Anti-fgfr2b antibodies and uses thereof

Abstract

The present application provides a human FGFR2 antibody and application thereof. Specifically, the present application discloses an antibody specifically binding to human FGFR2, which has higher selective binding to FGFR2b subtype, and weaker binding to other subtypes such as FGFR2c, and has strong killing activity to tumor cells endogenously expressing FGFR2b such as KATOIII and SNU-16, and can be used for developing drugs and diagnostic reagents for treating FGFR2b positive tumors.

Description

Anti-FGFR2b antibodies and their applications

[0001] This disclosure claims priority to 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 into this disclosure. Technical Field

[0002] The present invention relates to the field of antibodies, and in particular to FGFR2b antibodies and applications thereof. Background Art

[0003] The fibroblast growth factor (FGF) family consists of 22 known FGFs, which are divided into seven subfamilies based on activity and sequence similarity. Currently, only four fibroblast growth factor receptors (FGFRs) 1-4 and their subtypes are known to bind to FGF. When FGFRs bind to ligands and heparin, they induce FGFR dimerization, autophosphorylation of the intracellular tyrosine kinase domain, and activation of multiple intracellular signal transduction pathways such as proliferation (STATs, RAS / p38 / JNKs, and RAS / MAPK / ERK), survival (STATs and PI3K / AKT), and cytoskeleton regulation (PLC / Ca 2+ ), promoting cell proliferation, survival, and differentiation, and participating in the regulation of physiological processes in the body, such as embryonic development, organ development, wound healing, and angiogenesis. Different FGFR2 subtypes have different ligands. FGFR2b is a high-affinity receptor for the FGF7 subfamily. Different subtypes are expressed in different tissues. FGFR2b is primarily expressed in epithelial tissues (such as the epithelial cells on the surface of the colorectal lumen, the epithelial cells from the base to the lower third of the esophagus, the epithelial parietal cells on the surface of the gastric lumen, the squamous epithelium on the surface of the uterus, and vascular smooth muscle cells). FGFR2c is primarily expressed in interstitial tissues.

[0004] When FGFR is overexpressed or mutated, it causes abnormalities in the FGFR signaling pathway, promoting tumor development and progression by promoting cell proliferation, survival, migration, and angiogenesis. FGFR2 is overexpressed and mutated in a variety of solid tumors, with the FGFR2b isoform expressed in gastric cancer, squamous cell carcinoma of the non-small cell lung, 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 worldwide, with an annual incidence of over one million patients and a mortality rate second only to lung and breast cancer. However, current treatment options for gastric cancer are very limited, especially for the high proportion of HER2-negative patients, who lack effective treatment options. Approximately 30% of patients with HER2-negative advanced gastric and gastroesophageal junction cancer are FGFR2b-positive. Immunohistochemistry and sequencing have confirmed that the FGFR2b subtype, but not the FGFR2c subtype, is specifically overexpressed in gastric cancer tissues. FGFR2b expression is undetectable in adjacent tissues and normal organs (except the skin). Therefore, FGFR2b may serve as a therapeutic target for these gastric cancer patients. Furthermore, FGFR2b is highly expressed (2+ / 3+ IHC) in 31% of non-small cell lung cancer (NSCLC), 13% of triple-negative breast cancer, 40% of ovarian cancer, 4% of pancreatic cancer, and 22% of intrahepatic bile duct carcinoma. Therefore, the development of therapeutics targeting FGFR2b is of great significance for the treatment of various cancers. Summary of the Invention

[0005] In a first aspect, the present disclosure provides 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:

[0006] (1) Inhibit the binding of FGF7 to FGFR2;

[0007] (2) inhibiting FGF7-induced tumor cell proliferation;

[0008] (3) not more than 5×10 -8 M binds to FGFR2 with affinity; and

[0009] (4) Binds to FGFR2b but not to FGFR2c.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region comprising the following combinations of LCDRs and HCDRs:

[0011] (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;

[0012] (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;

[0013] (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;

[0014] (4) 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, 53, 48;

[0015] (5) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 58, 59, 60, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 55, 56, 57;

[0016] (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;

[0017] (7) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 68, 69 and 70, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 65, 66 and 67;

[0018] (8) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 74, 75, 39, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 71, 72, 73;

[0019] (9) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 79, 80, 81, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 76, 77, 78;

[0020] (10) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 85, 86, 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 82, 83, 84;

[0021] (11) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 90, 86, 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 87, 88, 89;

[0022] (12) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 94, 86, 60, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 91, 92, 93;

[0023] (13) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 98, 86, 64, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 95, 96, 97;

[0024] (14) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 102, 103, 70, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 99, 100, 101;

[0025] (15) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 43, 44, 45, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 40, 127, 42;

[0026] (16) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 43, 44, 45, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 40, 128, 42;

[0027] (17) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 43, 44, 45, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 40, 129, 42;

[0028] (18) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 49, 50, 51, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 46, 135, 48;

[0029] (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;

[0030] (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;

[0031] (21) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO. 58, 59, 60, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO. 55, 146, 57;

[0032] (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

[0033] (23) The sequence of the six CDRs has 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 has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0034] 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:

[0035] 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, respectively, or

[0036] 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 set forth 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 set forth in any of the preceding items.

[0037] In a second 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 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In a seventh aspect, the present disclosure provides a vector comprising the aforementioned isolated nucleic acid fragment.

[0043] 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 (e.g., Escherichia coli), fungi (e.g., yeast), insect cells or mammalian cells (e.g., CHO cell line or 293T cell line).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In a thirteenth aspect, 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, 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 the preparation of a drug for treating tumors or cancer; the tumor or cancer is a tumor or cancer expressing FGFR2.

[0049] 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 treating tumors or cancers; the tumors or cancers are tumors or cancers expressing FGFR2.

[0050] 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.

[0051] In a sixteenth aspect, the present disclosure provides a method for detecting FGFR2b expression in a biological sample using the aforementioned FGFR2 antibody or antigen-binding fragment thereof, or multispecific antibody.

[0052] In a seventeenth aspect, the present disclosure provides a use of the aforementioned FGFR2 antibody or antigen-binding fragment thereof, or multispecific antibody in preparing an FGFR2b detection reagent. Beneficial effects:

[0053] 1. The FGFR2 antibodies provided herein have high selectivity for subtypes, with strong binding activity to FGFR2b and weak binding to FGFR2c, resulting in enhanced tumor killing specificity and reduced nonspecific killing of normal organs;

[0054] 2. The FGFR2 antibodies provided in the present disclosure have a high affinity for FGFR2b and can exert a strong tumor-killing effect at a lower dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1. Binding reaction of chimeric antibody to human FGFR2(β)b protein;

[0056] Figure 2. Binding reaction of chimeric antibodies to SNU-16 cells;

[0057] Figure 3. Chimeric antibodies inhibit cell proliferation induced by the ligand FGF7;

[0058] Figure 4. In vitro killing activity of chimeric antibodies;

[0059] Figure 5. Binding reaction of humanized antibodies to human FGFR2(β)b protein;

[0060] Figure 6. Binding reaction of humanized antibody to KATOIII membrane protein;

[0061] Figure 7. Binding reaction of humanized antibodies to SNU-16 cells;

[0062] Figure 8. Humanized antibodies inhibit cell proliferation induced by the ligand FGF7;

[0063] Figure 9. In vitro killing activity of humanized antibodies;

[0064] Figure 10. Chimeric antibody regulates SNU-16-#232 tumor growth and weight change curve of tumor-bearing mice;

[0065] Figure 11. Humanized antibody regulates KATOIII-#729 tumor growth and body weight change curve of tumor-bearing mice;

[0066] Figure 12. Chimeric antibodies regulate SNU-16-#232 tumor growth and body weight change curves of tumor-bearing mice. DETAILED DESCRIPTION

[0067] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0068] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0069] Definitions and Explanations of Terms

[0070] Unless otherwise defined herein, scientific and technical terms used in connection with this 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.

[0071] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that other elements may be present in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0072] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0073] The term FGFR (Fibroblast Growth Factor Receptors) is used herein to refer to fibroblast growth factor receptors, which generally include four types: FGFR1-FGFR4 (also known as CD331-334). FGFRs are members of the tyrosinase receptor family and are type I transmembrane proteins. They consist of an extracellular domain containing a ligand-binding site comprised of two or three immunoglobulin-like domains (IgI to IgIII); a single transmembrane domain; and an intracellular domain containing the tyrosine kinase domain. FGFRs typically function as dimers. The extracellular domain comprises three Ig-like domains, D1 (IgI), D2 (IgII), and D3 (IgIII). D1 and the acidic box form the autoinhibitory region; D2 and D3 are responsible for ligand binding (D2 binds to heparan sulfate on the cell surface, while D3 has two forms, IIIb and IIIc, due to alternative splicing; only the IIIc isoform has been identified in FGFR4). Based on the number of Ig-like domains, FGFRs can be divided into two types: the α type, which contains the IgI, IgII, and IgIII regions; and the β type, which contains only IgII and IgIII. For example, in this disclosure, FGFR2b includes "FGFR2(α)b" and "FGFR2(β)b."

[0074] 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 paracrinely. Among them, FGF7 can only bind to type IIIb FGFR2. FGFR binding 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. The types and frequencies of FGFR mutations vary among different cancer types.

[0075] 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 signals generated by FGF stimulation to cells or tissues expressing FGFR or activates signal transduction.

[0076] The terms "FGFR2b" and "FGFR2IIIb" are 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). The FGFR2IIIb protein typically binds to one or two or more FGFs selected from FGF1, FGF3, FGF7 (KGF), FGF10, FGF22, and FGF23. The FGFR2IIIb protein may bind to other FGFs and may not bind to mutant forms of the FGFs included in the above group.

[0077] The terms "FGFR2c" and "FGFR2IIIc" are 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). The FGFR2IIIc protein typically binds to one or two or more FGFs selected from FGF1, FGF2, FGF4, FGF6, FGF9, FGF17, FGF18, FGF21, and FGF23. The FGFR2IIIc protein may bind to other FGFs and may not bind to mutant forms of the FGFs included in the above group.

[0078] In some embodiments, the antibodies of the present disclosure 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 of the present disclosure bind to FGFR2b with an affinity or binding activity 5-fold, 10-fold, 100-fold, 1000-fold, or 10,000-fold greater than that of FGFR2c, which activity can be indicated by measuring the binding strength of the antibody to the protein by, for example, ELISA or FACS.

[0079] 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 typically retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. An epitope may include, but is 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.

[0080] 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.

[0081] 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.

[0082] The term "antibody" is used in the broadest sense herein and refers to a polypeptide or combination of polypeptides that comprises sufficient sequence from the variable region of the immunoglobulin heavy chain and / or sufficient sequence from the variable region of the immunoglobulin light chain, thereby being 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 comprising, 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 for transplanting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0083] The term "antibody" herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain 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 is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Differences in the amino acid composition and order of arrangement of the constant region of immunoglobulins' heavy chains result in varying antigenicity. Consequently, "immunoglobulins" can be classified into five classes, or isotypes, herein: IgM, IgD, IgG, IgA, and IgE, corresponding to their corresponding heavy chains: μ, δ, γ, α, and ε. Within the same class, Ig can be further divided into subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0084] The term "antibody" herein 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 receptor, IgNAR) found in cartilaginous fish such as sharks.

[0085] 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, cassowaries, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0086] The term "heavy chain antibody" herein refers to an antibody lacking the light chains 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.

[0087] The terms "nanobody" and "single-domain antibody" (sdAb) are used interchangeably and have the same meaning. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy-chain variable region by cloning the variable region of a heavy-chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy-chain antibody naturally lacking 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 solely of a single heavy-chain variable region.

[0088] The term "multi-specificity" herein refers to the ability of an antibody or its antigen-binding fragment to bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody can bind to. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and can therefore 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 and can therefore 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 (the actual number of complementary determining regions / binding sites in a single antibody molecule is referred to as "valence") in a single antibody. 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 the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but generally also includes proteins, e.g. antibodies that specifically bind three or more different epitopes, scaffolds, i.e. antibodies with three or more paratopes / binding sites.

[0089] 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, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0090] "Full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0091] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0092] The term "chimeric antibody" herein refers to an antibody having variable sequences of an immunoglobulin from one source organism (e.g., rat, mouse, rabbit, or alpaca) and constant regions of an immunoglobulin from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art.

[0093] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase 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.

[0094] 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 that are 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 onto human framework sequences.

[0095] The term "variable region" herein refers to the region of an antibody heavy or light chain involved in antigen binding. "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 native antibodies generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0096] The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) found in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT), while being considered to be outside the hypervariable region under a different set of criteria (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 CDRs can be abbreviated as HCDRs, and the light chain variable region can be abbreviated as LCDRs. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the sheet structure and, in some cases, form a portion of the sheet structure. The CDRs in each chain are held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and contribute to the formation of the antibody antigen-binding site with the CDRs from other antibody chains.

[0097] "CDRs" herein can be annotated and defined using 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). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0098] 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.

[0099] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, and has a more conserved amino acid sequence relative to the variable domains of antibodies. 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 native antibody constant region, while the latter only includes "a portion of the full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" is selected from the Fc or CH3 domain.

[0100] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region is selected from a constant kappa domain or a constant lambda domain.

[0101] 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. For example, 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, antibodies produced by host cells through 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) or the C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or absent. Typically, an IgG Fc region comprises the IgG CH2 and IgG CH3 domains, 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 typically extends from approximately amino acid residue position 231 to approximately amino acid residue 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 stretch of residues in the Fc region at the C-terminus of the CH2 domain (i.e., from approximately amino acid residue position 341 to approximately amino acid residue position 447 of IgG). The CH3 domain herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced in one chain and a corresponding "hole" introduced in the other chain; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). As described herein, such variant CH3 domains can 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.

[0102] 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 the Fc protein. "Inter-Fc variant interactions" refer to interactions between Fc variants engineered to form space-filling effects, electrostatic interactions, hydrogen bonding, hydrophobic interactions, and other interactions. These interactions contribute to the formation of stable heterodimeric proteins. Preferred mutational designs are "knob-into-hole" mutational designs.

[0103] Mutational design techniques for Fc variants have been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-into-hole" approach proposed by Cater et al.; the electrostatic steering approach used by Amgen researchers to form Fc-containing heterodimers (US20100286374A1); the SEEDbodies approach proposed by Jonathan H. Davis et al. to form heterodimers via IgG / Ig chain exchange; bispecific molecules formed using Genmab's DuoBody platform technology; Xencor researchers' integrated structural calculations and Fc amino acid mutations to create heterodimeric proteins with different modes of action; and Suzhou Alphamab's charge network-based Fc engineering approach (CN201110459100.7) to generate heterodimeric proteins. Other genetic engineering approaches based on Fc amino acid changes or functional modifications to achieve heterodimeric functional proteins are also being explored. 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". Preferably, the "knob-into-hole" model of Cater et al. is used 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. Preferably, the Fc region of human antibodies IgG1, IgG2, IgG3, and IgG4, more preferably the Fc region of human antibody IgG1. 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).

[0104] 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 better affinity than the parent antibody. An antibody that "exhibits decreased binding" to an FcR binds to at least one FcR with less affinity than the parent antibody. An antibody that exhibits decreased binding to an FcR may have little or no appreciable binding to an FcR, e.g., 0-20% of the binding to an FcR compared to a native sequence IgG Fc region.

[0105] "Enhanced affinity for FcγRIIIA" refers to an antibody that has a higher affinity for FcγRIIIA (also known 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).

[0106] The term "Affibody" refers to a small, non-immunoglobulin affinity protein with a triple-stranded β-sheet structure. Its microstructure is a compact globular structure and it can bind to specific target molecules with high affinity and low immunogenicity.

[0107] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0108] 1) Alanine (A), serine (S), threonine (T);

[0109] 2) Aspartic acid (D), glutamic acid (E);

[0110] 3) Asparagine (N), glutamine (Q);

[0111] 4) Arginine (R), Lysine (K), Histidine (H);

[0112] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0113] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0114] 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 that position. The percentage of identity between the two sequences varies as the number of identical positions shared by the sequences changes, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

[0115] Mathematical algorithms can be used to compare sequences between two sequences and calculate percent identity. For example, the Needlema and Wunsch algorithms (available at www.gcg.com) that have been integrated into the GAP program of the GCG software package are used, using a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program (available at www.gcg.com) in the GCG software package is used, using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossum 62 scoring matrix with 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.

[0116] Additionally or alternatively, the nucleic acid sequences and protein sequences described herein 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 the 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 alignments 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.

[0117] 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 an antibody heavy chain variable region and / or light chain variable region, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to 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.

[0118] The term "signal peptide" is used interchangeably to refer 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 may be heterologous or homologous to the protein to which they are attached. Exemplary, the signal sequence may comprise MGWSWILLFLLSVTAGVHS (SEQ ID NO: 158).

[0119] 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 can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes the nucleotide bases with the modification of the sugar or phosphate backbone bonded or chemically modified residue of derivatization.Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for in vitro and / or in vivo, such as in a host or patient, directly expressing the antibody of the present application.Such DNA (such as cDNA) or RNA (such as 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 the subject to produce antibodies in vivo.

[0120] As used herein, an "isolated" nucleic acid 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 cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0121] As used herein, the term "vector" 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 operatively linked. Such vectors are referred to herein as "expression vectors."

[0122] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0123] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0124] 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, flavorings, dyes, etc. and combinations thereof, which are known to those skilled in the art. Except in the case of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0125] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes 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 relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0126] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

[0127] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0128] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" 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 used herein.

[0129] Non-restrictive 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 comprises> 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 comprising the cancer overexpression of FGFR2 is higher than FGFR2IIIc. In some embodiments, the normalized level of FGFR2IIIb expressed by the cancer comprising FGFR2 amplification exceeds 2 times, 3 times, 5 times or 10 times the normalized level of FGFR2IIIc expression. In some embodiments, the expression level is normalized to GUSB. In some embodiments, cancer overexpresses FGFR2IIIb but does not comprise 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, the normalized level of gastric cancer expressing FGFR2IIIb comprising FGFR2 gene amplification is more than 2 times, 3 times, 5 times or 10 times the normalized level of FGFR2IIIc expression. 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.

[0130] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.

[0131] Detailed Description of the Invention

[0132] Antibodies that bind to human FGFR2

[0133] In one aspect, the present disclosure provides 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 comprises one or more of the following properties:

[0134] (1) Inhibit the binding of FGF7 to FGFR2;

[0135] (2) inhibiting FGF7-induced tumor cell proliferation;

[0136] (3) not more than 5×10 -8 M binds to FGFR2 with affinity; and

[0137] (4) Binds to FGFR2b but not to FGFR2c.

[0138] 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.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and / or a heavy chain variable region.

[0140] In some embodiments, the antibody or antigen-binding fragment thereof binds to human, murine, or monkey FGFR2.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof is chimeric, humanized, or fully human.

[0142] In some embodiments, the antibody or antigen-binding fragment thereof is afucosylated.

[0143] CDR

[0144] The CDR of an antibody is part of the variable region and is a key part that determines the specificity of the 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 encompasses CDR sequences obtained according to any CDR division method, and also encompasses variants thereof, wherein the variants include the amino acid sequence of the CDR after substitution, deletion and / or addition of 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 encompasses homologs thereof, wherein the homologs have a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity 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.

[0145] In the present disclosure, the antibody or antigen-binding fragment thereof comprises three LCDRs of the light chain variable region: LCDR1, LCDR2 and LCDR3.

[0146] In some embodiments, the LCDRs may comprise LCDRs in the light chain variable region sequence 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.

[0147] In some embodiments, the LCDRs may comprise LCDR1, LCDR2, and LCDR3 of the following sequence:

[0148] (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;

[0149] (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;

[0150] (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;

[0151] (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;

[0152] (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;

[0153] (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;

[0154] (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;

[0155] (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;

[0156] (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;

[0157] (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;

[0158] (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;

[0159] (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;

[0160] (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;

[0161] (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

[0162] (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).

[0163] In some embodiments, the antibody or antigen-binding fragment thereof comprises three HCDRs of the heavy chain variable region: HCDR1, HCDR2, and HCDR3.

[0164] In some embodiments, the HCDRs may comprise the HCDRs in the heavy chain variable region 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.

[0165] In some embodiments, the HCDRs may comprise HCDR1, HCDR2, and HCDR3 having the following sequences:

[0166] (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;

[0167] (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;

[0168] (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;

[0169] (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;

[0170] (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;

[0171] (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;

[0172] (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;

[0173] (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;

[0174] (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;

[0175] (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;

[0176] (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;

[0177] (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;

[0178] (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;

[0179] (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;

[0180] (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;

[0181] (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;

[0182] (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;

[0183] (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;

[0184] (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;

[0185] (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;

[0186] (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;

[0187] (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

[0188] (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 higher identity to any of the HCDRs in (1) to (22).

[0189] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region comprising the following combinations of LCDRs and HCDRs:

[0190] (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;

[0191] (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;

[0192] (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;

[0193] (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;

[0194] (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;

[0195] (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;

[0196] (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;

[0197] (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;

[0198] (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;

[0199] (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;

[0200] (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;

[0201] (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;

[0202] (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;

[0203] (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;

[0204] (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;

[0205] (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;

[0206] (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;

[0207] (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;

[0208] (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;

[0209] (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;

[0210] (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;

[0211] (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

[0212] (23) The sequence of the six CDRs has 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 has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0213] Antibody variable region

[0214] 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).

[0215] 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 thereto.

[0216] 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 thereto.

[0217] 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:

[0218] (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;

[0219] (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;

[0220] (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;

[0221] (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;

[0222] (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;

[0223] (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;

[0224] (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;

[0225] (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;

[0226] (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;

[0227] (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;

[0228] (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;

[0229] (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;

[0230] (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;

[0231] (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;

[0232] (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;

[0233] (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;

[0234] (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;

[0235] (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;

[0236] (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;

[0237] (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;

[0238] (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;

[0239] (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;

[0240] (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;

[0241] (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;

[0242] (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;

[0243] (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;

[0244] (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;

[0245] (28) the light chain variable region and the heavy chain variable region comprise the sequences shown in SEQ ID NO. 155 and SEQ ID NO. 156, respectively; or

[0246] (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 the above (1) to (28), 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 the above (1) to (28).

[0247] Antibody constant region

[0248] In the present disclosure, the antibody or antigen-binding fragment thereof may comprise a light chain and / or heavy chain constant region sequence.

[0249] In some embodiments, the heavy chain constant region can comprise the heavy chain constant region sequence of a human or murine antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0250] In some embodiments, the heavy chain constant region comprises the constant region sequence of human or murine 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 murine antibody IgG1, IgG2, IgG3 or IgG4.

[0251] In some embodiments, the heavy chain constant region comprises a heavy chain constant region derived from a human IgG1 antibody.

[0252] In some embodiments, the light chain constant region comprises the constant region of a human or murine lambda or kappa chain.

[0253] Multispecific antibodies

[0254] 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.

[0255] In some embodiments, the 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.

[0256] In some embodiments, the antigen-binding molecule for the other antigen is an antibody or an antigen-binding fragment thereof.

[0257] In some embodiments, the multispecific antigen-binding molecule can be bispecific, trispecific, or tetraspecific.

[0258] In some embodiments, the multispecific antigen-binding molecule can be bivalent, trivalent, tetravalent, pentavalent, or hexavalent.

[0259] Immunoconjugates

[0260] 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.

[0261] In some embodiments, the immunoconjugate further comprises a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

[0262] immune effector cells

[0263] 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.

[0264] 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.

[0265] In some embodiments, the immune effector cells are selected from T cells, NK cells, NKT cells, DN T cells, monocytes, macrophages, dendritic cells or mast cells.

[0266] 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).

[0267] In some embodiments, the immune effector cells are autologous immune effector cells or allogeneic immune effector cells.

[0268] On the other hand, 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 activating the immune effector cells to express the aforementioned CAR.

[0269] Nucleic acid molecules

[0270] In another aspect, the present disclosure provides one or more isolated nucleic acid molecules, which can 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.

[0271] carrier

[0272] On the other hand, the present 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 can include a promoter, a transcriptor, an enhancer, a replicon, a selection element and a reporter gene, etc. For example, the vector can include components that assist in entering the cell. In order to allow the nucleic acid molecule to replicate in the vector, the 5' end and the 3' end of the nucleic acid molecule can also include long terminal repeats (LTRs).

[0273] host cells

[0274] In another aspect, the present disclosure provides a host cell comprising the aforementioned isolated nucleic acid molecule or isolated vector.

[0275] 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).

[0276] The cell may include progeny of a single cell. Progeny may not necessarily be completely identical (in the form of total DNA complement or in genome) to the original parent cell due to natural, accidental, or deliberate mutation.

[0277] In some embodiments, the host cell is genetically modified such that the expression of fucosyltransferase is reduced or absent.

[0278] In some embodiments, the fucosyltransferase comprises an α-1,6-fucosyltransferase.

[0279] In some embodiments, the genetic modification comprises knocking out the FUT8 gene of the host cell.

[0280] Pharmaceutical composition

[0281] 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.

[0282] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, stabilizers, excipients, solubilizers, surfactants, emulsifiers and / or preservatives.

[0283] 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 and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0284] 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.

[0285] Combination or co-administration of drugs

[0286] On the other hand, the present disclosure provides a pharmaceutical combination, which refers to a product produced by mixing or combining multiple active ingredients, and can be divided into fixed and non-fixed combinations.

[0287] 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, and applied to a patient or subject simultaneously in the form of a single entity (such as a mixed injection) or fixed strength and dosage.

[0288] 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, and such administration provides therapeutically effective levels of the two or more active ingredients in the patient or subject.

[0289] In some embodiments, the additional active ingredient(s) comprises an anti-tumor agent.

[0290] In some embodiments, the pharmaceutical composition can be administered by various routes, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

[0291] Preparation method

[0292] 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 allowing the expression of the antigen-binding protein.

[0293] use

[0294] 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, product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition.

[0295] 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.

[0296] In another aspect, 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.

[0297] In some embodiments, the tumor or cancer is a tumor or cancer expressing FGFR2, such as gastric cancer, non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, or colorectal cancer.

[0298] 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.

[0299] Detection kit

[0300] 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, product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition.

[0301] In another aspect, the present disclosure provides a method for detecting FGFR2b expression in a biological sample using the aforementioned FGFR2 antibody or antigen-binding fragment thereof, or multispecific antibody.

[0302] In another aspect, the present disclosure provides a use of the aforementioned FGFR2 antibody or antigen-binding fragment thereof, or multispecific antibody in preparing an FGFR2b detection reagent.

[0303] 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.

[0304] Example

[0305] Example 1: Preparation of FGFR2-related antigens, control antibodies, and construction of stable cell lines

[0306] 1.1 Preparation of FGFR2 antigen

[0307] 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. The amino acid sequences of the extracellular domain (ECD) of FGFR2 of different species and subtypes were linked to human IgG1 Fc (hFc) (as shown in SEQ ID NOs. 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, product number: FGB-H5223), crab-eating macaque FGFR2(β)b-his (purchased from Acro, product number: FGB-C52H6), mouse FGFR2(β)b-his (purchased from Acro, product number: FGB-M52H5) and human FGFR2(β)c (purchased from Acro, product number: FGC-H5225) were purchased for identification and screening of antibodies.

[0308] Table 1 FGFR2 extracellular domain and hFc amino acid sequence

[0309] The corresponding nucleotide sequences were cloned into pTT5 vector (purchased from U-Bio, catalog number: VT2202), and plasmids were prepared according to established standard molecular biology methods.

[0310] The expression vector and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Thermofisher, Catalog No. A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma-Aldrich, Catalog No. G8270-5KG) were added. On the sixth day of transfection, the cell culture supernatant was collected.

[0311] Proteins containing His tags were purified from the cell culture supernatant using a Ni column (purchased from Cytiva, Catalog No. 17371206). The column was first equilibrated with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4) (purchased from Sangon, Catalog No. B548117-0500), and the clarified culture supernatant was then loaded onto the Ni column at a flow rate of 5 mL / min. After loading, the Ni column was washed with equilibration buffer at a volume of 3-5 times the Ni column bed volume. Proteins bound to the Ni column were eluted with an eluent containing 250 mM imidazole. After the sample was appropriately concentrated, it was 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 was determined using Nanodrop (purchased from Thermofisher), the antibody purity was determined using HPLC-SEC, and the protein endotoxin content was detected using an endotoxin detection kit (purchased from Andus). After passing the test, the samples were divided into aliquots for use and stored at -80°C.

[0312] Proteins containing Fc tags were purified from cell culture supernatants using a Protein A column (purchased from Cytiva, Catalog No. 17549802). The Protein A column was first equilibrated with 3-5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.4). The clarified culture supernatant was then loaded onto the column at a flow rate of 10 mL / min. After loading, the column was washed with equilibration buffer at a volume 3-5 times the column bed volume. Proteins bound to the Protein A column were eluted with elution buffer (50 mM NaAc-HAc, pH 3.5). The eluted protein was collected and adjusted to a neutral pH by adding Tris buffer (purchased from Sinopharm, Catalog No. 30188336). After the sample is appropriately concentrated, it is further purified using PBS-equilibrated gel chromatography Superdex200 to remove aggregates, collect the monomer peak, and then sterile filter it using a 0.22 μm 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, the samples are aliquoted for use and stored at -80°C.

[0313] 1.2 Preparation of control antibodies

[0314] Currently, the only FGFR2b-targeting antibody drug in clinical trials is Amgen's bemarituzumab, a first-in-class targeted antibody developed by Five Prime Therapeutics, FPA144. The anti-FGFR2b positive control antibody, bemarituzumab (FPA144), is based on the HuGAL-FR21 sequence from patent US8603987B2. Based on this sequence, the variable region of this antibody was linked to either the human hIgG1 antibody (heavy chain SEQ ID NO. 8 and light chain SEQ ID NO. 9, as shown in Table 2) or the murine IgG2a antibody constant region (heavy chain SEQ ID NO. 10 and light chain SEQ ID NO. 11, as shown in Table 2). These antibodies were used in the screening positive control antibodies (designated FPA144-hIgG1 and FPA144-mIgG2a).

[0315] The negative control antibody is an antibody against fluorescein isothiocyanate (FITC) that does not bind to FGFR2 (the sequence is shown in Table 2, and is designated as hIgG1 or mIgG2a).

[0316] The nucleotide sequences corresponding to the antibody heavy and light chains were cloned into the pTT5 vector to obtain plasmids expressing the antibody heavy and light chains, 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.

[0317] Table 2 Heavy and light chain sequences of control antibodies Note: Antibody constant regions are in italics.

[0318] 1.3 Construction of human FGFR2b stably transfected cell lines

[0319] 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 Youbao Bio, product number VT1465) and viral particles were prepared in HEK293T cells (purchased from the Chinese Academy of Sciences).

[0320] Table 3 Full-length amino acid sequences of different FGFR2b subtypes in different species Note: Signal peptide (single underline) + extracellular region + transmembrane region (double underline) + intracellular region (italic part)

[0321] 1.3.1 Construction of human FGFR2(α)b-HEK293T and human FGFR2(β)b-HEK293T stable cell lines

[0322] HEK293T cells (purchased from the Chinese Academy of Sciences) were infected with human FGFR2(α)b and human FGFR2(β)b lentiviruses, respectively. After selective culture for two weeks in DMEM medium (Gibco, Catalog No. 11995-065) supplemented with 10% (v / v) fetal bovine serum (Excell Bio, Catalog No. FSP500) and 5 μg / mL puromycin (Gibco, Catalog No. A11138-03), monoclonal cells were plated onto 96-well plates and cultured in a 37°C, 5% CO2 incubator. After approximately two weeks, a subset of monoclonal cells were selected for expansion. The expanded clones were labeled with the positive control antibody FPA144-hIgG1 and a secondary antibody and analyzed by flow cytometry. Monoclonal cell lines with good growth and high fluorescence intensity were selected for further expansion and cryopreservation in liquid nitrogen.

[0323] 1.3.2 Construction of human FGFR2(α)b-Ba / F3 stable transgenic cell line

[0324] Ba / F3 cells (purchased from Kangyuan Bochuang) were infected with human FGFR2(α)b lentivirus and selectively cultured for two weeks in RPMI 1640 medium (Gibco, Catalog No. 22400-089) supplemented with 1 μg / mL puromycin, 10% (v / v) fetal bovine serum, and 8 ng / mL IL-3 (Gibco, Catalog No. PMC0035). Afterward, monoclonal cells were plated onto 96-well plates and cultured in a 37°C, 5% CO2 incubator. After approximately two weeks, a subset of monoclonal cells were selected for expansion. The expanded clones were labeled with the positive control antibody FPA144-hIgG1 and a secondary antibody and analyzed by flow cytometry. Monoclonal cell lines with good growth and high fluorescence intensity were selected for further expansion and cryopreservation in liquid nitrogen. After cell expansion, 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.

[0325] Example 2: Generation of anti-FGFR2b murine monoclonal antibodies

[0326] 2.1 Animal immunization

[0327] The animal immunization experiment was divided into five groups. The experimental animals were 6-8 week old female Balb / c mice, SJL mice (purchased from Shanghai Slake Co., Ltd.), and MRL / LPR mice (purchased from Shanghai Weitonglihua Co., Ltd.). The animals were housed in an SPF environment. Orbital blood was collected from the mice before immunization to serve as negative serum.

[0328] The first immunization group consisted of five Balb / c mice. The immunogens were alternating between human FGFR2(α)b-hFc protein, mouse FGFR2(α)b-hFc protein, and human FGFR2(α)b-HEK293T cells. For the first immunization, human FGFR2(α)b-hFc protein was emulsified with TiterMax (Sigma-Aldrich, Catalog No. T2684), Alum (Thermo Fisher Scientific, Catalog No. 77161), and CpG (synthesized by a contract manufacturer, Catalog No. ODN1826). 0.1 mL of the emulsified solution was injected intraperitoneally and then subcutaneously and at multiple sites in the paw pad. Each mouse received a total of 50 μg of the immunogen. Boosters were administered weekly thereafter, with a total of 25 μg of the immunogen injected at each booster. For the first and third booster immunizations, mouse FGFR2b-hFc protein was mixed with Alum and CpG and then injected subcutaneously and at multiple sites in the paw pad. For the second and fifth booster immunizations, TiterMax was mixed with an equal volume of saline to form an emulsified mixture, and then 50 μL of the emulsified TiterMax was pre-injected into the peritoneal cavity of each mouse. 15 minutes later, 0.1 mL of HEK293T human FGFR2(α) b cells and CpG suspension was injected into the peritoneal cavity, and 5×10 6 The fourth and sixth booster immunizations were administered in the same manner as the initial immunization.

[0329] The second immunization group consisted of five MRL / LPR mice. Immunogens were alternating between human FGFR2(α)b-hFc protein, mouse FGFR2(α)b-hFc protein, and human FGFR2(α)b-HEK293T cells. The remaining steps were similar to those for the first immunization group.

[0330] 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 soles of the feet, i.e., each mouse was injected with a total of 50 μg of immunogen. Thereafter, booster immunizations were performed once a week, with proteins alternating 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 soles of the feet, and each mouse was injected with 25 μg of immunogen, for a total of six booster immunizations.

[0331] The fourth immunization group consisted of five SJL mice. Human FGFR2(β)b-hFc protein and mouse FGFR2(β)b-hFc protein were used as the immunogens, alternating between immunizations. The remaining steps were similar to those of the third immunization group.

[0332] The fifth immunization group consisted of 5 SJL mice. The immunogens were human FGFR2(β)b-hFc protein and human FGFR2(β)b-HEK293T cells, which were immunized alternately. During the initial immunization, human FGFR2(β)b-hFc protein was emulsified with TiterMax, Alum, and CpG, and then 0.1 mL was injected intraperitoneally and injected subcutaneously at multiple points, i.e., each mouse was injected with a total of 50 μg of immunogen. Thereafter, booster immunizations were performed every other week, and each mouse was injected with 25 μg of immunogen. During the first and third booster immunizations, 0.1 mL of HEK293T-hFGFR2(β)b cell and CpG suspension was injected intraperitoneally, and each mouse was injected with 5×10 6 The second booster immunization was administered in the same manner as the initial immunization. The fourth booster immunization was administered with human FGFR2(β)b-hFc protein emulsified with TiterMax and CpG and injected subcutaneously at multiple sites.

[0333] 2.2 Spleen cell fusion

[0334] Following booster immunizations in the above groups, mice were bled from their orbitals and serum was assayed for antibody titers against human FGFR2(β)b. Mice with high serum antibody titers were selected for spleen cell fusion. Three days prior to spleen cell fusion, a final booster immunization was performed with a total of 50 μg / mouse of the immunogen solution in saline injected subcutaneously, intrapedicularly, and intraperitoneally.

[0335] Spleens and lymph nodes were sterilely removed, ground, and filtered through a 40 μm cell strainer (BD Falcon). 5 mL of ACK Lysing Buffer (Gibco, Catalog No. A1049201) was added to lyse red blood cells to obtain a cell suspension. Cells were washed twice with Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Catalog No. 10569-010) by centrifugation at 1500 rpm. Cells were then mixed with mouse myeloma SP2 / 0 cells (ATCC) at a 2:1 ratio of viable cells per cell. Cell fusion was performed using the BTX ECM2001+ high-efficiency electrofusion method (see ECM2001+ELECTROFUSION PROTOCOL). The fused cells were diluted into DMEM supplemented with 20% fetal bovine serum (ExCell Bio, Catalog No. FND500) and 1× Hybri-Max HAT (Sigma, Catalog No. H0262-10VL). Percentages are by volume. After confluence, 5×10 cells were added to each well. 4 10 cells / 200 μL were added to a 96-well cell culture plate and cultured in a 37° C., 5% CO 2 incubator.

[0336] 2.3 Screening of hybridoma cells

[0337] After 7 days of fusion, the hybridoma cell supernatant was collected and tested for binding activity to human FGFR2(β)b, monkey FGFR2(β)b, mouse FGFR2(β)b protein, and human FGFR2(β)c protein. The hybridoma cells in the fusion plate wells corresponding to the supernatant that had binding activity to human FGFR2(β)b, monkey FGFR2(β)b, and mouse FGFR2(β)b proteins but did not bind to human FGFR2(β)c protein were subjected to limiting dilution with DMEM. The cells were observed under a microscope with the naked eye and the number of viable cells was counted. Approximately 200 cells were added to 2 mL of Medium D (purchased from STEMCELL, Catalog No. 03810), mixed well, and plated flatly in a 6-well cell culture plate. The cells were cultured at 37°C in 5% CO2.

[0338] After 7 days, single clones were picked and cultured in DMEM medium containing 10% (v / v) FBS and 1×HT (purchased from Sigma-Aldrich, catalog number: H0137-10VL) at 37°C and 5% CO2 for 2 days. The binding activity to human FGFR2(β)b protein was detected for preliminary screening. Positive single clones were selected and expanded to 24-well cell culture plates for further culture. After 3 days, the supernatant was tested for binding activity to 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-to-high expression) (purchased from Nanjing Kebai Biological) and for blocking activity of FGF7 binding to KATOIII cells (FGFR2b high expression) (purchased from Nanjing Kebai Biological). Based on the results of the 24-well plate sample test, 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.

[0339] 2.4 Identification of mouse monoclonal antibodies

[0340] 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.

[0341] The binding activity of antibodies to antigen proteins was tested using an enzyme-linked immunosorbent assay (ELISA). The antigen protein was diluted to a final concentration of 1 μg / mL in PBS buffer (purchased from Hyclone, Catalog No. SH30256.01), and then 50 μL was added to each well of a 96-well ELISA plate. Seal with plastic film and incubate overnight at 4°C. The next day, the plate was washed twice with PBST (PBS + 0.05% (v / v) Tween 20) and blocked with blocking buffer (PBS + 2% (v / v) BSA) for 2 hours at room temperature. The blocking buffer was discarded, and 50 μL of a 100 nM serially diluted control antibody was added to each well. After incubation at room temperature for 1 hour, the plate was washed three times with PBS. HRP (horseradish peroxidase)-conjugated goat anti-mouse IgG (H+L) secondary antibody (purchased from Jackson Immuno, Catalog No. 115-035-003) was added and incubated at room temperature for 1 hour, followed by five washes with PBS. TMB substrate (KPL, 5120-0077) (50 μL) was added to each well and incubated at room temperature for 10 minutes. Stop solution (1.0 M HCl) (50 μL) was then added to each well. OD450 nm values ​​were read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer).

[0342] Flow cytometry (FACS) was used to detect the binding activity of the antibody to the cells. The cells were cultured in T-175 cell culture flasks to 90% confluence. The culture medium was aspirated, the cells were washed once with PBS, and then treated with Versene (purchased from Gibco, product number 15040066) and collected. After counting the cells, the cells were washed twice with PBS and diluted to 2×10 6 Cells were plated at 50 μL per well in a 96-well FACS reaction plate. PBS was supplemented with 1% (v / v) fetal bovine serum as FACS buffer, and the cells were washed twice by centrifugation at 1500 rpm 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. Washed three times by centrifugation with FACS buffer, 50 μL of Alexa Fluor 647 fluorescently labeled goat anti-mouse IgG (H+L) secondary antibody (Jackson, Cat. No. 115-605-003) was added to each well and incubated at 4°C for 1 hour. Washed three times by centrifugation with FACS buffer. Cells were resuspended in 100 μL of FACS buffer and analyzed by FACS (FACS Canto II, BD Biosciences). The results showed that the seven mouse antibodies had strong binding activity to human FGFR2(β)b at both the protein and cellular levels.

[0343] Example 3: Identification of anti-FGFR2b chimeric antibodies

[0344] The variable region sequences of the above-mentioned 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 to obtain recombinant plasmids expressing VH-CH and VL-CL. Referring to the antibody preparation method in Section 1.2 of Example 1, 7 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 chimeric antibody variable region sequences, and Table 6 shows the IMGT analysis results of the chimeric antibody variable region sequences.

[0345] Table 4 FGFR2b chimeric antibody variable region, signal peptide, and constant region sequences

[0346] Table 5 Kabat analysis results of FGFR2b chimeric antibody variable region sequences

[0347] Table 6 IMGT analysis results of FGFR2b chimeric antibody variable region sequences

[0348] 3.1 Detection of the binding activity of chimeric antibodies to human FGFR2b

[0349] The binding activity of the chimeric antibodies to human FGFR2(β)b protein and SNU-16 cells was assayed using the ELISA and FACS methods described in Section 2.4 of Example 2. The results, as shown in Figures 1 and 2 , indicate that the seven chimeric antibodies exhibited strong binding activity to human FGFR2(β)b at both the protein and cellular levels.

[0350] 3.2 Detection of nonspecific binding activity of chimeric antibodies to human FGFR2(β)c

[0351] 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 test results are shown in Table 7. None of the seven chimeric antibodies bound to human FGFR2(β)c.

[0352] Table 7 Non-specific binding of chimeric antibodies to human FGFR2(β)c protein

[0353] 3.3 Detection of affinity of chimeric antibodies to human FGFR2(β)b protein

[0354] Surface plasmon resonance (SPR) analysis was used to determine the affinity of antibodies for human FGFR2(β)b protein. Antibodies were captured using a Protein A chip (Cytiva, Catalog No. 29-127-558). Sample and running buffer used HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (Cytiva, Catalog No. BR-1006-69). The flow-through cell was set to 25°C and the sample block to 16°C. Both were preconditioned with running buffer. In each cycle, the test antibody was first captured using the Protein A chip. A single concentration of human FGFR2(β)b-His antigen was then injected, and the binding and dissociation processes between the antibody and antigen were recorded. Finally, the chip was regenerated using 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 and diluted 1:1 for a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with a 10 mM glycine solution (pH 1.5) at a flow rate of 30 μL / min for 30 seconds. The bulk refractive index difference was corrected by subtracting the response value obtained from the reference channel. A blank injection (= double reference) was also subtracted. To calculate the apparent KD and other kinetic parameters, a Langmuir 1:1 model was used. The association rate (ka), dissociation rate (kd) and binding affinity (KD) of the antibody to human FGFR2 (β) b-His protein are shown in Table 8.

[0355] Table 8 Affinity of chimeric antibodies to human FGFR2(β)b protein

[0356] 3.4 Detection of chimeric antibodies to inhibit cell proliferation induced by ligand FGF7

[0357] The in-house constructed human FGFR2(α)b-Ba / F3 cell line relies on the FGFR2b-FGF7 signaling pathway for proliferation and growth. This cell line was used to detect the activity of anti-FGFR2b antibodies in blocking FGF7 binding and thus inhibiting cell proliferation. Human FGFR2(α)b-Ba / F3 cells in good growth condition were taken and resuspended in RPMI 1640 complete medium. The resuspended cells were plated at 1×10 per well. 4Cells / 50 μL were added to a 96-well plate (purchased from Corning, Cat. No. 3610). Antibodies were serially diluted in RPMI 1640 complete medium (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 per well was 100 μL.

[0358] After incubation in a carbon dioxide incubator for 3 days, 100 μL of CellTiter-Glo reagent (purchased from Promega, product number G9243) was added to each well of the 96-well plate. After incubation at room temperature for 10 minutes, the fluorescence value was read on an Envision instrument (purchased from PerkinElmer, model Envision2105).

[0359] The cell killing rate was calculated using the following formula: Tumor cell wells cultured in RPMI 1640 complete medium supplemented with 10 ng / mL FGF7 and 10 μg / mL Heparin served as positive controls, demonstrating uninhibited proliferation. Tumor cell wells cultured in RPMI 1640 medium alone served as blank negative controls. Cell proliferation inhibition rate = ((positive well reading - sample well reading) / (positive well reading - blank negative well reading)) × 100%. Results were calculated and graphed using GraphPad Prism 9.0 software.

[0360] 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.

[0361] Table 9 Chimeric antibodies inhibit cell proliferation induced by ligand FGF7

[0362] 3.5 Detection of the killing activity of chimeric antibodies in vitro

[0363] Healthy human peripheral blood mononuclear cells (PBMCs) were thawed and resuspended in RPMI 1640 complete medium containing rhIL-2 (purchased from R&D, product number 202-IL-050) at a final concentration of 50 ng / mL. The cells were then incubated overnight in a CO2 incubator. 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, product number 11835-030) and plated at 1×10 cells per well. 4 Individual FGFR2(α)b-Ba / F3 cells and 2×10 5PBMCs were added to a 96-well plate (Corning, Cat. No. 3599) at a ratio of 1:1 (50 μL) to 1:1. Antibodies were serially diluted in phenol red-free 1640 medium containing 5% inactivated serum (starting at 400 nM, with 5-fold dilutions over eight concentration steps). According to the experimental design, 50 μL of the corresponding antibody dilution was added to each well, resulting in a final volume of 100 μL per well. The cell culture plates were incubated in a CO2 incubator for 4 hours.

[0364] 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, product number CK12, and the usage method was referred to the kit instructions), and the percentage of target cell killing was calculated.

[0365] Results were calculated and graphed using GraphPad Prism 9.0 software. The cell killing rate was calculated using the following formula: The wells in which all tumor cells were lysed were defined as the positive control, with 100% killing. The wells in which the antibody concentration was 0 were defined as the blank negative control. Cell killing rate = ((reading value of the sample well - reading value of the blank negative well) / (reading value of the positive well - reading value of the blank negative well)) × 100%.

[0366] 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 seven 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.

[0367] Table 10 In vitro killing activity of chimeric antibodies

[0368] Example 4: Humanization of anti-FGFR2b monoclonal antibody

[0369] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy and light chain variable region germline genes with high homology to murine antibodies as templates. The CDRs of the murine antibodies were then transplanted into the corresponding human templates, resulting in a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the antibody's three-dimensional structure, we backmutated buried residues, residues that directly interact with the CDR regions, and residues in the framework regions that significantly influence VL and VH conformations to generate humanized monoclonal antibodies. Tables 11 and 12 list the humanized light chain and heavy chain template sequences, respectively, used in the Examples.

[0370] Table 11 Humanized light chain template

[0371] Table 12 Humanized heavy chain template

[0372] 4.1 Humanization of mab01

[0373] The humanized light chain templates for the murine 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 murine antibody mab01 were transplanted into their humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region sequences of the humanized mab01 antibody were backmutated to their murine counterparts to maintain the original affinity. The CDR amino acid residues are assigned and annotated using the Kabat numbering system. Detailed mutation design is shown in Table 13.

[0374] Table 13 Humanized antibody mutation design of mab01 Note: Q45K means the 45th Q is mutated to K, and so on. The mutated amino acids are numbered in natural order.

[0375] The specific sequence of the variable region of the mab01 humanized antibody is as follows:

[0376] The amino acid sequence of Hab01.L3 is shown in SEQ ID NO.118:

[0377] The amino acid sequence of Hab01.L4 is shown in SEQ ID NO.119:

[0378] The amino acid sequence of Hab01.H1 is shown in SEQ ID NO.120:

[0379] The amino acid sequence of Hab01.H2 is shown in SEQ ID NO.121:

[0380] The present invention selected different light chain and heavy chain sequences from the mutation designs of the light chain and heavy chain variable regions of the humanized antibody mab01 described above, and cross-combined them to ultimately obtain the mab01 humanized antibody. The amino acid sequences of the variable regions of each antibody are as follows:

[0381] Table 14 Amino acid sequences corresponding to the variable regions of the mab01 humanized antibody

[0382] According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibodies are shown in Table 15.

[0383] Table 15 Kabat analysis results of mab01 humanized antibody CDR sequences

[0384] 4.2 Humanization of mab02

[0385] The humanized light chain templates for the murine antibody mab02 were IGKV2-40*01 / IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-69*02 and IGHJ6*01. CDR amino acid residues were assigned and annotated using the Kabat numbering system. The mutation strategy was described in Section 4.1 above. Detailed mutation design is shown in Table 16.

[0386] Table 16 Humanized antibody mutation design of mab02 Note: P49S means the 49th amino acid is mutated to S, and so on. The mutated amino acids are numbered in natural order.

[0387] The specific sequence of the variable region of the mab02 humanized antibody is as follows:

[0388] The amino acid sequence of Hab02.L1 is shown in SEQ ID NO.122:

[0389] The amino acid sequence of Hab02.L2 is shown in SEQ ID NO.123:

[0390] The amino acid sequence of Hab02.H1a is shown in SEQ ID NO.124:

[0391] The amino acid sequence of Hab02.H2a is shown in SEQ ID NO.125:

[0392] The amino acid sequence of Hab02.H3a is shown in SEQ ID NO.126:

[0393] The present invention selected different light chain and heavy chain sequences from the aforementioned humanized antibody mab02 light chain and heavy chain variable region mutation designs for cross-combination, ultimately obtaining a variety of mab02 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0394] Table 17 Amino acid sequences corresponding to the variable regions of the mab02 antibody

[0395] According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibodies are shown in Table 18.

[0396] Table 18 Kabat analysis results of mab02 humanized antibody CDR sequences

[0397] 4.3 Humanization of mab03

[0398] The humanized light chain templates for the murine 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. CDR amino acid residues are assigned and annotated using the Kabat numbering system. The mutation strategy is described in Section 4.1 above. Detailed mutation design is shown in Table 19.

[0399] 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.

[0400] The specific sequence of the variable region of the mab03 humanized antibody is as follows:

[0401] The amino acid sequence of Hab03.L3 is shown in SEQ ID NO.130:

[0402] The amino acid sequence of Hab03.L5 is shown in SEQ ID NO.131:

[0403] The amino acid sequence of Hab03.L6 is shown in SEQ ID NO.132:

[0404] The amino acid sequence of Hab03.H2b is shown in SEQ ID NO.133:

[0405] The amino acid sequence of Hab03.H10a is shown in SEQ ID NO.134:

[0406] The present invention selected different light chain and heavy chain sequences from the aforementioned humanized antibody mab03 light chain and heavy chain variable region mutation designs for cross-combination, ultimately obtaining a variety of mab03 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0407] Table 20 Amino acid sequences corresponding to the variable regions of the mab03 antibody

[0408] According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibodies are shown in Table 21.

[0409] Table 21 Kabat analysis results of mab03 humanized antibody CDR sequences

[0410] 4.4 Humanization of mab04

[0411] The humanized light chain templates for the murine antibody mab04 were IGKV1-33*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-3*01 and IGHJ6*01. The CDR amino acid residues were assigned and annotated using the Kabat numbering system. The mutation strategy was described in Section 4.1 above. Detailed mutation design is shown in Table 22.

[0412] Table 22 Humanized antibody mutation design of mab04 Note: A43S indicates that the 43rd amino acid position is mutated to S, and so on. The mutated amino acids are numbered in natural order.

[0413] The specific sequence of the variable region of the mab04 humanized antibody is as follows:

[0414] The amino acid sequence of Hab04.L2 is shown in SEQ ID NO.136:

[0415] The amino acid sequence of Hab04.H1a is shown in SEQ ID NO.137:

[0416] The amino acid sequence of Hab04.H2a is shown in SEQ ID NO.138:

[0417] The amino acid sequence of Hab04.H4 is shown in SEQ ID NO.139:

[0418] The present invention selected different light chain and heavy chain sequences from the aforementioned humanized antibody mab04 light chain and heavy chain variable region mutation designs for cross-combination, ultimately obtaining a variety of mab04 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0419] Table 23 Amino acid sequences corresponding to the variable regions of the mab04 antibody

[0420] According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibodies are shown in Table 24.

[0421] Table 24 Kabat analysis results of mab04 humanized antibody CDR sequences

[0422] 4.5 Humanization of mAb05

[0423] The humanized light chain templates for the murine 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 are assigned and annotated using the Kabat numbering system. The mutation strategy is described in Section 4.1 above. Detailed mutation design is shown in Table 25.

[0424] Table 25 Humanized antibody back mutation design of mab05 Note: K42H indicates that the 42nd K mutated to H, and so on. The mutated amino acids are numbered in natural order.

[0425] The specific sequence of the variable region of the mab05 humanized antibody is as follows:

[0426] The amino acid sequence of Hab05.L1 is shown in SEQ ID NO.142:

[0427] The amino acid sequence of Hab05.L2 is shown in SEQ ID NO.143:

[0428] The amino acid sequence of Hab05.L3 is shown in SEQ ID NO.144:

[0429] The amino acid sequence of Hab05.H1a is shown in SEQ ID NO.145:

[0430] The present invention selected different light chain and heavy chain sequences from the aforementioned humanized antibody mab05 light chain and heavy chain variable region mutation designs for cross-combination, ultimately obtaining a variety of mab05 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0431] Table 26 Amino acid sequences corresponding to the variable regions of the mab05 antibody

[0432] According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibodies are shown in Table 27.

[0433] Table 27 Kabat analysis results of mab05 humanized antibody CDR sequences

[0434] 4.6 Humanization of mab06

[0435] The humanized light chain templates for the murine antibody mab06 were IGKV1-33*01 / IGKV2-29*02 and IGKJ4*01, and the humanized heavy chain templates were IGHV1-46*01 and IGHJ1*01. The CDR amino acid residues were assigned and annotated using the Kabat numbering system. The mutation strategy was described in Section 4.1 above. Detailed mutation design is shown in Table 28.

[0436] Table 28 Humanized antibody mutation design of mab06 Note: Q45K indicates that the 45th Q mutated to a K, and so on. The mutated amino acids are numbered in natural order.

[0437] The specific sequence of the variable region of the mab06 humanized antibody is as follows:

[0438] The amino acid sequence of Hab06.L1 is shown in SEQ ID NO.147:

[0439] The amino acid sequence of Hab06.L2 is shown in SEQ ID NO.148:

[0440] The amino acid sequence of Hab06.H2 is shown in SEQ ID NO.149:

[0441] The amino acid sequence of Hab06.H3 is shown in SEQ ID NO.150:

[0442] The amino acid sequence of Hab06.H4a is shown in SEQ ID NO.151:

[0443] The present invention selected different light chain and heavy chain sequences from the aforementioned humanized antibody mab06 light chain and heavy chain variable region mutation designs for cross-combination, ultimately obtaining multiple mab06 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0444] Table 29 Amino acid sequences corresponding to the variable regions of the mab06 antibody

[0445] According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibodies are shown in Table 30.

[0446] Table 30 Kabat analysis results of mab06 humanized antibody CDR sequences

[0447] 4.7 Humanization of mab07

[0448] The humanized light chain templates for the murine antibody mab07 were IGKV6-21*01 / IGKV2-30*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-3*01 and IGHJ6*01. The CDR amino acid residues were assigned and annotated using the Kabat numbering system. The mutation strategy was described in Section 4.1 above. Detailed mutation design is shown in Table 31.

[0449] Table 31 Humanized antibody mutation design of mab07 Note: L46W indicates that the 46th amino acid mutated from L to W, and so on. The mutated amino acids are numbered in natural order.

[0450] The specific sequence of the variable region of the mab07 humanized antibody is as follows:

[0451] The amino acid sequence of Hab07.L1 is shown in SEQ ID NO.153:

[0452] The amino acid sequence of Hab07.L2 is shown in SEQ ID NO.154:

[0453] The amino acid sequence of Hab07.L3 is shown in SEQ ID NO.155:

[0454] The amino acid sequence of Hab07.H1 is shown in SEQ ID NO.156:

[0455] The amino acid sequence of Hab07.H3 is shown in SEQ ID NO.157:

[0456] The present invention selected different light chain and heavy chain sequences from the aforementioned humanized antibody mab07 light chain and heavy chain variable region mutation designs for cross-combination, ultimately obtaining a variety of mab07 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0457] Table 32 Amino acid sequences corresponding to the variable regions of the mab07 antibody

[0458] According to the Kabat numbering system, the results of CDR sequence analysis of the above humanized antibodies are shown in Table 33.

[0459] Table 33 Kabat analysis results of mab07 humanized antibody CDR sequences

[0460] Example 5: Identification of anti-FGFR2b humanized antibodies

[0461] The nucleic acid sequences encoding the humanized antibodies VH and VL were recombined into the expression vector pTT5 containing a signal peptide and the CH and CL sequences of the human IgG1 antibody, respectively, to obtain recombinant plasmids expressing VH-CH and VL-CL. The humanized antibodies were obtained by referring to the antibody preparation method described in Section 1.2 of Example 1.

[0462] 5.1 Detection of Binding Activity of Humanized Antibodies to Human FGFR2b

[0463] The binding activity of the antibody to KATOIII and SNU-16 cell surface membrane proteins was tested using a cell-based ELISA. Cells were cultured in T-175 cell culture flasks to 90% confluence. The culture medium was aspirated, the cells were washed once with PBS, and then treated with Versene and harvested. After counting the cells, the cells were washed twice with PBS and diluted to 4 × 10 5 Cells were plated at a concentration of 100 μL per well of a 96-well cell culture plate and cultured overnight in an incubator. The next day, the culture medium was discarded, the cells were washed twice with PBS, and 50 μL of fixative solution was added to each well. The cells were fixed in a fume hood for 30 minutes. After washing twice with PBST, the assay was performed according to the ELISA method.

[0464] The binding activity of the humanized antibody to human FGFR2(β)b protein was detected using the ELISA and FACS methods described in Section 2.4 of Example 2. The test results are shown in Figures 5, 6, and 7, indicating that the humanized antibody has strong binding activity to human FGFR2(β)b at both the protein and cellular levels.

[0465] 5.2 Detection of nonspecific binding activity of humanized antibodies

[0466] 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 test results are shown in Table 34, and none of the humanized antibodies bound to human FGFR2(β)c.

[0467] Table 34 Non-specific binding activity of humanized antibodies to human FGFR2 (β) c protein

[0468] 5.3 Detection of affinity of humanized antibodies to human FGFR2(β)b protein

[0469] The affinity of the humanized antibodies to human FGFR2(β)b protein was determined using the method of Reference Example 3.3. The results are shown in Table 35. The affinity of some humanized antibodies can retain the affinity of the chimeric antibody.

[0470] Table 35 Affinity of humanized antibodies to human FGFR2 (β) b protein

[0471] 5.4 Detection of humanized antibodies to inhibit cell proliferation induced by ligand FGF7

[0472] Refer to the experimental method in Section 3.4 of Example 3, and 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.

[0473] Table 36 Humanized antibodies inhibit cell proliferation induced by ligand FGF7

[0474] 5.5 Detection of the killing activity of humanized antibodies in vitro

[0475] The in vitro cytotoxicity of the humanized antibodies was tested using the experimental method described in Section 3.5 of Example 3, and the results are shown in Table 37 and Figure 9. The experimental results showed that the humanized antibodies had a significant cytotoxic effect on human FGFR2(α)b-Ba / F3 cells.

[0476] Table 37 In vitro killing activity of humanized antibodies

[0477] Example 6: Endocytic effect of anti-FGFR2b antibodies

[0478] The KATOIII cells used in this experiment (purchased from Nanjing Kebai Biotechnology, catalog number: CBP60483) were cultured in RPMI 1640 + 10% FBS (purchased from Gibco, catalog number: 10491; Gibco, catalog number: 10091-148, respectively). KATOIII cells were resuspended in flow cytometry buffer (purchased from Biolegend, catalog number: 420201) and the cell density was adjusted to 2×10 6 50 μL of cell suspension was added to each well of a round-bottom 96-well plate (purchased from Corning, catalog number: 3799) so that the number of cells in each well was 1×10 5. 50 μL of serially diluted antibody (starting concentration 100 nM, 3-fold dilution) was then added, and the cells were incubated at 4°C for 1 hour. After 1 hour, unbound antibody was washed away, and the cells were resuspended in flow cytometry buffer. The control group was continued to be incubated at 4°C, while the endocytosis group was incubated at 37°C for 4 hours. After 4 hours, the cells were washed twice with PBS, and Alexa Fluor 647 fluorescently labeled goat anti-human IgG Fcγ secondary antibody (purchased from Jackson Immuno, cat. no. 109-605-098) was added. The cells were incubated at 4°C for 1 hour, washed twice with PBS, and the Alexa Fluor 647 fluorescence signal (MFI) was analyzed by FACS. GraphPad Prism 9.0 software was used for calculation and graphing. The wells containing the isotype negative control antibody were defined as negative controls. Endocytosis efficiency = (control group MFI - endocytosis group MFI) / control group MFI × 100%.

[0479] The results are shown in Table 38. In this experiment, the negative control had no obvious endocytic activity in KATO III cells at a saturated concentration of 100 nM. The seven anti-FGFR2b antibodies of the present invention all had a certain degree of endocytic activity in KATO III cells (endocytic rate 33.5% to 43.5%), and the endocytic activity of the anti-FGFR2b monoclonal antibodies of the present invention was better than that of the control antibody FPA144-hIgG1 (endocytic rate 30.5%).

[0480] Table 38 Endocytic activity of anti-FGFR2b antibodies in KATOIII cells

[0481] Example 7: In vivo efficacy of anti-FGFR2b antibodies

[0482] 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 Laboratory Animal Technology Co., Ltd.), and this model was used to evaluate the anti-tumor efficacy of candidate molecules in vivo.

[0483] 7.1 Chimeric Antibodies Inhibit Growth of Human Gastric Cancer SNU-16-#232 Tumors

[0484] The inoculation time of human gastric cancer cells SNU-16-#232 (self-extracted primary cells, purchased from Nanjing Kebai Biotechnology) was designated as day 0 of the experiment. On day 0 of the experiment, human gastric cancer cells SNU-16-#232 were harvested and inoculated to the desired number and in the logarithmic growth phase (confluence was approximately 80%, and fresh culture medium was replaced with cells one day before inoculation). First, the cell suspension was collected into a 50mL centrifuge tube and centrifuged at 300g for 7 minutes. The cells were resuspended in an appropriate amount of serum-free RPMI1640 culture medium (purchased from Gibco, catalog number: 61870-036). 500μL of the cell suspension was counted on a cell counter (Beckman, SIC-TP-573). Finally, based on the cell count results, the cell density was adjusted to 200×10 using serum-free culture medium. 6 cells / mL, 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, product number: 356237) in equal proportions, and 100 μL of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.

[0485] After inoculation of gastric cancer cells SNU-16-#232, tumor growth was monitored. When the mean tumor volume was between 100-150 mm 3 Mice with appropriate tumor volumes were randomly divided into groups of 8 mice per group. They were then treated with intravenous administration twice a week for a total of 6 times. The tumor volume and mouse body weight changes were measured. The specific dosing regimen is shown in Table 39, wherein hIgG1 was the isotype control, FPA144-hIgG1 was the positive molecule, mab04, mab07, and mab06 were candidate antibodies. The candidate antibodies were dosed at the same molar amount per unit body weight as the isotype control hIgG1, and the equimolar dose was converted according to the formula m=n*M, where m is the drug mass, n is the drug molar mass, and M is the drug molecular weight. The doses in this section were calculated based on this principle.

[0486] The results are shown in Table 40 and Figure 10. As of 18 days after drug withdrawal, each group showed a certain degree of efficacy, and the efficacy trend remained stable. Among them, mab06 and mab04 showed better efficacy than the positive control.

[0487] Table 39: In vivo efficacy and dosing regimen of chimeric antibodies in human gastric cancer SNU-16-#232 tumor-bearing mice

[0488] Table 40: Pharmacological efficacy of chimeric antibodies in regulating human gastric cancer SNU-16-#232 tumors

[0489] 7.2 Humanized Antibodies Inhibit Tumor Growth in Human Gastric Cancer KATOIII-#729

[0490] The inoculation time of human gastric cancer cells KATOIII-#729 (self-extracted primary cells, purchased from Nanjing Kebai Biological) was set as day 0 of the experiment. On day 0 of the experiment, human gastric cancer cells KATOIII-#729 that had been 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 culture medium in the cell culture flask was removed and washed twice with phosphate buffered saline (PBS, purchased from Hyclone, catalog number: SH30256.01). 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. The flask 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 flask were gently blown off the culture flask. The digested cell suspension was collected into a 50 mL centrifuge tube and centrifuged at 350 g for 5 minutes. An appropriate amount of serum-free RPMI1640 medium was aspirated to resuspend the cells and filtered through a 70 μm mesh. 500 μL of the cell suspension was counted on a cell counter. Finally, based on the cell counting results, the cell density was adjusted to 100 × 10 6 cells / mL, 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 gel in equal proportions, and 200 μL of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.

[0491] After inoculation of gastric cancer cells KATOIII-#729, tumor growth was monitored. When the mean tumor volume was 200 mm 3 Mice with appropriate tumor volumes were randomly divided into groups of 7 mice per group. Each group was administered with tail vein injection twice a week for a total of 6 times. The tumor volumes and weight changes of the mice were measured. The specific administration schedule is shown in Table 41.

[0492] 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.

[0493] Table 41: Efficacy and dosing regimen of humanized antibodies in mice bearing human gastric cancer KATOIII-#729

[0494] Table 42: Pharmacological efficacy of humanized antibodies in regulating human gastric cancer KATOIII-#729 tumors

[0495] 7.3 Chimeric Antibodies Inhibit Growth of Human Gastric Cancer SNU-16-#232 Tumors

[0496] Refer to the experimental method in 7.1 above, with 7 mice in each group and the specific dosing regimen as shown in Table 43.

[0497] The results are shown in Table 44 and Figure 12. The efficacy of each drug-dosing group was stable, and the chimeric molecule mab01 was superior to the positive control FPA144-hIgG1.

[0498] Table 43: Efficacy and dosing regimen of chimeric antibodies in mice bearing human gastric cancer SNU-16-#232

[0499] Table 44 Chimeric antibody regulation of human gastric cancer SNU-16-#232 tumor efficacy results

[0500] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of this application, those skilled in the art may make various changes or modifications to this application, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. 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) No more than 5×10 -8 M binds to FGFR2 with affinity; and (4) Binds to FGFR2b but not to FGFR2c.

2. 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).

3. 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).

4. 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.

5. 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 one of 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 thereto; and, (2) The heavy chain variable region comprises a sequence as shown in any one of 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 sequences shown.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The antibody or antigen-binding fragment thereof has a combination of a light chain variable region and a heavy chain variable region 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 comprise the sequences shown in SEQ ID NO.155 and SEQ ID NO.156, respectively; 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.

7. 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.

8. 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.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antibody or its antigen-binding fragment 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.

10. 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.

11. 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.

12. 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 a 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 antigen other than FGFR2 comprises: 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.

13. 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.

14. 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.

15. 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 cell is preferably selected from cytotoxic T cells (CTL), regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

16. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the multispecific antigen-binding molecule of claim 12, or the chimeric antigen receptor of claim 14.

17. A vector, wherein The vector comprises the nucleic acid molecule of claim 16.

18. 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 lines or 293T cell lines); 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.

19. 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 of claim 18, and isolating the antibody, antigen-binding fragment, or multispecific antigen-binding molecule expressed by the cell.

20. 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 an immune effector cell. Optionally, the method further comprises initiating the immune effector cell to express the CAR.

21. 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 a product prepared according to the method of 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.

22. A method for treating a tumor or cancer, wherein: The method comprises 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, squamous cell carcinoma of non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

23. 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, squamous cell carcinoma of non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

24. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 15, the isolated nucleic acid molecule of claim 16, or 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 of claim 21, for treating a tumor or cancer; the tumor or cancer is a tumor or cancer that overexpresses FGFR2, such as gastric cancer, squamous cell carcinoma of non-small cell lung cancer, triple-negative breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, intrahepatic bile duct carcinoma, and colorectal cancer.

25. 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 by the method according to claim 19, the immune effector cell prepared by the method according to claim 20, or the pharmaceutical composition according to claim 21.

26. 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.

27. 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.

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