenic cell line

After Ba/F3 cells (purchased from Kangyuan Bochuang) were infected with human FGFR2(α)b lentivirus, they were selectively cultured in RPMI 1640 medium (purchased from Gibco, catalog number: 22400-089) containing 1 μg/mL puromycin, 10% (v/v) fetal bovine serum and 8 ng/mL IL-3 (purchased from Gibco, catalog number: PMC0035) for 2 weeks, and then monoclonal cells were plated on 96-well plates and placed in a 37°C, 5% CO ₂ incubator for culture. After about 2 weeks, some monoclonal clones were selected for amplification. The amplified clones were labeled with positive control antibody FPA144-hIgG1 and secondary antibodies, and flow cytometry analysis was performed to select monoclonal cell lines with good growth and high fluorescence intensity to continue to expand the culture and freeze in liquid nitrogen. After cell proliferation, IL-3 was removed from the culture medium, and 10 ng/mL FGF7 (purchased from R&D system, catalog number: 251-KG-050) and 10 μg/mL heparin sodium (purchased from Sigma-Aldrich, catalog number: H3149-500KU-9) were added to allow the cell line to proliferate dependent on the FGFR2b-FGF7 signaling pathway. After the cells resumed growth, the culture was expanded and frozen in liquid nitrogen.

Example 2: Generation of anti-FGFR2b murine monoclonal antibodies

2.1 Animal immunization

The animal immunization experiment was divided into five groups. The experimental animals were 6-8 week old Balb/c mice, SJL mice (purchased from Shanghai Slake Company), and MRL/LPR mice (purchased from Shanghai Weitong Lihua Company). They were female and kept in SPF environment. Blood was collected from the eye sockets of mice before immunization as negative serum.

The first immunization group consisted of 5 Balb/c mice. The immunogen was human FGFR2(α)b-hFc protein, and mouse FGFR2(α)b-hFc protein and human FGFR2(α)b-HEK293T cells were immunized alternately. During the first immunization, human FGFR2(α)b-hFc protein was emulsified with TiterMax (purchased from Sigma-Aldrich, catalog number: T2684), Alum (purchased from Thermo, catalog number: 77161) and CpG (commissioned by Sangon, catalog number: ODN1826), and then 0.1 mL was injected intraperitoneally, and then subcutaneously and at multiple points in the sole of the foot, that is, each mouse was injected with 50 μg of immunogen. After that, booster immunization was performed once a week, and mice were injected with a total of 25 μg of immunogen during the booster immunization. During the first and third booster immunizations, mouse FGFR2b-hFc protein was mixed with Alum and CpG and then injected subcutaneously and at multiple points in the sole of the foot. For the second and fifth booster immunizations, TiterMax was mixed with an equal volume of saline to emulsify, and then each mouse was intraperitoneally injected with 50 μL of the emulsified TiterMax; 15 minutes later, 0.1 mL of HEK293T human FGFR2 (α) b cells and CpG suspension was intraperitoneally injected, with each mouse injected with 5×10 ⁶ cells. The fourth and sixth booster immunizations were the same as the initial immunization method.

The second immunization group consisted of 5 MRL/LPR mice. The immunogens were human FGFR2(α)b-hFc protein, mouse FGFR2(α)b-hFc protein, and human FGFR2(α)b-HEK293T cells, which were immunized alternately. The remaining steps were similar to those of the first immunization group.

The third immunization group consisted of 5 Balb/c mice. The immunogens were human FGFR2(β)b-hFc protein and mouse FGFR2(β)b-hFc protein, which were immunized alternately. During the initial immunization, 0.1 mL of human FGFR2(β)b-hFc protein was emulsified with TiterMax, Alum and CpG and injected intraperitoneally, and then injected subcutaneously and at multiple points in the sole of the foot, i.e., each mouse was injected with a total of 50 μg of the immunogen. Thereafter, booster immunizations were performed once a week, with proteins alternately immunized during immunization. The adjuvants were Alum and CpG mixed or TiterMax, Alum and CpG emulsified and then injected subcutaneously and at multiple points in the sole of the foot, and each mouse was injected with 25 μg of the immunogen, for a total of six booster immunizations.

The fourth immunization group, 5 SJL, the immunogens were human FGFR2(β)b-hFc protein and mouse FGFR2(β)b-hFc protein alternately immunized. The rest of the steps refer to the third immunization group.

The fifth immunization group, 5 SJL. The immunogens were human FGFR2(β)b-hFc protein and human FGFR2(β)b-HEK293T cells alternating immunization. In the first immunization, human FGFR2(β)b-hFc protein was emulsified with TiterMax, Alum and CpG, and then 0.1mL was injected intraperitoneally and multiple points were injected subcutaneously, that is, each mouse was injected with a total of 50μg of immunogen. Thereafter, booster immunization was performed every other week, and each mouse was injected with 25μg of immunogen. In the first and third booster immunizations, 0.1mL of HEK293T-hFGFR2(β)b cell and CpG suspension was injected intraperitoneally, and 5×10 ⁶ cells were injected per mouse. In the second booster immunization, the steps were the same as the first immunization. In the fourth booster immunization, human FGFR2(β)b-hFc protein was emulsified with TiterMax and CpG and then injected subcutaneously at multiple points.

2.2 Spleen cell fusion

After booster immunization of the above immunization groups, blood was collected from the mouse orbits to detect the binding titer of antibodies in the mouse serum to human FGFR2(β)b, and mice with high antibody titers in the serum were selected for spleen cell fusion. The final booster immunization was performed 3 days before spleen cell fusion, and 50 μg/mouse of the immunogen solution prepared with physiological saline was injected subcutaneously, in the sole of the foot, and intraperitoneally.

The spleen and lymph nodes were sterilely taken, ground and filtered with a 40 μm cell strainer (purchased from BD Falcon), 5 mL ACK Lysing Buffer (purchased from Gibco, catalog number: A1049201) was added, and the red blood cells were lysed to obtain a cell suspension. The cells were washed twice by centrifugation at 1500 rpm with DMEM medium (purchased from Gibco, catalog number: 10569-010), and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 2:1 in the number of live cells, and cell fusion was performed using the BTX ECM2001+ high-efficiency electric fusion method (see ECM2001+ELECTROFUSION PROTOCOL). The fused cells were diluted into DMEM medium containing 20% fetal bovine serum (purchased from ExCell Bio, catalog number: FND500) and 1×Hybri-Max HAT (purchased from Sigma, catalog number: H0262-10VL), and the percentages are volume percentages. The fused cells were added to a 96-well cell culture plate at 5×10 ⁴ cells/200 μL per well and cultured in a 37°C, 5% CO ₂ incubator.

2.3 Screening of hybridoma cells

After 7 days of fusion, the hybridoma cell supernatant was taken and the binding activity with human FGFR2(β)b, monkey FGFR2(β)b, mouse FGFR2(β)b protein and human FGFR2(β)c protein was detected. The hybridoma cells in the fusion plate wells corresponding to the supernatant that had binding activity with human FGFR2(β)b, monkey FGFR2(β)b, mouse FGFR2(β)b protein but not binding with human FGFR2(β)c protein were limitedly diluted with DMEM. The cells under the microscope were observed with the naked eye and the number of living cells was counted. About 200 cells were taken in 2 mL Medium D (purchased from STEMCELL, product number 03810), mixed and spread on a 6-well cell culture plate, and cultured at 37°C, 5% _CO2 .

After 7 days, single clones were picked up and placed in DMEM medium containing 10% (v/v) FBS and l×HT (purchased from Sigma-Aldrich, product number: H0137-10VL), and cultured at 37°C, 5% CO2 for ₂ days. The binding activity with human FGFR2(β)b protein was detected for preliminary screening, and positive single clones were selected and expanded to 24-well cell culture plates for further culture. After 3 days, the supernatant was detected for binding activity with human FGFR2(β)b protein, monkey FGFR2(β)b protein, mouse FGFR2(β)b protein, human FGFR2(β)c protein and SNU-16 cells (FGFR2b medium and high expression) (purchased from Nanjing Kebai Biological) and the activity of blocking the binding of FGF7 to KATOIII cells (FGFR2b high expression) (purchased from Nanjing Kebai Biological). According to the test results of the 24-well plate samples, the target clone was selected and the optimal clone was expanded and cultured in _DMEM medium containing 10% (v/v) FBS at 37°C and 5% CO2 to produce and purify mouse monoclonal antibodies.

2.4 Identification of mouse monoclonal antibodies

The monoclonal antibodies obtained above were identified by ELISA, FACS, BIAcore, etc., and 7 candidate antibodies were obtained, namely F4-mab01, F7-mab02, F8-mab03, F8-mab04, F9-mab05, F10-mab06, and F10-mab07.

The binding activity of the antibody and the antigen protein was detected by enzyme-linked immunosorbent assay (ELISA). The antigen protein was diluted to a final concentration of 1 μg/mL with PBS buffer (purchased from Hyclone, catalog number SH30256.01), and then added to a 96-well ELISA plate at 50 μL per well. Seal with plastic film and incubate overnight at 4°C. The next day, wash the plate twice with PBST (PBS + 0.05% (v/v) Tween20), add blocking solution (PBS + 2% (v/v) BSA) and block at room temperature for 2 hours. Pour out the blocking solution, add 100nM gradient dilution control antibody 50 μL per well. After incubation at room temperature for 1 hour, wash the plate 3 times with PBS. Add HRP (horseradish peroxidase) labeled goat anti-mouse IgG (H+L) secondary antibody (purchased from Jackson Immuno, catalog number: 115-035-003), incubate at room temperature for 1 hour, and wash the plate 5 times with PBS. Add 50 μL of TMB substrate (KPL, 5120-0077) to each well, incubate at room temperature for 10 minutes, and then add 50 μL of stop solution (1.0 M HCl) to each well. Use an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer) to read the OD450nm value.

Flow cytometry (FACS) was used to detect the binding activity of antibodies to cells. The cells were expanded and cultured in a T-175 cell culture flask to 90% confluence. The culture medium was aspirated, washed once with PBS, and then treated and collected with Versene (purchased from Gibco, catalog number 15040066). After cell counting, the cells were washed twice with PBS and diluted to 2×10 ⁶ cells/mL, and 50 μL was added to a 96-well FACS reaction plate per well. 1% (v/v) fetal bovine serum was added to PBS as FACS buffer, and the cells were centrifuged and washed twice at 1500rpm at 4°C. 100 μL of diluted positive control antibody FPA144-hIgG1 was added to each well and incubated at 4°C for 1 hour. Wash three times with FACS buffer by centrifugation, add 50 μL of Alexa Fluor647 fluorescently labeled goat anti-mouse IgG (H+L) secondary antibody (purchased from Jackson, catalog number: 115-605-003) to each well, and incubate at 4°C for 1 hour. Wash three times with FACS buffer by centrifugation. Suspend the cells with 100 μL of FACS buffer and detect with FACS (FACS CantoII, purchased from BD). The test results show that the seven mouse antibodies have strong binding activity with human FGFR2 (β) b at both the protein level and the cell level.

Example 3: Identification of anti-FGFR2b chimeric antibodies

The variable region sequences of the above 7 mouse antibodies were obtained by sequencing, and the nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of the antibodies were recombined into the expression vector pTT5 with a signal peptide and the heavy chain constant region (CH) and light chain constant region (CL) sequences of the human antibody IgG1, respectively, to obtain recombinant plasmids expressing VH-CH and VL-CL. Referring to the antibody preparation method in Section 1.2 of Example 1, 7 strains of human-mouse chimeric antibodies were obtained: mab01, mab02, mab03, mab04, mab05, mab06, and mab07. Table 4 shows the VH and VL sequences of the chimeric antibodies, the signal peptide sequences, and the CH and CL sequences of the human antibody IgG1, Table 5 shows the Kabat analysis results of the variable region sequences of the chimeric antibodies, and Table 6 shows the IMGT analysis results of the variable region sequences of the chimeric antibodies.

Table 4 FGFR2b chimeric antibody variable region, signal peptide, constant region sequences

Table 5 Kabat analysis results of FGFR2b chimeric antibody variable region sequences

Table 6 IMGT analysis results of FGFR2b chimeric antibody variable region sequences

3.1 Detection of the binding activity of chimeric antibodies to human FGFR2b

The binding activity of chimeric antibodies to human FGFR2(β)b protein and SNU-16 cells was detected by ELISA and FACS in Section 2.4 of Reference Example 2. The test results are shown in Figures 1 and 2, and the seven chimeric antibodies have strong binding activity to human FGFR2(β)b at both the protein level and the cell level.

3.2 Detection of nonspecific binding activity of chimeric antibodies to human FGFR2(β)c

The binding activity of the chimeric antibodies to human FGFR2(β)c protein was detected by ELISA method in Section 2.4 of Reference Example 2. The detection results are shown in Table 7, and none of the 7 chimeric antibodies binds to human FGFR2(β)c.

Table 7 Non-specific binding of chimeric antibodies to human FGFR2 (β) c protein

3.3 Detection of affinity of chimeric antibodies to human FGFR2(β)b protein

Surface plasmon resonance (SPR) analysis was used to determine the affinity of the antibody to human FGFR2 (β) b protein. Protein A chips (purchased from Cytiva, catalog number: 29-127-558) were used to capture the antibody. The sample and running buffer were HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (purchased from Cytiva, catalog number: BR-1006-69). The flow-through pool was set to 25°C. The sample block was set to 16°C. Both were pre-treated with running buffer. In each cycle, the antibody to be tested was first captured with the Protein A chip, then a single concentration of human FGFR2(β)b-His antigen protein was injected, the binding and dissociation processes of the antibody and antigen protein were recorded, and finally the chip was regenerated with Glycine pH 1.5 (Cytiva; BR-1003-54). Binding was measured by injecting different concentrations of human FGFR2(β)b-His protein in solution for 240 seconds, with a flow rate of 30 μL/min, starting from 200 nM, diluted 1:1, for a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds and triggered by switching from sample solution to running buffer. The surface was regenerated by washing with 10 mM glycine solution (pH 1.5) at a flow rate of 30 μL/min for 30 seconds. Bulk refractive index differences were corrected by subtracting the response values obtained with the reference channel. Blank injections (= double reference) were also subtracted. To calculate the apparent KD and other kinetic parameters, the Langmuir 1:1 model was used. The binding rate (ka), dissociation rate (kd) and binding affinity (KD) of the antibody to human FGFR2 (β) b-His protein are shown in Table 8.

Table 8 Affinity of chimeric antibodies to human FGFR2(β)b protein

3.4 Detection of chimeric antibodies inhibiting cell proliferation induced by ligand FGF7

The human FGFR2(α)b-Ba/F3 cell line constructed in-house depends on the FGFR2b-FGF7 signaling pathway for proliferation and growth. The cell line was used to detect the activity of anti-FGFR2b antibodies in blocking FGF7 binding and inhibiting cell proliferation. Human FGFR2(α)b-Ba/F3 cells with good growth status were taken and resuspended in RPMI 1640 complete medium. The resuspended cells were added to a 96-well plate (purchased from Corning, Cat. No. 3610) at 1×10 ⁴ cells/50μL per well. The antibodies were diluted in RPMI 1640 complete medium (antibody starting concentration was 400nM, 5-fold dilution, 8 concentration gradients), and 50μL of the corresponding antibody dilution was added to each well according to the experimental design, so that the final volume of each well was 100μL.

After incubation in a carbon dioxide incubator for 3 days, add 100 μL of CellTiter-Glo reagent (purchased from Promega, product number G9243) to each well of the 96-well plate. After incubation at room temperature for 10 minutes, read the fluorescence value on an Envision instrument (purchased from PerkinElmer, model Envision2105).

The cell killing rate calculation formula is as follows: the tumor cell wells cultured with RPMI 1640 complete medium supplemented with 10 ng/mL FGF7 and 10 μg/mL Heparin are defined as positive controls with uninhibited proliferation, and the tumor cell wells cultured with only RPMI 1640 medium are defined as blank negative controls. Cell proliferation inhibition rate = ((positive well reading value - sample well reading value) / (positive well reading value - blank negative well reading value)) × 100%. The results were calculated and plotted using GraphPad Prism 9.0 software.

The results are shown in Table 9 and Figure 3. In this experiment, the negative isotype control hIgG1 had no significant effect on FGF ligand-induced cell proliferation. The seven chimeric antibodies were able to inhibit the proliferation of human FGFR2(α)b-Ba/F3 cells induced by the ligand FGF7 in vitro.

Table 9 Chimeric antibodies inhibit cell proliferation induced by ligand FGF7

3.5 Detection of the killing activity of chimeric antibodies in vitro

Healthy human peripheral blood mononuclear cells (PBMC) were thawed and resuspended in RPMI 1640 complete medium containing rhIL-2 (purchased from R&D, catalog number 202-IL-050) with a final concentration of 50 ng/mL. The cells were then incubated in a _CO2 incubator overnight. The next day, human FGFR2(α)b-Ba/F3 cells and PBMCs were resuspended in phenol red-free 1640 medium containing 5% inactivated serum (purchased from Gibco, catalog number 11835-030), and added to a 96-well plate (purchased from Corning, catalog number 3599) at a ratio of 1×10 ⁴ human FGFR2(α)b-Ba/F3 cells and 2×10 ⁵ PBMCs per well (cell mixture volume of 50 μL), and the antibodies were gradiently diluted in phenol red-free 1640 medium containing 5% inactivated serum (antibody starting concentration was 400 nM, 5-fold dilution, 8 concentration gradients), and 50 μL of the corresponding antibody dilution was added to each well according to the experimental design, so that the final volume of each well was 100 μL. The cell culture plate was placed in a CO ₂ incubator and incubated for 4 hours.

After 4 hours, the LDH value released by cells in each well in the supernatant culture medium was detected using an LDH detection kit (purchased from Dojindo, catalog number CK12, and the method of use was referred to the kit manual) to calculate the percentage of target cell killing.

The results were calculated and plotted using GraphPad Prism 9.0 software. The formula for calculating the cell killing rate is as follows: the wells with all tumor cell lysis were defined as the positive control with 100% killing, and the wells with an antibody concentration of 0 were defined as the blank negative control. The cell killing rate = ((sample well reading value - blank negative well reading value) / (positive well reading value - blank negative well reading value)) × 100%.

The results are shown in Table 10 and Figure 4. In this experiment, the negative isotype control hIgG1 had no obvious cytotoxic effect, while the 7 chimeric antibodies had a significant killing effect on human FGFR2(α)b-Ba/F3 cells, and the killing effect was better than that of the control antibody FPA144-hIgG1.

Table 10 In vitro killing activity of chimeric antibodies

Example 4: Humanization of anti-FGFR2b monoclonal antibody

By comparing the IMGT (http://imgt.cines.fr) human antibody heavy and light chain variable region germline gene database, the heavy chain and light chain variable region germline genes with high homology to the mouse antibody were selected as templates, and the CDRs of the mouse antibody were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the three-dimensional structure of the antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues in the framework region that have an important influence on the conformation of VL and VH were reversely mutated to obtain humanized monoclonal antibodies. Table 11 and Table 12 respectively record the humanized light chain template sequence and heavy chain template sequence used in the examples.

Table 11 Humanized light chain template

Table 12 Humanized heavy chain template

4.1 Humanization of mab01

The humanized light chain templates of the mouse antibody mab01 are IGKV2-40*01/IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ1*01. The CDRs of the mouse antibody mab01 are transplanted into their humanized templates to obtain the corresponding humanized versions. As needed, the key amino acids in the FR region sequence of the humanized antibody of mab01 are back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity. The CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system. The specific mutation design is shown in Table 13.

Table 13 Humanized antibody mutation design of mab01

Note: Q45K means the 45th Q is mutated to K, and so on. The numbering of the mutated amino acids is the natural sequence numbering.

The specific sequence of the variable region of mab01 humanized antibody is as follows:

The amino acid sequence of Hab01.L3 is shown in SEQ ID NO.118:

The amino acid sequence of Hab01.L4 is shown in SEQ ID NO.119:

The amino acid sequence of Hab01.H1 is shown in SEQ ID NO.120:

The amino acid sequence of Hab01.H2 is shown in SEQ ID NO.121:

The present invention selects different light chain and heavy chain sequences from the above-mentioned humanized antibody light chain and heavy chain variable region mutation designs for cross-combination, and finally obtains the humanized antibody mab01. The amino acid sequences of the variable regions of each antibody are as follows:

Table 14 Amino acid sequences corresponding to the variable regions of mab01 humanized antibody

According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibody are shown in Table 15.

Table 15 Kabat analysis results of mab01 humanized antibody CDR sequences

4.2 Humanization of mab02

The humanized light chain templates of the mouse antibody mab02 are IGKV2-40*01/IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The CDR amino acid residues are determined and annotated by the Kabat numbering system, and the mutation method is referred to Section 4.1 above. The specific mutation design is shown in Table 16.

Table 16 Humanized antibody mutation design of mab02

Note: P49S means the 49th P is mutated to S, and so on. The numbering of the mutated amino acids is the natural sequence numbering.

The specific sequence of the variable region of the mab02 humanized antibody is as follows:

The amino acid sequence of Hab02.L1 is shown in SEQ ID NO.122:

The amino acid sequence of Hab02.L2 is shown in SEQ ID NO.123:

The amino acid sequence of Hab02.H1a is shown in SEQ ID NO.124:

The amino acid sequence of Hab02.H2a is shown in SEQ ID NO.125:

The amino acid sequence of Hab02.H3a is shown in SEQ ID NO.126:

The present invention selects different light chain and heavy chain sequences from the above-mentioned mab02 humanized antibody light chain and heavy chain variable region mutation designs for cross-combination, and finally obtains a variety of mab02 humanized antibodies. The variable region amino acid sequences of each antibody are as follows:

Table 17 Amino acid sequences corresponding to the variable regions of the mab02 antibody

According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibody are shown in Table 18.

Table 18 Kabat analysis results of mab02 humanized antibody CDR sequences

4.3 Humanization of mab03

The humanized light chain templates of the mouse antibody mab03 are IGKV3-20*01/IGKV1-33*01/IGKV2-29*02 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01/IGHV1-69*02 and IGHJ6*01. The CDR amino acid residues are determined and annotated by the Kabat numbering system. The mutation method refers to Section 4.1 above. The specific mutation design is shown in Table 19.

Table 19 Humanized antibody mutation design of mab03

Note: A43S means the 43rd position A is mutated to S, and so on. The numbering of the mutated amino acids is the natural sequence numbering

The specific sequence of the variable region of the mab03 humanized antibody is as follows:

The amino acid sequence of Hab03.L3 is shown in SEQ ID NO.130:

The amino acid sequence of Hab03.L5 is shown in SEQ ID NO.131:

The amino acid sequence of Hab03.L6 is shown in SEQ ID NO.132:

The amino acid sequence of Hab03.H2b is shown in SEQ ID NO.133:

The amino acid sequence of Hab03.H10a is shown in SEQ ID NO.134:

The present invention selects different light chain and heavy chain sequences from the above-mentioned mab03 humanized antibody light chain and heavy chain variable region mutation designs for cross-combination, and finally obtains a variety of mab03 humanized antibodies. The variable region amino acid sequences of each antibody are as follows:

Table 20 Amino acid sequences corresponding to the variable regions of the mAb03 antibody

According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibody are shown in Table 21.

Table 21 Kabat analysis results of mab03 humanized antibody CDR sequences

4.4 Humanization of mab04

The humanized light chain templates of the mouse antibody mab04 are IGKV1-33*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system. The mutation method refers to Section 4.1 above. The specific mutation design is shown in Table 22.

Table 22 Humanized antibody mutation design of mab04

Note: A43S means the 43rd position A mutated to S, and so on. The numbering of the mutated amino acids is the natural sequence numbering.

The specific sequence of the variable region of the mab04 humanized antibody is as follows:

The amino acid sequence of Hab04.L2 is shown in SEQ ID NO.136:

The amino acid sequence of Hab04.H1a is shown in SEQ ID NO.137:

The amino acid sequence of Hab04.H2a is shown in SEQ ID NO.138:

The amino acid sequence of Hab04.H4 is shown in SEQ ID NO.139:

The present invention selects different light chain and heavy chain sequences from the above-mentioned mab04 humanized antibody light chain and heavy chain variable region mutation designs for cross-combination, and finally obtains a variety of mab04 humanized antibodies. The variable region amino acid sequences of each antibody are as follows:

Table 23 Amino acid sequences corresponding to the variable regions of the mAb04 antibody

According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibody are shown in Table 24.

Table 24 Kabat analysis results of mab04 humanized antibody CDR sequences

4.5 Humanization of mab05

The humanized light chain templates of the mouse antibody mab05 are IGKV1-33*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system. The mutation method refers to Section 4.1 above. The specific mutation design is shown in Table 25.

Table 25 Humanized antibody back mutation design of mab05

Note: K42H means the 42nd K mutates to H, and so on. The numbering of the mutated amino acids is the natural sequence numbering.

The specific sequence of the variable region of the mab05 humanized antibody is as follows:

The amino acid sequence of Hab05.L1 is shown in SEQ ID NO.142:

The amino acid sequence of Hab05.L2 is shown in SEQ ID NO.143:

The amino acid sequence of Hab05.L3 is shown in SEQ ID NO.144:

The amino acid sequence of Hab05.H1a is shown in SEQ ID NO.145:

The present invention selects different light chain and heavy chain sequences from the above-mentioned mab05 humanized antibody light chain and heavy chain variable region mutation designs for cross-combination, and finally obtains a variety of mab05 humanized antibodies. The variable region amino acid sequences of each antibody are as follows:

Table 26 Amino acid sequences corresponding to the variable regions of mab05 antibody

According to the Kabat numbering system, the results of the CDR sequence analysis of the above humanized antibody are shown in Table 27.

Table 27 Kabat analysis results of mab05 humanized antibody CDR sequence

4.6 Humanization of mab06

The humanized light chain templates of the mouse antibody mab06 are IGKV1-33*01/IGKV2-29*02 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ1*01. The CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system. The mutation method refers to Section 4.1 above. The specific mutation design is shown in Table 28.

Table 28 Humanized antibody mutation design of mab06

Note: Q45K means the 45th Q mutates to K, and so on. The numbering of the mutated amino acids is the natural sequence numbering.

The specific sequence of the variable region of mab06 humanized antibody is as follows:

The amino acid sequence of Hab06.L1 is shown in SEQ ID NO.147:

The amino acid sequence of Hab06.L2 is shown in SEQ ID NO.148:

The amino acid sequence of Hab06.H2 is shown in SEQ ID NO.149:

The amino acid sequence of Hab06.H3 is shown in SEQ ID NO.150:

The amino acid sequence of Hab06.H4a is shown in SEQ ID NO.151:

The present invention selects different light chain and heavy chain sequences from the above-mentioned mab06 humanized antibody light chain and heavy chain variable region mutation designs for cross-combination, and finally obtains a variety of mab06 humanized antibodies, and the variable region amino acid sequences of each antibody are as follows:

Table 29 Amino acid sequences corresponding to the variable regions of mAb06 antibody

According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibody are shown in Table 30.

Table 30 Kabat analysis results of mab06 humanized antibody CDR sequences

4.7 Humanization of mab07

The humanized light chain templates of the mouse antibody mab07 are IGKV6-21*01/IGKV2-30*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system. The mutation method refers to Section 4.1 above. The specific mutation design is shown in Table 31.

Table 31 Humanized antibody mutation design of mab07

Note: L46W means that the 46th L mutated to W, and so on. The numbering of the mutated amino acids is the natural sequence numbering.

The specific sequence of the variable region of the mab07 humanized antibody is as follows:

The amino acid sequence of Hab07.L1 is shown in SEQ ID NO.153:

The amino acid sequence of Hab07.L2 is shown in SEQ ID NO.154:

The amino acid sequence of Hab07.L3 is shown in SEQ ID NO.155:

The amino acid sequence of Hab07.H1 is shown in SEQ ID NO.156:

The amino acid sequence of Hab07.H3 is shown in SEQ ID NO.157:

The present invention selects different light chain and heavy chain sequences from the above-mentioned mab07 humanized antibody light chain and heavy chain variable region mutation designs for cross-combination, and finally obtains a variety of mab07 humanized antibodies. The variable region amino acid sequences of each antibody are as follows:

Table 32 Amino acid sequences corresponding to the variable regions of the mAb07 antibody

According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibody are shown in Table 33.

Table 33 Kabat analysis results of mab07 humanized antibody CDR sequences

Example 5: Identification of anti-FGFR2b humanized antibodies

The nucleic acid sequences encoding the humanized antibodies VH and VL were recombined into the expression vector pTT5 with a signal peptide and the CH and CL sequences of the humanized antibody IgG1 to obtain recombinant plasmids expressing VH-CH and VL-CL. The humanized antibodies were obtained by referring to the antibody preparation method in Section 1.2 of Example 1.

5.1 Detection of binding activity of humanized antibodies to human FGFR2b

The binding activity of antibodies to KATOIII and SNU-16 cell surface membrane proteins was detected by cell-based ELISA. The cells were expanded and cultured in T-175 cell culture flasks to 90% confluence. The culture medium was aspirated, washed once with PBS, and then treated and collected with Versene. After cell counting, the cells were washed twice with PBS and diluted to 4×10 ⁵ cells/mL, added to 96-well cell culture plates at 100 μL per well, and placed in an incubator for overnight culture. The next day, the culture medium was discarded, PBS was washed twice, 50 μL of fixative was added to each well, and the plates were fixed in a fume hood for 30 minutes. After washing twice with PBST, the experiment was continued according to the ELISA method.

The binding activity of the humanized antibody to human FGFR2(β)b protein was detected by ELISA and FACS methods in Section 2.4 of Reference Example 2. The test results are shown in Figures 5, 6 and 7, and the humanized antibody has strong binding activity to human FGFR2(β)b at both the protein level and the cell level.

5.2 Detection of non-specific binding activity of humanized antibodies

The binding of humanized antibodies to human FGFR2(β)c protein was detected by ELISA method in Section 2.4 of Reference Example 2. The detection results are shown in Table 34, and none of the humanized antibodies bind to human FGFR2(β)c.

Table 34 Non-specific binding activity of humanized antibodies to human FGFR2 (β) c protein

5.3 Detection of affinity of humanized antibodies to human FGFR2(β)b protein

The affinity of the humanized antibody to human FGFR2(β)b protein was determined by the method of Reference Example 3.3. The results are shown in Table 35, and the affinity of some humanized antibodies can retain the affinity of the chimeric antibody.

Table 35 Affinity of humanized antibodies to human FGFR2 (β) b protein

5.4 Detection of humanized antibodies inhibiting cell proliferation induced by ligand FGF7

Referring to the experimental method in Section 3.4 of Example 3, the results are shown in Table 36 and Figure 8. The experimental results show that the humanized antibodies can inhibit the proliferation of human FGFR2(α)b-Ba/F3 cells induced by the ligand FGF7.

Table 36 Humanized antibodies inhibit cell proliferation induced by ligand FGF7

5.5 Detection of in vitro killing activity of humanized antibodies

The in vitro killing activity of the humanized antibody was detected by the experimental method in Section 3.5 of Reference Example 3, and the results are shown in Table 37 and Figure 9. The experimental results show that the humanized antibody can produce a significant killing effect on human FGFR2(α)b-Ba/F3 cells.

Table 37 In vitro killing activity of humanized antibodies

Example 6: Endocytosis effect of anti-FGFR2b antibody

The culture medium of KATOIII cells (purchased from Nanjing Kebai Biological, catalog number: CBP60483) used in this experiment is RPMI 1640 + 10% FBS (purchased from Gibco, catalog number: 10491; Gibco, catalog number: 10091-148). Resuspend KATOIII cells with flow buffer (purchased from Biolegend, catalog number: 420201) and adjust the cell density to 2×10 ⁶ cells/mL. Add 50 μL of cell suspension per well to a round-bottom 96-well plate (purchased from Corning, catalog number: 3799) so that the number of cells in each well is 1×10 ^5. Then add 50 μL of gradient diluted antibodies (starting concentration 100nM, 3-fold dilution) and incubate at 4°C for 1 hour. After 1 hour, wash off the unbound antibodies, resuspend the cells with flow buffer, and continue to place the control group at 4°C, and the endocytosis group at 37°C for 4 hours. After 4 hours, the cells were washed twice with PBS and then Alexa Fluor 647 fluorescently labeled goat anti-human IgG Fcγ secondary antibody (purchased from Jackson Immuno, catalog number: 109-605-098) was added. After incubation at 4°C for 1 hour, the cells were washed twice with PBS and the Alexa Fluor 647 fluorescence signal (MFI) was analyzed by FACS. The GraphPad Prism 9.0 software was used to calculate and plot the data. The wells with the isotype negative control antibody were defined as negative controls. Endocytosis efficiency = (control group MFI - endocytosis group MFI) / control group MFI × 100%.

The results are shown in Table 38. In this experiment, under the condition of saturated concentration of 100 nM, the negative control had no obvious endocytosis activity in KATOIII cells. The 7 anti-FGFR2b antibodies of the present invention all had a certain degree of endocytosis activity in KATOIII cells (endocytosis rate 33.5% to 43.5%), and the endocytosis activity of the anti-FGFR2b monoclonal antibodies of the present invention was better than that of the control antibody FPA144-hIgG1 (endocytosis rate 30.5%).

Table 38 Endocytic activity of anti-FGFR2b antibodies in KATOIII cells

Example 7: In vivo efficacy of anti-FGFR2b antibodies

FGFR2b-positive human gastric cancer cells SNU-16-#232 and KATO-III-#729 were selected to establish an in vivo model in mice (CB17-SCID: female, 6-8 weeks, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), and this model was used to evaluate the anti-tumor efficacy of candidate molecules in vivo.

7.1 Chimeric Antibodies Inhibit Tumor Growth in Human Gastric Cancer SNU-16-#232

The inoculation time of human gastric cancer cells SNU-16-#232 (self-extracted primary cells, purchased from Nanjing Kebai Biological) was taken as day 0 of the experiment. On day 0 of the experiment, human gastric cancer cells SNU-16-#232 that had been amplified and cultured to the required number and were in the logarithmic growth phase (confluence was about 80%, and fresh culture medium was replaced for the cells one day before inoculation) were collected and inoculated. First, the cell suspension was collected into a 50 mL centrifuge tube and centrifuged at 300 g for 7 minutes. An appropriate amount of serum-free RPMI1640 medium (purchased from Gibco, catalog number: 61870-036) was aspirated to resuspend the cells. 500 μL of the cell suspension was counted in a cell counter (Beckman, SIC-TP-573). Finally, according to the cell counting results, the cell density was adjusted to 200×10 ⁶ cells/mL using serum-free medium. The cells were placed on ice and transferred to the SPF animal room through a transfer window for inoculation modeling. Before inoculation, the above cell suspension was mixed with Matrigel matrix glue (purchased from Corning Bio, catalog number: 356237) in equal proportions, and 100 μL of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.

After inoculation of gastric cancer cells SNU-16-#232, tumor growth was monitored. When the overall tumor volume average was 100-150 mm ³ , mice with appropriate tumor volume were selected for random grouping, with 8 mice in each group. Subsequently, intravenous administration was performed twice a week for a total of 6 times, and the tumor volume and mouse body weight changes were measured. The specific administration regimen is shown in Table 39, wherein hIgG1 is an isotype control, FPA144-hIgG1 is a positive molecule, mab04, mab07, and mab06 are candidate antibodies, and the candidate antibodies are selected to have the same molar dosage per unit body weight as the isotype control hIgG1, and converted into an equimolar dosage according to the formula m=n*M, wherein m is the dosage mass, n is the drug molar mass, and M is the drug molecular weight. The dosages in this section are all calculated based on this principle.

The results are shown in Table 40 and Figure 10. As of 18 days after drug withdrawal, each group showed a certain efficacy, and the trend of efficacy remained stable; among them, mab06 and mab04 showed better efficacy than the positive control.

Table 39: Efficacy and dosing regimen of chimeric antibodies in human gastric cancer SNU-16-#232 tumor-bearing mice

Table 40 Chimeric antibody regulation of human gastric cancer SNU-16-#232 tumor efficacy results

7.2 Humanized antibodies inhibit the growth of human gastric cancer KATOIII-#729 tumors

The inoculation time of human gastric cancer cells KATOIII-#729 (self-extracted primary cells, purchased from Nanjing Kebai Biological) was taken as the 0th day of the experiment. On the 0th day of the experiment, human gastric cancer cells KATOIII-#729 that were amplified and cultured to the required number and in the logarithmic growth phase (confluence was about 80%, and the cells were replaced with fresh culture medium one day before inoculation) were collected and inoculated. First, the culture medium in the cell culture flask was removed, and washed twice with phosphate buffered saline (PBS, purchased from Hyclone, catalog number: SH30256.01), and then an appropriate amount of 0.25% trypsin digestion solution (purchased from Gibco, catalog number: 25200-072) was added. The bottom of the flask was gently shaken to ensure that the trypsin digestion solution was evenly covered on the cell surface, and it was placed in a 37°C environment for digestion for 5 minutes, and then 10% fetal bovine serum (Fetal Bovine Serum) was added. The digestion reaction was terminated with complete medium containing serum, FBS, purchased from Gibco, catalog number: 10091-148/2418958P), and the cells adhering to the bottom of the bottle were gently blown off the culture bottle. The digested cell suspension was collected into a 50mL centrifuge tube, centrifuged at 350g for 5 minutes, and an appropriate amount of serum-free RPMI1640 medium was taken to resuspend the cells, and filtered through a 70μm mesh. 500μL of the cell suspension was counted on a cell counter. Finally, according to the cell counting results, the cell density was adjusted to 100× ¹⁰⁶ cells/mL using serum-free medium, placed on ice, and transferred to the SPF animal room through a transfer window for inoculation modeling. Before inoculation, the above cell suspension was mixed with Matrigel matrix glue in equal proportions, and 200μL of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.

After inoculation of gastric cancer cells KATOIII-#729, tumor growth was monitored. When the overall tumor volume average was around 200 ^mm3 , mice with appropriate tumor volume were selected and randomly divided into groups, with 7 mice in each group. Each group was administered via the tail vein, with treatment twice a week for a total of 6 times. The tumor volume and mouse weight changes were measured. The specific dosing schedule is shown in Table 41.

The results are shown in Table 42 and Figure 11. After administration, the candidate molecules all showed certain pharmacodynamics, among which the humanized candidate molecules Hab07-L1H1 and Hab04-L2H1a were significantly better than FPA144-hIgG1.

Table 41: Efficacy and dosing regimen of humanized antibodies in human gastric cancer KATOIII-#729 tumor-bearing mice

Table 42 Humanized antibody regulation of human gastric cancer KATOIII-#729 tumor efficacy results

7.3 Chimeric Antibodies Inhibit Tumor Growth in Human Gastric Cancer SNU-16-#232

Referring to the experimental method in 7.1 above, there were 7 mice in each group and the specific dosing regimen was shown in Table 43.

The results are shown in Table 44 and Figure 12. The efficacy of each drug administration group was stable, and the chimeric molecule mab01 was superior to the positive control FPA144-hIgG1.

Table 43 chimeric antibody efficacy and dosing regimen in human gastric cancer SNU-16-#232 tumor-bearing mice

Table 44 Chimeric antibody regulation of human gastric cancer SNU-16-#232 tumor efficacy results

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

Claims (27)

An antibody or antigen-binding fragment thereof that specifically binds to fibroblast growth factor receptor 2 (FGFR2), wherein the antibody or antigen-binding fragment thereof has one or more of the following properties: (1) Inhibit the binding of FGF7 to FGFR2; (2) Inhibit FGF7-induced tumor cell proliferation; (3) binds to FGFR2 with an affinity of no greater than 5×10 ⁻⁸ M; and (4) Binds to FGFR2b but not to FGFR2c. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) wherein: The light chain variable region comprises: (1) LCDR1 as shown in SEQ ID NO.37, LCDR2 as shown in SEQ ID NO.38, and LCDR3 as shown in SEQ ID NO.39; (2) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44, and LCDR3 as shown in SEQ ID NO.45; (3) LCDR1 as shown in SEQ ID NO.49, LCDR2 as shown in SEQ ID NO.50, and LCDR3 as shown in SEQ ID NO.51; (4) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50, and LCDR3 as shown in SEQ ID NO.51; (5) LCDR1 as shown in SEQ ID NO.58, LCDR2 as shown in SEQ ID NO.59, and LCDR3 as shown in SEQ ID NO.60; (6) LCDR1 as shown in SEQ ID NO.63, LCDR2 as shown in SEQ ID NO.59, and LCDR3 as shown in SEQ ID NO.64; (7) LCDR1 as shown in SEQ ID NO.68, LCDR2 as shown in SEQ ID NO.69, and LCDR3 as shown in SEQ ID NO.70; (8) LCDR1 as shown in SEQ ID NO.74, LCDR2 as shown in SEQ ID NO.75, and LCDR3 as shown in SEQ ID NO.39; (9) LCDR1 as shown in SEQ ID NO.79, LCDR2 as shown in SEQ ID NO.80, and LCDR3 as shown in SEQ ID NO.81; (10) LCDR1 as shown in SEQ ID NO.85, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.51; (11) LCDR1 as shown in SEQ ID NO.90, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.51; (12) LCDR1 as shown in SEQ ID NO.94, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.60; (13) LCDR1 as shown in SEQ ID NO.98, LCDR2 as shown in SEQ ID NO.86, and LCDR3 as shown in SEQ ID NO.64; (14) LCDR1 as shown in SEQ ID NO.102, LCDR2 as shown in SEQ ID NO.103 and LCDR3 as shown in SEQ ID NO.70; or (15) LCDRs whose sequences have 1, 2, 3 amino acid substitutions, deletions and/or insertions or have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any of the groups of LCDRs in (1) to (14). The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises: (1) HCDR1 as shown in SEQ ID NO.34, HCDR2 as shown in SEQ ID NO.35, and HCDR3 as shown in SEQ ID NO.36; (2) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.41, and HCDR3 as shown in SEQ ID NO.42; (3) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.47, and HCDR3 as shown in SEQ ID NO.48; (4) HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.53 and HCDR3 as shown in SEQ ID NO.48; (5) HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.56 and HCDR3 as shown in SEQ ID NO.57; (6) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.61, and HCDR3 as shown in SEQ ID NO.62; (7) HCDR1 as shown in SEQ ID NO.65, HCDR2 as shown in SEQ ID NO.66, and HCDR3 as shown in SEQ ID NO.67; (8) HCDR1 as shown in SEQ ID NO.71, HCDR2 as shown in SEQ ID NO.72, and HCDR3 as shown in SEQ ID NO.73; (9) HCDR1 as shown in SEQ ID NO.76, HCDR2 as shown in SEQ ID NO.77, and HCDR3 as shown in SEQ ID NO.78; (10) HCDR1 as shown in SEQ ID NO.82, HCDR2 as shown in SEQ ID NO.83, and HCDR3 as shown in SEQ ID NO.84; (11) HCDR1 as shown in SEQ ID NO.87, HCDR2 as shown in SEQ ID NO.88 and HCDR3 as shown in SEQ ID NO.89; (12) HCDR1 as shown in SEQ ID NO.91, HCDR2 as shown in SEQ ID NO.92, and HCDR3 as shown in SEQ ID NO.93; (13) HCDR1 as shown in SEQ ID NO.95, HCDR2 as shown in SEQ ID NO.96 and HCDR3 as shown in SEQ ID NO.97; (14) HCDR1 as shown in SEQ ID NO.99, HCDR2 as shown in SEQ ID NO.100, and HCDR3 as shown in SEQ ID NO.101; (15) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.127, and HCDR3 as shown in SEQ ID NO.42; (16) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.128 and HCDR3 as shown in SEQ ID NO.42; (17) HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.129 and HCDR3 as shown in SEQ ID NO.42; (18) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.135, and HCDR3 as shown in SEQ ID NO.48; (19) HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.140 and HCDR3 as shown in SEQ ID NO.48; (20) HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.141, and HCDR3 as shown in SEQ ID NO.48; (21) HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.146, and HCDR3 as shown in SEQ ID NO.57; (22) HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.152 and HCDR3 as shown in SEQ ID NO.62; or (23) HCDRs whose sequences have 1, 2, 3 amino acid substitutions, deletions and/or insertions or have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with any of the HCDRs in (1) to (22). The antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region in combination of the following LCDRs and HCDRs: (1) LCDR1 as shown in SEQ ID NO.37, LCDR2 as shown in SEQ ID NO.38 and LCDR3 as shown in SEQ ID NO.39, and HCDR1 as shown in SEQ ID NO.34, HCDR2 as shown in SEQ ID NO.35 and HCDR3 as shown in SEQ ID NO.36; (2) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.41 and HCDR3 as shown in SEQ ID NO.42; (3) LCDR1 as shown in SEQ ID NO.49, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.47 and HCDR3 as shown in SEQ ID NO.48; (4) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.53 and HCDR3 as shown in SEQ ID NO.48; (5) LCDR1 as shown in SEQ ID NO.58, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.60, and HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.56 and HCDR3 as shown in SEQ ID NO.57; (6) LCDR1 as shown in SEQ ID NO.63, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.64, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.61 and HCDR3 as shown in SEQ ID NO.62; (7) LCDR1 as shown in SEQ ID NO.68, LCDR2 as shown in SEQ ID NO.69 and LCDR3 as shown in SEQ ID NO.70, and HCDR1 as shown in SEQ ID NO.65, HCDR2 as shown in SEQ ID NO.66 and HCDR3 as shown in SEQ ID NO.67; (8) LCDR1 as shown in SEQ ID NO.74, LCDR2 as shown in SEQ ID NO.75 and LCDR3 as shown in SEQ ID NO.39, and HCDR1 as shown in SEQ ID NO.71, HCDR2 as shown in SEQ ID NO.72 and HCDR3 as shown in SEQ ID NO.73; (9) LCDR1 as shown in SEQ ID NO.79, LCDR2 as shown in SEQ ID NO.80 and LCDR3 as shown in SEQ ID NO.81, and HCDR1 as shown in SEQ ID NO.76, HCDR2 as shown in SEQ ID NO.77 and HCDR3 as shown in SEQ ID NO.78; (10) LCDR1 as shown in SEQ ID NO.85, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.82, HCDR2 as shown in SEQ ID NO.83 and HCDR3 as shown in SEQ ID NO.84; (11) LCDR1 as shown in SEQ ID NO.90, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.87, HCDR2 as shown in SEQ ID NO.88 and HCDR3 as shown in SEQ ID NO.89; (12) LCDR1 as shown in SEQ ID NO.94, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.60, and HCDR1 as shown in SEQ ID NO.91, HCDR2 as shown in SEQ ID NO.92 and HCDR3 as shown in SEQ ID NO.93; (13) LCDR1 as shown in SEQ ID NO.98, LCDR2 as shown in SEQ ID NO.86 and LCDR3 as shown in SEQ ID NO.64, and HCDR1 as shown in SEQ ID NO.95, HCDR2 as shown in SEQ ID NO.96 and HCDR3 as shown in SEQ ID NO.97; (14) LCDR1 as shown in SEQ ID NO.102, LCDR2 as shown in SEQ ID NO.103 and LCDR3 as shown in SEQ ID NO.70, and HCDR1 as shown in SEQ ID NO.99, HCDR2 as shown in SEQ ID NO.100 and HCDR3 as shown in SEQ ID NO.101; (15) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.127 and HCDR3 as shown in SEQ ID NO.42; (16) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.128 and HCDR3 as shown in SEQ ID NO.42; (17) LCDR1 as shown in SEQ ID NO.43, LCDR2 as shown in SEQ ID NO.44 and LCDR3 as shown in SEQ ID NO.45, and HCDR1 as shown in SEQ ID NO.40, HCDR2 as shown in SEQ ID NO.129 and HCDR3 as shown in SEQ ID NO.42; (18) LCDR1 as shown in SEQ ID NO.49, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.135 and HCDR3 as shown in SEQ ID NO.48; (19) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.140 and HCDR3 as shown in SEQ ID NO.48; (20) LCDR1 as shown in SEQ ID NO.54, LCDR2 as shown in SEQ ID NO.50 and LCDR3 as shown in SEQ ID NO.51, and HCDR1 as shown in SEQ ID NO.52, HCDR2 as shown in SEQ ID NO.141 and HCDR3 as shown in SEQ ID NO.48; (21) LCDR1 as shown in SEQ ID NO.58, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.60, and HCDR1 as shown in SEQ ID NO.55, HCDR2 as shown in SEQ ID NO.146 and HCDR3 as shown in SEQ ID NO.57; (22) LCDR1 as shown in SEQ ID NO.63, LCDR2 as shown in SEQ ID NO.59 and LCDR3 as shown in SEQ ID NO.64, and HCDR1 as shown in SEQ ID NO.46, HCDR2 as shown in SEQ ID NO.152 and HCDR3 as shown in SEQ ID NO.62; or, (23) A sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the six CDRs described in any one of (1) to (22), or a sequence of six CDRs having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity. The antibody or antigen-binding fragment thereof according to claim 4, wherein: (1) the light chain variable region comprises a sequence as shown in any of the sequences shown in SEQ ID NO. 19, 21, 23, 25, 27, 29, 31, 118, 119, 122, 123, 130, 131, 132, 136, 142, 143, 144, 147, 148, 153, 154 or 155, or a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown; and, (2) The heavy chain variable region comprises a sequence as shown in any of the sequences shown in SEQ ID NO. 18, 20, 22, 24, 26, 28, 30, 120, 121, 124, 125, 126, 133, 134, 137, 138, 139, 145, 149, 150, 151, 156 or 157, or a sequence that has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof has a light chain variable region and a heavy chain variable region combination as shown below: (1) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.19 and SEQ ID NO.18, respectively; (2) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.21 and SEQ ID NO.20, respectively; (3) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.23 and SEQ ID NO.22, respectively; (4) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.25 and SEQ ID NO.24, respectively; (5) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.27 and SEQ ID NO.26, respectively; (6) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.29 and SEQ ID NO.28, respectively; (7) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.31 and SEQ ID NO.30, respectively; (8) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.118 and SEQ ID NO.120, respectively; (9) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.118 and SEQ ID NO.121, respectively; (10) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.119 and SEQ ID NO.120, respectively; (11) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.122 and SEQ ID NO.126, respectively; (12) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.123 and SEQ ID NO.124, respectively; (13) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.123 and SEQ ID NO.125, respectively; (14) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.130 and SEQ ID NO.133, respectively; (15) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.131 and SEQ ID NO.134, respectively; (16) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.132 and SEQ ID NO.134, respectively; (17) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.136 and SEQ ID NO.137, respectively; (18) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.136 and SEQ ID NO.138, respectively; (19) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.136 and SEQ ID NO.139, respectively; (20) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.142 and SEQ ID NO.145, respectively; (21) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.143 and SEQ ID NO.145, respectively; (22) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.144 and SEQ ID NO.145, respectively; (23) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.147 and SEQ ID NO.149, respectively; (24) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.148 and SEQ ID NO.150, respectively; (25) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.147 and SEQ ID NO.151, respectively; (26) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.153 and SEQ ID NO.156, respectively; (27) The light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO.154 and SEQ ID NO.157, respectively; (28) The light chain variable region and the heavy chain variable region respectively comprise the sequences shown in SEQ ID NO.155 and SEQ ID NO.156; or (29) The light chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the light chain variable region shown in any one of (1) to (28) above, and the heavy chain variable region comprises a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chain variable region shown in any one of (1) to (28) above. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is chimeric, humanized or fully human. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof binds to human, mouse or monkey FGFR2. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprises the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or comprises a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antigen-binding fragment is selected from one or more of F(ab) ₂ , Fab', Fab, Fv, scFv, nanobody or affibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment is defucosylated. A multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and an antigen-binding molecule that binds to an antigen other than FGFR2, or an antigen-binding molecule that binds to an FGFR2 epitope different from the FGFR2 epitope bound by the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11; optionally, the other antigens other than FGFR2 comprise: CD3 (preferably CD3ε), CD16, NKG2D, NKp46, NKp30, CD137, CD258, PD-1, PD-L1, 4-1BB, CD40, CD64, EGFR, VEGF, HER2, HER1, HER3, IGF-1R, phosphatidylserine (PS), C-Met, HSA, MSLN, blood-brain barrier receptor, GPC3, PSMA, CD33, GD2, ROR1, ROR2, FRα or Gucy2C; Preferably, the antigen-binding molecule of the other antigen is an antibody or an antigen-binding fragment thereof; Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific; Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent. An immunoconjugate, wherein the immunoconjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the multispecific antigen-binding molecule according to claim 12. A chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises at least a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the multispecific antigen-binding molecule according to claim 12. An immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor of claim 14, or comprises a nucleic acid molecule encoding the chimeric antigen receptor of claim 14; preferably, the immune effector cell is selected from T cells, NK cells, NKT cells, DNT cells, monocytes, macrophages, dendritic cells or mast cells, and the T cells are preferably selected from cytotoxic T cells (CTLs), regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the multispecific antigen-binding molecule according to claim 12, or the chimeric antigen receptor according to claim 14. A vector, wherein the vector comprises the nucleic acid molecule according to claim 16. A host cell, wherein the host cell comprises the vector of claim 17; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (such as Escherichia coli), fungi (such as yeast), insect cells or mammalian cells (such as CHO cell line or 293T cell line); preferably, the host cell is genetically modified so that the expression of fucosyltransferase is reduced or deleted; preferably, the fucosyltransferase comprises α-1,6-fucosyltransferase; preferably, the genetic modification comprises knocking out the FUT8 gene of the host cell. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or the multispecific antigen-binding molecule according to claim 12, wherein the method comprises culturing the cell according to claim 18, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell. A method for preparing the immune effector cell of claim 15, wherein the method comprises introducing a nucleic acid molecule encoding the CAR of claim 14 into the immune effector cell, and optionally, the method further comprises initiating the immune effector cell to express the CAR. A pharmaceutical composition, wherein the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the multispecific antigen-binding molecule according to claim 12, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, or the product prepared by the method according to claim 19 or 20; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent. A method for treating a tumor or cancer, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the multispecific antigen-binding molecule according to claim 12, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared according to the method of claim 19, the immune effector cell prepared according to the method of claim 20, or the pharmaceutical composition according to claim 21; the tumor or cancer is a tumor or cancer expressing FGFR2, such as gastric cancer, non-small cell lung cancer squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the multispecific antigen-binding molecule according to claim 12, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, or the product prepared according to the method of claim 19 or 20, or the pharmaceutical composition according to claim 21 in the preparation of a drug for treating a tumor or cancer; the tumor or cancer is a tumor or cancer expressing FGFR2, such as gastric cancer, non-small cell lung cancer squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the multispecific antigen-binding molecule according to claim 12, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared according to the method of claim 19, the immune effector cell prepared according to the method of claim 20, or the pharmaceutical composition according to claim 21, for treating a tumor or cancer; the tumor or cancer is a tumor or cancer that overexpresses FGFR2, such as gastric cancer, non-small cell lung cancer squamous cell carcinoma, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer. A kit, wherein the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the multispecific antigen-binding molecule according to claim 12, the immune effector cell according to claim 15, the isolated nucleic acid molecule according to claim 16, the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared according to the method of claim 19, the immune effector cell prepared according to the method of claim 20, or the pharmaceutical composition according to claim 21. A method for detecting the expression of FGFR2b in a biological sample, the method comprising contacting the biological sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 under conditions where a complex can be formed between the antibody or antigen-binding fragment thereof and FGFR2b; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of FGFR2b in the sample. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 in the preparation of a FGFR2b detection reagent.