Anti-fgfr2b antibodies and antibody drug conjugates

Abstract

This invention provides anti-FGFR2b antibodies and antibody-drug conjugates. The antibodies or antigen-binding fragments thereof of this invention comprise HCDR1 with amino acid sequences as shown in SEQ ID NO: 25, 1, 9, or 17; HCDR2 with amino acid sequences as shown in SEQ ID NO: 45, 2, 10, 18, 26, 33, 43, or 44; HCDR3 with amino acid sequences as shown in SEQ ID NO: 27, 3, 11, or 19; LCDR1 with amino acid sequences as shown in SEQ ID NO: 51, 5, 13, 21, 29, 37, or 38; LCDR2 with amino acid sequences as shown in SEQ ID NO: 53, 6, 14, 22, 30, 39, or 52; and LCDR3 with amino acid sequences as shown in SEQ ID NO: 31, 7, 15, or 23. The antibody-drug conjugates of this invention have a good inhibitory effect on tumor cell proliferation.

Description

Anti-FGFR2b antibodies and antibody-drug conjugates Technical Field

[0001] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to FGFR2b and an antibody-drug conjugate thereof. Background Art

[0002] Gastric cancer is the second most common malignant tumor in China. Its incidence, including gastroesophageal junction cancer, ranks fifth in the world, and its mortality rate ranks third in the world. Advanced gastric cancer is treated with sequential chemotherapy, with a platinum-fluoropyrimidine doublet as the first line, but the median survival of patients is less than 1 year. Approved targeted therapies for the treatment of gastric cancer include trastuzumab (for HER2-positive patients, first line), ramucirumab (anti-angiogenic drug, second line), and nivolumab or pembrolizumab (anti-PD-1 antibody, third line). In addition, gastric cancer is highly heterogeneous, and precise treatment strategies need to be formulated according to different gastric cancer classifications.

[0003] Fibroblast growth factors (FGFs) and their receptors (FGFRs) play important regulatory roles in embryonic development, tissue homeostasis, and metabolism. FGF family members bind to only four known tyrosine kinase receptors, fibroblast growth factor receptors 1-4 (FGFR1-4) and their isoforms, with individual FGFs binding to different FGFRs to varying degrees.

[0004] FGFR2, a member of the FGFR family, undergoes alternative splicing at the mRNA level to generate two typical splice isoforms, designated FGFR2-IIIb (FGFR2b) and FGFR2-IIIc (FGFR2c). The immunoglobulin-like domains and intracellular tyrosine kinase domains of FGFR2b and FGFR2c are nearly identical, differing primarily in the latter half of the third immunoglobulin-like domain. Furthermore, the two have distinct expression profiles and ligand-binding specificities. FGFR2b is primarily expressed on the surface of epithelial cells and specifically interacts with FGF-7, FGF-10, and FGF-22, while FGFR2c is primarily expressed in mesenchymal cells and can bind to a variety of FGF ligands, including FGF1, FGF2, FGF4, FGF6, FGF9, FGF17, and FGF18. FGFR2 is hypothesized to play a crucial role in epithelial-mesenchymal transition.

[0005] However, potential FGFR2 overexpression, missense mutation activation, or abnormal protein fusion have been reported in a variety of cancer types, including endometrial cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, bladder cancer, and bile duct cancer. For example, FGFR2b is gene amplified or overexpressed in gastric cancer cells. FGFR2b overexpression accounts for approximately 30% of current gastric cancer / gastroesophageal junction cancer patients, who usually have a poor prognosis and very limited treatment options after progression on first-line chemotherapy. There is a huge unmet clinical need worldwide. Furthermore, there is still a significant unmet medical need for improved anticancer drugs that are effective in cancers expressing FGFR2b.

[0006] Antibody-drug conjugates (ADCs) are targeted therapeutics that combine a cytotoxic small molecule with a monoclonal antibody via a chemical linker. Leveraging the antibody's targeting properties, they deliver therapeutic drugs to antigen-positive cancer cells. FGFR2b is also a potential target for ADCs, but currently, no FGFR2b ADCs are available in this field.

[0007] In summary, FGFR2b may be a potential target for tumor treatment. This invention aims to develop novel monoclonal antibody drugs or antibody-drug conjugates targeting FGFR2b, blocking the FGF / FGFR2b signaling pathway for the treatment of cancer patients with FGFR2b overexpression (such as advanced gastric cancer / gastroesophageal junction cancer), effectively improving the quality of life of cancer patients and prolonging their survival. Summary of the Invention

[0008] The purpose of the present application is to provide an antibody (i.e., an anti-FGFR2b antibody) or an antigen-binding fragment thereof that can specifically bind to FGFR2b, a polynucleotide molecule encoding the antibody or antigen-binding fragment thereof of the present application, an expression vector and a host cell for expressing the antibody or antigen-binding fragment thereof of the present application, and uses of the antibody or antigen-binding fragment thereof of the present application.

[0009] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, comprising a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, 9, 17 or 25; a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, 10, 18, 26, 33, 43, 44 or 45; a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3, 11, 19 or 27; a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 5, 13, 21, 29, 37, 38 or 51; a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 6, 14, 22, 30, 39, 52 or 53; and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 7, 15, 23 or 31.

[0010] In one aspect, the present invention provides an anti-FGFR2b antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region:

[0011] In another aspect, the present invention provides an isolated anti-FGFR2b antibody or antigen-binding fragment thereof having one or more of the following properties:

[0012] (I) the epitope of human FGFR2b protein bound by it is identical to, completely overlaps with, or partially overlaps with that of the antibody or antigen-binding fragment thereof described herein;

[0013] (II) competing with the antibodies or antigen-binding fragments thereof described herein for binding to an epitope of human FGFR2b protein.

[0014] In some embodiments, the antibodies described herein are monoclonal antibodies.

[0015] In yet another aspect, the present invention provides a polynucleotide molecule encoding an anti-FGFR2b antibody or antigen-binding fragment thereof described herein.

[0016] In yet another aspect, the present invention provides an expression vector comprising the polynucleotide molecule described herein, preferably, the vector is a eukaryotic expression vector.

[0017] In another aspect, the present invention provides a host cell comprising the polynucleotide molecule described herein or the expression vector described herein. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0018] In another aspect, the present invention provides a method for preparing the anti-FGFR2b antibody or antigen-binding fragment thereof described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0019] The present invention also provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any embodiment of the present invention and a coupling portion.

[0020] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, polynucleotide molecule, expression vector, host cell or immunoconjugate described in any embodiment herein, and a pharmaceutically acceptable carrier and / or excipient.

[0021] In another aspect, the present invention provides use of the antibodies or antigen-binding fragments thereof, polynucleotide molecules, expression vectors, host cells, immunoconjugates or pharmaceutical compositions described herein in the preparation of a medicament for treating and / or preventing a disease or condition mediated by FGFR2b, preferably the disease or condition is cancer; more preferably, the cancer is selected from endometrial cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, bladder cancer and bile duct cancer.

[0022] The present invention also provides an article of manufacture comprising a first container containing a composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, immunoconjugate, or pharmaceutical composition described in any embodiment herein.

[0023] In yet another aspect, the present invention provides a method for detecting the presence of FGFR2b in a sample using the antibody or antigen-binding fragment thereof described herein, or a detection composition containing the antibody or antigen-binding fragment thereof.

[0024] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1: ELISA results of anti-FGFR2b chimeric antibody binding to FGFR1c protein (upper panel) and FGFR3c protein (lower panel), respectively.

[0026] Figure 2: ELISA results of anti-FGFR2b chimeric antibody binding to Cyno FGFR2b protein.

[0027] Figure 3: ELISA results of binding of anti-FGFR2b chimeric antibody to mouse FGFR2b protein.

[0028] Figure 4: Results of anti-FGFR2b chimeric antibody blocking activity experiment (FGF7 / FGFR2b).

[0029] Figure 5: Results of anti-FGFR2b chimeric antibody blocking activity experiment (FGF10 / FGFR2b).

[0030] Figure 6: Anti-FGFR2b chimeric antibody ADCC activity experimental results.

[0031] Figure 7: Experimental results of cell proliferation inhibition activity of anti-FGFR2b chimeric antibody.

[0032] Figure 8: Experimental results of ERK phosphorylation inhibition activity of anti-FGFR2b chimeric antibody.

[0033] Figure 9: Anti-FGFR2b humanized antibody cell binding activity assay results. Top: hu-032; Bottom: hu-042.

[0034] Figure 10: Anti-FGFR2b humanized antibody ADCC activity assay results. Top panel: hu-032; bottom panel: hu-042.

[0035] Figure 11: Anti-FGFR2b humanized antibody cell proliferation inhibition assay results. Top: hu-032; Bottom: hu-042.

[0036] Figure 12: Experimental results of ERK phosphorylation inhibitory activity of anti-FGFR2b humanized antibody.

[0037] Figure 13: Anti-FGFR2b ADC endocytosis activity experimental results.

[0038] Figure 14: In vitro cytotoxicity test results of anti-FGFR2b ADC.

[0039] Figure 15: Results of cell killing assays with different linkers-toxins.

[0040] Figure 16: hu-042-016 ADC cell killing assay results.

[0041] Figure 17: Anti-FGFR2b ADC bystander activity results.

[0042] Figure 18: In vivo efficacy test results of anti-FGFR2b ADCs with different linkers.

[0043] Figure 19: In vivo efficacy test results of anti-FGFR2b ADC (DAR4).

[0044] Figure 20: In vivo pharmacodynamic study results of anti-FGFR2b antibody-drug conjugate on human gastric cancer subcutaneous xenografts.

[0045] Figure 21: In vivo pharmacodynamic study results of anti-FGFR2b antibody-drug conjugate on human breast cancer subcutaneous xenografts.

[0046] Figure 22: In vivo pharmacodynamic study results of anti-FGFR2b antibody-drug conjugate on human lung cancer subcutaneous xenografts. DETAILED DESCRIPTION

[0047] The present invention provides antibodies or functional fragments thereof that specifically bind to FGFR2b, antibody-drug conjugates (ADCs) or pharmaceutical compositions thereof, uses of the antibodies, functional fragments, pharmaceutical compositions, and ADCs in the treatment and / or prevention of FGFR2b-mediated diseases, and methods of using the antibodies, functional fragments, pharmaceutical compositions, and ADCs to treat or prevent FGFR2b-mediated diseases.

[0048] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new technical solution.

[0049] definition

[0050] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0051] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined as follows. Unless otherwise clearly defined elsewhere in this article, the scientific and technological terms used herein all have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form unless otherwise clearly provided in the text.

[0052] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0053] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0054] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when explicitly stated to the contrary, such as "only one" or "exactly one" or when used in a claim, "consisting of..." will refer to only one of the listed numbers or one of the elements of the list.

[0055] Unless expressly stated otherwise, as used herein, the words "a" and "an" should be understood to mean "at least one."

[0056] "FGFR2IIIb" or "FGFR2b" are used interchangeably to refer to the splice form of fibroblast growth factor receptor 2IIIb. Exemplary FGFR2b sequences include Homo sapiens (human) FGFR2b protein (e.g., precursor sequence with signal peptide, Genbank accession number: NP_075259.4); Rattus norvegicus (rat) FGFR2b protein (e.g., full sequence, Genbank accession number: NP_001103363.1); and Mus musculus (mouse) FGFR2b protein (e.g., full sequence, Genbank accession number: NP_963895.2).

[0057] "FGFR2IIIc" or "FGFR2c" are used interchangeably to refer to the splice form of fibroblast growth factor receptor 2IIIc. Exemplary FGFR2c sequences include human FGFR2c protein (e.g., precursor sequence, Genbank accession number: NP_000132.3); Rattus norvegicus (rat) FGFR2c protein (full sequence, Genbank accession number: NP_001103362.1); and Mus musculus (mouse) FGFR2c protein (full sequence, Genbank accession number: NP_034337.2).

[0058] The term "percent (%) amino acid sequence identity," or simply "identity," is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences.

[0059] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which result in the selective damage, destruction, or elimination from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0060] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-and-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0061] The terms "activity" or "biological activity", or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize FGFR2b activity in vivo or in vitro, IC 50 , the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies well known in the art include, for example, cross-reactivity (i.e., usually with non-human homologs of the target peptide, or with cross-reactivity of other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured or assessed using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other source).

[0062] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0063] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0064] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0065] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises at least four peptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). 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 subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0066] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When the binding activity to the antigen is expressed on a molar basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0067] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0068] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or a light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0069] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. However, a bivalent antibody can be bispecific.

[0070] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0071] The term "murine antibody" or "hybridoma antibody" as used herein refers to a monoclonal antibody against human FGFR2b prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with an FGFR2b antigen, and then a hybridoma expressing an antibody having the desired sequence or functional properties is isolated.

[0072] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0073] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0074] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0075] An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, for example to enhance or diminish effector function or binding to an Fc receptor.

[0076] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0077] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0078] The term "cross-reactivity" as used herein refers to binding to antigenic fragments of the same target molecule of human, monkey, and / or murine origin (mouse or rat). Thus, "cross-reactivity" should be understood as interspecies reactivity with the same molecule X expressed in different species. The cross-reactivity specificity of monoclonal antibodies recognizing human FGFR2b, monkey, and / or murine FGFR2b (mouse or rat) can be determined by FACS analysis.

[0079] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents the equilibrium dissociation constant, which is the dissociation rate constant and the association rate constant (k dis and k on Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0080] The term "not binding" to a protein or cell means that the protein or cell does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the K of the protein or cell is less than 1%. D 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0081] The term "high affinity" for IgG antibodies refers to the K D 1.0×10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype refers to a K D is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0082] The ability to "compete for binding" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human FGFR2b and anti-FGFR2b antibody) to any detectable extent (e.g., inhibition of at least 85%, or at least 90%, or at least 95%).

[0083] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively bind cell membrane surface antigens to antibodies.

[0084] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies, which, when bound to surface antigens, trigger the classic complement pathway, including formation of the membrane attack complex and target cell lysis.

[0085] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," and "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited otherwise, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0086] "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner. Recombinant constructs will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.

[0087] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0088] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

[0089] The term "immunoconjugate" refers to an antibody that is connected to a portion having a desired activity through a linker or directly. The connection can be a covalent connection or a non-covalent connection. The desired activity can be, for example, cell killing activity or detection (such as luminescence) activity. The portion having the desired activity can be an anti-tumor agent, an immunomodulator, a cytotoxic drug, a fluorescent substance, a luminescent substance, an enzyme, a small nucleic acid molecule, and any combination thereof. Suitable small nucleic acids may include siRNA or antisense oligonucleotides (ASOs), etc. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) as described in any embodiment herein, or an antibody or antigen-binding fragment thereof labeled with a radioactive atom or spin label as described in any embodiment herein.

[0090] The terms "antibody drug conjugate (ADC)", "antibody drug conjugate (ADC)" and "ADC" are used interchangeably and generally refer to one or more therapeutic compounds (e.g., Dxd) linked to one or more antibodies or antigen-binding fragments and are defined by the following general formula: Ab-(LD)(s), where Ab = antibody or antigen-binding fragment, L = linker, D = Drug unit, and s = the number of drug moieties per antibody or antigen-binding fragment, which can be any number from 1 to 10. In some embodiments, the linker L can include a cleavable moiety between the antibody or antigen-binding fragment and the Drug unit.

[0091] The term "drug" as used herein generally refers to any compound having a desired biological activity and a reactive functional group for preparing the conjugates of the present invention. The desired biological activity includes the diagnosis, cure, alleviation, treatment, or prevention of human or other animal diseases. As new drugs are continuously discovered and developed, these new drugs should also be included in the drugs of the present invention. Specifically, the drugs include, but are not limited to, cytotoxic drugs, cell differentiation factors, stem cell trophic factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs, or drugs for infectious diseases. More specifically, the drugs include, but are not limited to, microtubule inhibitors or DNA or RNA damaging agents.

[0092] The term "cytotoxic drug" or "cytotoxin" refers to substances that inhibit cell function and / or cause cell death or destruction. Cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations. However, due to their lack of selectivity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, radioactive isotopes, chemotherapeutic agents, antibiotics, and nucleolytic enzymes.

[0093] "Tubulin inhibitors" refer to a class of compounds that exert anti-tumor effects by inhibiting tubulin polymerization or promoting tubulin assembly, thereby interfering with cell mitosis. Non-limiting examples include: maytansines, calicheamicins, taxanes, vincristine, colchicine, dolastatin, auristatin, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF).

[0094] In some embodiments, the microtubule inhibitors include but are not limited to dolastatin, auristatin, and maytansine; the DNA damaging agents include but are not limited to calicheamicins, duocarmycins, anthramycin derivatives PBD (pyrrolobenzodiazepine), camptothecin derivatives SN-38, and topoisomerase I inhibitors.

[0095] In some embodiments, the auristatin drugs include but are not limited to monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF) or their derivatives, the maytansine drugs include but are not limited to DM1, DM3, DM4 or their derivatives ("Progress in Research on Warhead Molecules of Antibody Drug Conjugates", Hu Xinyue et al., "China Pharmaceutical Biotechnology", December 2017, Vol. 12, No. 6; "Progress in Research on Maytansine Antibody Drug Conjugates", Zhou Lei et al., "China Journal of New Drugs", Vol. 25, No. 22, 2016, pp. 2521-2530), and the topoisomerase I inhibitors include but are not limited to exitecan, topotecan, irinotecan, and 9-nitrocamptothecin.

[0096] In the present invention, the term "linker," "linker," "linker," or "L" generally refers to any chemical moiety capable of covalently joining a compound (typically a drug unit) to another moiety (such as an antibody or antigen-binding fragment). The linker may be susceptible to or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage under conditions that allow the compound or antibody to remain active.

[0097] According to the properties, the linkers suitable for the present invention can be linkers that can be cleaved by tissue proteases, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; MC-GGFG linkers, TRX linkers, galactoside-containing linkers, etc.

[0098] In a specific embodiment, the linker includes but is not limited to maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit, vc), alanine-phenylalanine (ala-phe), p-Aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

[0099] The term "load" or "drug load" or "payable load" refers to the average number of therapeutically active substances or active pharmaceutical ingredients per antibody within the ADC molecule ("payable load" is used interchangeably herein with "therapeutically active substance or active pharmaceutical ingredient"). Drug load can range from 1-20 therapeutically active substances or active pharmaceutical ingredients per antibody.

[0100] The term "drug / antibody ratio" or "DAR" refers to the ratio of the therapeutically active substance or active pharmaceutical ingredient (D) conjugated to the antibody to the antibody. The ADCs described herein typically have a DAR of 1-10, and in certain embodiments have a DAR of 1-8, 2-8, 2-6, 2-5, 3-8, 4-6, and 2-4. Representative DAR values ​​are 2, 3, 4, 5, 6, 7, 8, 9, and 10, typically expressed as a combination of the letter D and a number, wherein the number represents the numerical value of the DAR, for example, D2 represents a drug / antibody ratio with a DAR value of 2. In some embodiments, the DAR is an average DAR, i.e., the average DAR as determined by a detection method (e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC). Quantitative DAR values ​​can also be determined. The DAR may be limited by the number of attachment sites on the antibody. For example, where the attachment site is a cysteine ​​thiol, the antibody may have only one or a few cysteine ​​thiol groups or may have only one or a few sufficiently reactive thiol groups through which a linker unit may be attached.

[0101] "Activation," "stimulation," and "treatment" as applied to a cell or receptor may have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context dictates otherwise or explicitly. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response," e.g., a response of a cell, tissue, organ, or organism, includes a change in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, rate of gene expression, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0102] As used herein, the terms "treatment" or "treating" of any disease or condition refer, in one embodiment, to ameliorating the disease or condition (i.e., slowing or preventing or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, "treatment" or "treating" refers to regulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless expressly described herein, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0103] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey.

[0104] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in either order.

[0105] "Therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of the FGFR2b antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, e.g., an amount to treat, cure, prevent, or ameliorate a related medical condition or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0106] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0107] The term "cancer" is used herein to refer to a group of cells that exhibit an abnormally high level of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. The term "tumor" as used herein refers to one or more cells comprising a cancer. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.

[0108] Anti-FGFR2b antibodies

[0109] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to FGFR2b. The terms "anti-FGFR2b antibody," "anti-FGFR2b," "FGFR2b antibody," or "antibody that binds to FGFR2b" refer to antibodies that are capable of binding to FGFR2b protein or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting FGFR2b.

[0110] In some embodiments, the present invention provides antibodies that bind to FGFR2b protein. In some embodiments, the present invention provides antibodies that block the FGF / FGFR2b signaling pathway.

[0111] In some embodiments, the antibodies of the present invention bind to human or cynomolgus monkey FGFR2b protein. In some embodiments, the antibodies of the present invention bind to human FGFR2b and block the interaction between human FGFR2b and FGF protein.

[0112] In some embodiments, the amino acid sequence of HCDR1 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence shown in SEQ ID NO: 1, 9, 17 or 25, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 1, 9, 17 or 25. In some embodiments, the amino acid sequence of HCDR1 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence shown in SEQ ID NO: 17 or 25, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 17 or 25.

[0113] In some embodiments, the amino acid sequence of HCDR2 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence of SEQ ID NO: 2, 10, 18, 26, 33, 43, 44 or 45, or has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 2, 10, 18, 26, 33, 43, 44 or 45. In some embodiments, the amino acid sequence of HCDR2 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence of SEQ ID NO: 18, 26, 33, 43, 44 or 45, or has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 18, 26, 33, 43, 44 or 45.

[0114] In some embodiments, the amino acid sequence of HCDR3 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence shown in SEQ ID NO: 3, 11, 19 or 27, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 3, 11, 19 or 27. In some embodiments, the amino acid sequence of HCDR3 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence shown in SEQ ID NO: 19 or 27, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 19 or 27.

[0115] In some embodiments, the amino acid sequence of LCDR1 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence of SEQ ID NO: 5, 13, 21, 29, 37, 38 or 51, or has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 5, 13, 21, 29, 37, 38 or 51. In some embodiments, the amino acid sequence of LCDR1 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence of SEQ ID NO: 21, 29, 37, 38 or 51, or has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 21, 29, 37, 38 or 51.

[0116] In some embodiments, the amino acid sequence of LCDR2 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence of SEQ ID NO: 6, 14, 22, 30, 39, 52, or 53, or has 1, 2, or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 6, 14, 22, 30, 39, 52, or 53. In some embodiments, the amino acid sequence of LCDR2 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence of SEQ ID NO: 39, 52, or 53, or has 1, 2, or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 39, 52, or 53.

[0117] In some embodiments, the amino acid sequence of LCDR3 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence shown in SEQ ID NO: 7, 15, 23 or 31, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 7, 15, 23 or 31. In some embodiments, the amino acid sequence of LCDR3 comprised by the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention is selected from the amino acid sequence shown in SEQ ID NO: 23 or 31, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 23 or 31.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention comprises: a HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 9, 17, or 25, or having 1, 2, or 3 amino acid differences therefrom; a HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 33, 43, 44, or 45, or having 1, 2, or 3 amino acid differences therefrom; a HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 11, 19, or 27, or having 1, 2, or 3 amino acid differences therefrom; and a HCDR4 having an amino acid sequence selected from the group consisting of SEQ ID NO: 4. NO: 5, 13, 21, 29, 37, 38 or 51, or a LCDR1 whose amino acid sequence has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 5, 13, 21, 29, 37, 38 or 51; a LCDR2 whose amino acid sequence is selected from the group consisting of SEQ ID NO: 6, 14, 22, 30, 39, 52 or 53, or a LCDR2 whose amino acid sequence has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 6, 14, 22, 30, 39, 52 or 53; and a LCDR3 whose amino acid sequence is selected from the group consisting of SEQ ID NO: 7, 15, 23 or 31, or a LCDR3 whose amino acid sequence has 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 7, 15, 23 or 31.

[0119] In some embodiments, in the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b of the present invention, the amino acid sequence of HCDR1 is selected from the amino acid sequence of SEQ ID NO: 17 or 25, or has 1, 2, or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 17 or 25; the amino acid sequence of HCDR2 is selected from the amino acid sequence of SEQ ID NO: 18, 26, 33, 43, 44, or 45, or has 1, 2, or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 18, 26, 33, 43, 44, or 45; the amino acid sequence of HCDR3 is selected from the amino acid sequence of SEQ ID NO: 19 or 27, or has 1, 2, or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 19 or 27; the amino acid sequence of LCDR1 is selected from the amino acid sequence of SEQ ID NO: 21, 29, 37, 38, or 51, or has 1, 2, or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 21, 29, 37, 38, or 51; the amino acid sequence of LCDR2 is selected from the amino acid sequence of SEQ ID NO: The amino acid sequence of LCDR3 is selected from the amino acid sequence shown in SEQ ID NO: 23 or 31, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 23 or 31.

[0120] In some embodiments, the heavy chain variable region of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises:

[0121] HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; or

[0122] HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; or

[0123] HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or

[0124] HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; or

[0125] The HCDR1, HCDR2, and HCDR3 amino acid sequences are as shown in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19; or

[0126] HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 43, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 43, and SEQ ID NO: 27; or

[0127] HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27; or

[0128] The HCDR1, HCDR2 and HCDR3 amino acid sequences are as shown in SEQ ID NO: 25, SEQ ID NO: 45 and SEQ ID NO: 27, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 45 and SEQ ID NO: 27.

[0129] In some embodiments, the light chain variable region of the anti-FGFR2b antibody or antigen-binding fragment thereof of the present invention comprises:

[0130] LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; or LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; or

[0131] LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; or

[0132] LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; or

[0133] LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31; or

[0134] LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23; or

[0135] LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23; or

[0136] LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23; or

[0137] LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31; or

[0138] LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 31, respectively; or LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 31.

[0139] In some embodiments, the antibodies described herein comprise:

[0140] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; or

[0141] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or

[0142] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; or

[0143] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or

[0144] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or

[0145] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or

[0146] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or

[0147] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or

[0148] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or

[0149] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or

[0150] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 43, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or

[0151] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or

[0152] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 31, respectively; or

[0153] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 45, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or

[0154] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 45, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 31, respectively; or

[0155] a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 43 and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 31, respectively.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region:

[0157] the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; or

[0158] the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 16; or

[0159] the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:20, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:20; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:24, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:24; or

[0160] The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:28, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as shown in SEQ ID NO:28; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:32, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as shown in SEQ ID NO:32.

[0161] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0162] a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 34, 35, 36, 46, 47, 48, 49, or 50; and

[0163] A light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 40, 41, 42, 54, 55, 56, 57, 58, 58, 60, 61 or 62.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0165] a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 34, 35, 36, 46, 47, 48, 49 or 50; and

[0166] A light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 40, 41, 42, 54, 55, 56, 57, 58, 58, 60, 61 or 62.

[0167] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0168] The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 34 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or

[0169] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 35 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or

[0170] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 36 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or

[0171] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 46, 47 or 48 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 54; or

[0172] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 46, 47 or 48 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 55; or

[0173] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 48 or 49 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 56; or

[0174] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 48 or 49 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 57; or

[0175] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 48 or 49 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 58; or

[0176] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 49 or 50 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 59; or

[0177] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 49 or 50 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 60; or

[0178] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 49 or 50 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 61; or

[0179] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 49 or 50, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 62.

[0180] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0181] A heavy chain comprising the amino acid sequence shown in any one of SEQ ID NO: 63, and

[0182] A light chain comprising the amino acid sequence shown in any one of SEQ ID NO: 64, 65 or 66.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0184] a heavy chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NO: 63, and

[0185] A light chain comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 64, 65 or 66.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0187] The heavy chain of the amino acid sequence shown in SEQ ID NO: 63 and the light chain of the amino acid sequence shown in SEQ ID NO: 64, or

[0188] The heavy chain of the amino acid sequence shown in SEQ ID NO: 63 and the light chain of the amino acid sequence shown in SEQ ID NO: 65, or

[0189] The heavy chain has an amino acid sequence as shown in SEQ ID NO: 63, and the light chain has an amino acid sequence as shown in SEQ ID NO: 66.

[0190] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0191] Any suitable method for producing antibodies can be used to produce antibodies of the present invention. Any suitable form of FGFR2b can be used as an immunogen (antigen) to produce antibodies. By way of example and not limitation, any FGFR2b variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing monoclonal anti-human FGFR2b antibodies of mouse origin can be produced by methods well known in the art.

[0192] Antibodies derived from rodents (e.g., mice) may induce unwanted antibody immunogenicity when used as therapeutic agents in vivo. Repeated use can lead to immune responses against the therapeutic antibodies in the human body. Such immune responses can lead to loss of therapeutic efficacy at the very least and, in severe cases, potentially lethal allergic reactions. One approach to reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies, in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the retention of intact rodent variable regions in chimeric antibodies may still induce unwanted immunogenicity in patients. Grafting the complementarity determining region (CDR) loops of rodent variable domains onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences (Jones et al. (1986) n nature 321:522; Verhoeyen et al. (1988) Science 239:1534).

[0193] In some embodiments, chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal hybridoma antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0194] In some embodiments, the chimeric FGFR2b antibodies of the present invention can be prepared by operatively linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the constant region comprised by the chimeric antibodies of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0195] In some embodiments, the chimeric FGFR2b antibodies of the present invention can be obtained by "mixing and matching" chimeric light chain and chimeric heavy chain expression plasmids to transfect expression cells, and the FGFR2b binding of such "mixed and matched" antibodies can be tested using the above-mentioned binding assays and other conventional binding assays (e.g., ELISA).

[0196] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of antibodies and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0197] Unless otherwise indicated, the boundaries of the CDRs of the antibodies of the invention can be determined by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).

[0198] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0199] Antibodies with different specificities (that is, different binding sites for different antigens) have different CDRs. However, although CDR is different between antibodies, there are only a limited number of amino acid positions in the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the CDR sequence remainder can be determined. Therefore, the present invention also contemplates the variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0200] >Chi-006-VH

[0201] HCDR1: SYNIH (SEQ ID NO: 1)

[0202] HCDR2: YIYPDNGDTNYNQKFKG (SEQ ID NO: 2)

[0203] HCDR3: DWTY (SEQ ID NO: 3)

[0204] >Chi-006-VL

[0205] LCDR1: SATSIVNHMY (SEQ ID NO: 5)

[0206] LCDR2: FTSNLAS (SEQ ID NO: 6)

[0207] LCDR3: QQWSSTPFT (SEQ ID NO: 7)

[0208] >Chi-018-VH

[0209] HCDR1: DYYIH (SEQ ID NO: 9)

[0210] HCDR2:WIYHENGDTEFAPKFQG (SEQ ID NO: 10)

[0211] HCDR3: KITY (SEQ ID NO: 11)

[0212] >Chi-018-VL

[0213] LCDR1:KASQNVGTNVA(SEQ ID NO:13)

[0214] LCDR2:SASYRFS(SEQ ID NO:14)

[0215] LCDR3:QQYYSSPYT(SEQ ID NO:15)

[0216] >Chi-032-VH

[0217] HCDR1:SYNMH(SEQ ID NO:17)

[0218] HCDR2:YIYPENGDTNYNQRFKG(SEQ ID NO:18)

[0219] HCDR3:ADY(SEQ ID NO:19)

[0220] >Chi-032-VL

[0221] LCDR1:SASSSVNHMY(SEQ ID NO:21)

[0222] LCDR2:LTSNLAS(SEQ ID NO:22)

[0223] LCDR3:QQWSSNPFT(SEQ ID NO:23)

[0224] >Chi-042-VH

[0225] HCDR1:DYNMH(SEQ ID NO:25)

[0226] HCDR2:YIYPNNGDTSYNQKFKG(SEQ ID NO:26)

[0227] HCDR3:GLLD(SEQ ID NO:27)

[0228] >Chi-042-VL

[0229] LCDR1:GASENIYGALN(SEQ ID NO:29)

[0230] LCDR2:GATNLAD(SEQ ID NO:30)

[0231] LCDR3: QNVLSTPYT (SEQ ID NO: 31)

[0232] The humanized antibodies of the present invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, such as those described in U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0233] The amino acid sequences of the light chain and heavy chain variable regions and CDRs of the anti-human FGFR2b humanized antibody of the present invention are as follows:

[0234] The HCDR sequence of hu-032-HC is as follows:

[0235] HCDR1: SYNMH (SEQ ID NO: 17)

[0236] HCDR2: YIYPENGDTNYNQKFQG (SEQ ID NO: 18) or YIYPENGDTNYAQKFQG (SEQ ID NO: 33)

[0237] HCDR3: ADY (SEQ ID NO: 19)

[0238] hu-032-HC-1

[0239] hu-032-HC-2

[0240] hu-032-HC-3

[0241] The LCDR sequence of hu-032-LC is as follows:

[0242] LCDR1: RASSSVNHMY (SEQ ID NO: 21) or RASSSVNHLA (SEQ ID NO: 37) or RASSSVNHMA (SEQ ID NO: 38)

[0243] LCDR2:LTSNRAT(SEQ ID NO:39)

[0244] LCDR3: QQWSSNPFT (SEQ ID NO: 23)

[0245] hu-032-LC-1

[0246] hu-032-LC-2

[0247] hu-032-LC-3

[0248] The HCDR sequence of hu-042-HC is as follows:

[0249] HCDR1: DYNMH (SEQ ID NO: 25)

[0250] HCDR2: IIYPNNGDTSYAQKFQG (SEQ ID NO: 43) or YIYPNNGDTSYNQKFQG (SEQ ID NO: 44) or YIYPNNGDTSYAQKFQG (SEQ ID NO: 45)

[0251] HCDR3: GLLD (SEQ ID NO: 27)

[0252] hu-042-HC-1

[0253] hu-042-HC-2

[0254] hu-042-HC-3

[0255] hu-042-HC-4

[0256] hu-042-HC-5

[0257] The LCDR sequence of hu-042-LC is as follows:

[0258] LCDR1: RASENIYGALN (SEQ ID NO: 51)

[0259] LCDR2: GATSLQS (SEQ ID NO: 52) or GATSLAS (SEQ ID NO: 53)

[0260] LCDR3: QNVLSTPYT (SEQ ID NO: 31)

[0261] hu-042-LC-1

[0262] hu-042-LC-2

[0263] hu-042-LC-3

[0264] hu-042-LC-4

[0265] hu-042-LC-5

[0266] hu-042-LC-6

[0267] hu-042-LC-7

[0268] hu-042-LC-8

[0269] hu-042-LC-9

[0270] The numbers of the humanized anti-FGFR2b antibodies of the present invention and the sources of their heavy chain and light chain variable regions are shown in Tables 1 and 2.

[0271] Table 1: Numbering of humanized anti-FGFR2b antibody 032 and sources of its heavy and light chain variable regions

[0272] Table 2: Numbering of humanized anti-FGFR2b antibody 042 and sources of its heavy and light chain variable regions

[0273] The light and heavy chain amino acid sequences of antibodies hu-042-012, hu-042-015, and hu-042-016 are shown below.

[0274] >hu-042-012-HC, hu-042-015-HC, hu-042-016-HC

[0275] >hu-042-012-LC

[0276] >hu-042-015-LC

[0277] >hu-042-016-LC

[0278] In some embodiments, the amino acid changes include amino acid deletions, insertions, or substitutions. In some embodiments, the anti-FGFR2b antibodies or antigen-binding fragments thereof of the present invention include those having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above-described antibodies (particularly in the CDR regions depicted in the above sequences). In some embodiments, the antibodies of the present invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions when compared to the CDR regions depicted in the specific sequences. In some embodiments, the antibodies of the present invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the framework regions when compared to the framework regions in the specific sequences.

[0279] In some embodiments, the polynucleotide molecules encoding the antibodies of the present invention include polynucleotide molecules that have been mutated by nucleotide deletion, insertion or substitution, but still have at least about 60, 70, 80, 90, 95 or 100% identity with the CDR corresponding coding regions depicted in the sequences described above.

[0280] In some embodiments, the FGFR2b antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody, or a modified form thereof, as described in the following sections.

[0281] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0282] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0283] In some embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0284] Antibody expression

[0285] In another aspect, the present invention provides a polynucleotide molecule encoding an anti-FGFR2b antibody or antigen-binding fragment thereof as described herein. The polynucleotide molecule may comprise a polynucleotide molecule encoding an amino acid sequence of the light chain variable region and / or the heavy chain variable region of the antibody, or a polynucleotide molecule encoding an amino acid sequence of the light chain and / or the heavy chain of the antibody.

[0286] In another aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the vector is a eukaryotic expression vector. In some embodiments, the polynucleotide molecule as described herein is contained in one or more expression vectors.

[0287] In another aspect, the present invention provides a host cell comprising the polynucleotide molecule as described herein or the expression vector as described herein. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0288] In another aspect, the present invention provides a method for preparing an anti-FGFR2b antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell as described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0289] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These especially include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.

[0290] In one embodiment, the present invention provides a method for preparing an anti-FGFR2b antibody, wherein the method comprises introducing an expression vector into a mammalian host cell and then culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown, to produce the antibody. The antibody can be recovered from the culture medium using standard protein purification methods.

[0291] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies. Likewise, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are less likely to be immunogenic because their carbohydrate structures are normal components of natural human serum IgG.

[0292] Antibody-drug conjugates (ADCs)

[0293] Also provided herein is an antibody-drug conjugate (ADC), comprising the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b according to any embodiment of the present invention and a coupling moiety.

[0294] In some embodiments, the conjugated moiety comprises an anti-tumor agent, an immunomodulator, a cytotoxic agent, a fluorescent substance, a luminescent substance, an enzyme, a small nucleic acid molecule (including siRNA or antisense oligonucleotide (ASO)), and any combination thereof.

[0295] In some embodiments, the antibody-drug conjugate comprises multiple coupling moieties, and the multiple coupling moieties can be a combination of different therapeutically active substances or pharmaceutically active ingredients, or a combination of the same therapeutically active substance or pharmaceutically active ingredient.

[0296] In some embodiments, the conjugation moiety is covalently linked to the anti-FGFR2b antibody or antigen-binding fragment thereof in a non-site-specific manner or in a site-specific manner via a linker.

[0297] Based on the obtained antibodies that specifically bind to human FGFR2b, the inventors prepared a series of antibody-drug conjugates (ADCs) using different linkers and cytotoxic drug components, and verified their activity. They found that the ADCs of the present invention have a good inhibitory effect on the proliferation of cells overexpressing human FGFR2b and have a good effect on inhibiting tumor growth in transplanted tumor models.

[0298] In some embodiments, the present invention provides an antibody drug conjugate having a structure of Ab-(LD)m, wherein Ab is an antibody or antigen-binding fragment thereof that specifically binds to FGFR2b according to any embodiment of the present invention; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient, and m represents the average number of LD units coupled to Ab, and m ranges from 1 to 8, preferably m is 4, and more preferably is about 3.0, 3.5, 3.6, 3.7, 3.8, 4.0, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8 or 8.0.

[0299] In some embodiments, the amino acid sequence of LCDR1 of the antibody or antigen-binding fragment thereof that specifically binds to FGFR2b is selected from SEQ ID NO: 37 or 51, the amino acid sequence of LCDR2 is selected from SEQ ID NO: 39 or 53, the amino acid sequence of LCDR3 is selected from SEQ ID NO: 23 or 31, the amino acid sequence of HCDR1 is selected from SEQ ID NO: 17 or 25, the amino acid sequence of HCDR2 is selected from SEQ ID NO: 18 or 45, and the amino acid sequence of HCDR3 is selected from SEQ ID NO: 19 or 27.

[0300] In some embodiments, the heavy chain variable region of the antibody that specifically binds to FGFR2b comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 45, and SEQ ID NO: 27, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively.

[0301] In some embodiments, the antibody that specifically binds to FGFR2b is hu-042-012, hu-042-015, and hu-042-016 described herein.

[0302] In some embodiments, the antibody can be conjugated to the drug directly or through a linker.

[0303] In some embodiments, the drug moiety D is covalently linked to the anti-FGFR2b antibody or antigen-binding fragment thereof via a linker L in a non-site-specific manner or a site-specific manner.

[0304] The linker may comprise one or more linker components. In some embodiments, the structure of L is as shown in formula (A):

[0305] in:

[0306] L1 represents a group or bond formed by the reaction of a functional group capable of reacting with a thiol group and a thiol group;

[0307] L2 and Y are each independently selected from -(CH2) p -、-(OCH2CH2) p -and-(CH2CH2O) p -;

[0308] L3 is a hydrophilic group;

[0309] L4 is an amino acid group;

[0310] R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl;

[0311] X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-;

[0312] Z is selected from C and S;

[0313] n is selected from 0, 1 and 2;

[0314] p is selected from integers from 1 to 10.

[0315] In some embodiments, the functional group capable of reacting with a thiol group described in L1 is selected from maleimide, halogen, halogen-substituted functional group (such as halogen-substituted aldehyde group, halogen-substituted -S(O)2-), aldehyde group, alkenyl group, alkynyl group, alkanone group, sulfonyl group, silane group, isocyanate group and norbornene group.

[0316] In some embodiments, the functional group capable of reacting with a thiol group described in L1 is selected from:

[0317] and their derivatives;

[0318] Wherein: A is a halogen; the wavy line indicates the position where L1 and L2 are connected.

[0319] Preferably, the functional group described in L1 is:

[0320] In some embodiments, L2 is -(CH2) p -, and p is an integer of 1-4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-.

[0321] In some embodiments, X is -NH- or -NH(CH2) n C(=O)-; preferably, X is -NH- or -NHC(=O)-.

[0322] In some embodiments, Y is -(CH2) p -, p is 1, 2 or 3; preferably, Y is a methylene group.

[0323] In some embodiments, Z is C.

[0324] In some embodiments, L3 is selected from monosaccharides, disaccharides and five-membered or six-membered saturated heterocycles containing 1-2 nitrogen atoms and their derivatives, which are divalent hydrophilic groups with two monovalent radical centers produced by removing two hydrogen atoms; preferably, the monosaccharide is selected from trisaccharides, tetrasaccharides, pentoses, hexoses and heptoses; preferably, the disaccharide is selected from maltose, sucrose and lactose; preferably, the five-membered or six-membered saturated heterocycles containing 1-2 nitrogen atoms or their derivatives are selected from piperazinyl, piperidinyl and pyrrolidinyl.

[0325] In some embodiments, the antibody-drug conjugate according to the present invention is characterized in that L3 is selected from glucose, galactose, mannose, glucuronic acid, galactonic acid, mannuronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and N-acetylmuramic acid, which are divalent hydrophilic groups with two monovalent radical centers generated by removing two hydrogen atoms.

[0326] In some embodiments, the antibody-drug conjugate according to the present invention is characterized in that L3 is a structure represented by formula L3-1:

[0327] Where:

[0328] R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4;

[0329] R2, R3 and R4 are each independently selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups;

[0330] t is an integer from 1 to 20;

[0331] The wavy lines indicate where L3 connects to X and Y.

[0332] In some embodiments, L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine ​​and tyrosine; said amino acid group is optionally substituted with one or more -OR 20 Replaced by, where R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t -C1-C6 alkyl, sulfonic acid and phosphoric acid groups, wherein t is an integer from 1 to 20.

[0333] In some embodiments, L4 is selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine-glycine, glycine-glycine-phenylalanine-glycine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine and lysine-phenylalanine, preferably selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA) and glycine-glycine-phenylalanine-glycine (GGFG); each peptide segment is optionally substituted by one or more substituents selected from the following group: -OR 20 and -(OCH2CH2) p O-C1-C6 alkyl, wherein R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O) t - C1-C6 alkyl, sulfonic acid and phosphoric acid groups, t is an integer from 1 to 20, and p is an integer from 1 to 6.

[0334] In some embodiments, L4 is selected from:

[0335] The wavy line indicates the position where L4 is connected to the rest of formula (A).

[0336] In some embodiments, R6 is H, C1-C3 alkoxy, or -(OCH2CH2) p O-C1-C3 alkyl, wherein p is an integer from 1 to 6; preferably, R6 is H, methoxy or -(OCH2CH2) p OCH3, wherein p is an integer of 1-6 or 1-4.

[0337] In some embodiments, the structure of the linker L is as described in any of the following structures:

[0338] In each formula, R2, R3, R4 and R6 are as described in any embodiment herein, R" is H or C1-C6 alkyl; the wavy line indicates the position where L is attached to the rest of the ADC.

[0339] In some embodiments, the linker is selected from maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-amino Benzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

[0340] In some embodiments, the drug moiety D comprises a cytotoxin, a plant toxin, a small molecule toxin, a radioisotope, etc. In some embodiments, the drug moiety D comprises a tubulin inhibitor, a DNA damaging agent.

[0341] In some preferred embodiments, the microtubule inhibitors include dolastatin, auristatin, and maytansine; the DNA damaging agents include calicheamicins, duocarmycins, anthramycin derivatives PBD (pyrrolobenzodiazepine), and topoisomerase I inhibitors.

[0342] In some preferred embodiments, the auristatin drugs include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF) or their derivatives, the maytansine drugs include but are not limited to DM1, DM3, DM4 or their derivatives; the topoisomerase I inhibitors include but are not limited to exitecan, topotecan, irinotecan, 9-nitrocamptothecin, and the camptothecin derivative SN-38.

[0343] In some embodiments, the drug moiety D may also be vitamin A precursor, folic acid, etc. The drug moiety D is not limited to the above categories, but also includes all drugs that can be used in ADC.

[0344] In some embodiments, the linker-drug (LD) moiety is selected from the following structures: VCMMAE, Deruxtecan, CPD2, CPD4, CPD5, CPD6, or SN-38Comp5:

[0345] It should be understood that the structures shown above for MMAE, Deruxtecan, CPD2, CPD4, CPD5, CPD6, or SN-38Comp5 are coupled to Ab via the reaction of their maleimide groups with the sulfhydryl groups of the antibody.

[0346] In some embodiments, the antibody drug conjugate is selected from the structure shown in Formula ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI or ADC-VII:

[0347] wherein m represents the average number of linker-drug (LD) units coupled to the Ab, and m ranges from 1 to 8, preferably m is 4; preferably, the amino acid sequence of LCDR1 of the Ab is selected from SEQ ID NO: 37 or 51, the amino acid sequence of LCDR2 is selected from SEQ ID NO: 39 or 53, the amino acid sequence of LCDR3 is selected from SEQ ID NO: 23 or 31, the amino acid sequence of HCDR1 is selected from SEQ ID NO: 17 or 25, the amino acid sequence of HCDR2 is selected from SEQ ID NO: 18 or 45, and the amino acid sequence of HCDR3 is selected from SEQ ID NO: 19 or 27. More preferably, the heavy chain variable region of the Ab comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 25, SEQ ID NO: 45, and SEQ ID NO: 27, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively. More preferably, the Ab is hu-042-012, hu-042-015, and hu-042-016 as described herein.

[0348] It should be understood that in the antibody-drug conjugates described herein, the position of connection between the drug portion D and the remainder of the linker-drug conjugate generally does not affect the biological activity of the drug D itself. Those skilled in the art can readily determine the active site of drug D based on existing techniques and covalently link it to the remainder of the linker-drug conjugate at a position distal to the active site. Covalent linkage methods are well known in the art and include acylamino groups (-NRCO-, where R is H or C1-C4 alkyl), ester bonds (-COO-), and -S-.

[0349] In some embodiments, the ADC described herein is selected from ADC-02, ADC-03, ADC-04, ADC-05, ADC-07, ADC-08, ADC-09, ADC-10, ADC-12, ADC-14, ADC-17, ADC-18, ADC-19, ADC-20, and ADC-21 described herein.

[0350] The ADCs herein can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group of an antibody with a bivalent linker reagent via a covalent bond to form an antibody-linker via a covalent bond, followed by reaction with a drug; and (2) reacting a nucleophilic group of a drug moiety with a bivalent linker reagent via a covalent bond to form a drug-linker, followed by reaction with a nucleophilic group of an antibody.

[0351] Pharmaceutical compositions and pharmaceutical preparations

[0352] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-FGFR2b antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, or an immunoconjugate as described herein, and a pharmaceutically acceptable carrier or excipient. It should be understood that the anti-FGFR2b antibody or pharmaceutical composition provided herein can be incorporated into a formulation with suitable carriers, excipients, and other agents for co-administration, thereby providing improved transfer, delivery, tolerance, and the like.

[0353] The term "pharmaceutical composition" refers to a preparation that permits the active ingredient contained therein to exist in biologically effective form, and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0354] Pharmaceutical formulations comprising the anti-FGFR2b antibodies described herein can be prepared by mixing an anti-FGFR2b antibody of the invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized formulation.

[0355] In yet another aspect, the present invention provides a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the pharmaceutical composition described herein, or the immunoconjugate described herein, and one or more additional therapeutic agents.

[0356] Medical uses and treatments

[0357] Any anti-FGFR2b antibody provided herein can be used in therapeutic methods. It should also be understood that when discussing "antibodies," compositions comprising antibodies are also included. The anti-FGFR2b antibodies of the present invention can be used in therapeutic or prophylactic methods described in any embodiment of the present invention in a therapeutically effective amount or a prophylactically effective amount.

[0358] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the immunoconjugate described herein, or the pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing a disease or condition mediated by FGFR2b, preferably the disease or condition is cancer, more preferably the cancer is selected from endometrial cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, bladder cancer, and bile duct cancer.

[0359] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof, a polynucleotide molecule, an expression vector, a host cell, an immunoconjugate, or a pharmaceutical composition as described herein, for use in treating and / or preventing a disease or condition mediated by FGFR2b, preferably a cancer, more preferably a cancer selected from endometrial cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, bladder cancer, and bile duct cancer.

[0360] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by FGFR2b, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, an immunoconjugate as described herein, or a pharmaceutical composition or drug combination as described herein, preferably the disease or condition is cancer, more preferably the cancer is selected from endometrial cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, bladder cancer, and bile duct cancer. In some embodiments, the present invention provides a method for treating a disease and / or condition requiring blocking FGFR2b, comprising administering to a subject in need thereof an anti-FGFR2b antibody as described herein.

[0361] In some embodiments, the cancer or tumor described herein can be selected from endometrial cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, bladder cancer, and bile duct cancer.

[0362] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration.

[0363] Methods for diagnosis and detection

[0364] In another aspect, the present invention provides a method for detecting the presence of FGFR2b in a sample using the antibodies or antigen-binding fragments thereof described herein. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues. The method includes the steps of contacting the sample with an antibody or antigen-binding fragment thereof described herein or a detection composition containing the antibody or antigen-binding fragment thereof, and detecting the presence of a conjugate or binding signal generated by the binding of the antibody or antigen-binding fragment thereof to FGFR2b. When used for detection purposes, the antibodies or antigen-binding fragments thereof described herein may be labeled to indicate whether the conjugate has been formed.

[0365] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0366] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0367] Example

[0368] The present invention is illustrated by the following examples, but is not intended to be limiting thereof. The present invention has been described in detail herein, and specific embodiments thereof are disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.

[0369] Example 1: Preparation of FGFR2b extracellular segment recombinant protein

[0370] The recombinant protein Human FGFR2b-His used for mouse immunization or antibody activity detection was purchased from Kactus Biotechnology (Cat. No.: Kactus-FGR-HM1BB). The protein contains Arg152-Glu378 (Accession# P21802-3). The recombinant protein Cyno FGFR2b-His used for antibody species cross-binding activity detection was purchased from Kactus Biotechnology (Cat. No.: Kactus-FGF-CM1BB), which contains Pro154-Lys368 (Accession#A0A2K5TL84-1); the recombinant protein mouse FGFR2b-His used for antibody species cross-binding activity detection was purchased from Kactus Biotechnology (Cat. No.: Kactus-FGF-MM1BB), which contains Pro39-Glu263 (Accession#P21803-2); the recombinant protein human FGFR2c-His used for antibody specificity detection was purchased from Kactus Biotechnology (Cat. No.: Kactus-FGR-HMIBC), which contains Arg152-Glu377 (Accession#P21802-1).

[0371] Example 2: Preparation of mouse hybridoma cells

[0372] 2.1 Mouse immunization

[0373] Balb / c, A / J, or C57 mice were immunized with purchased human FGFR2b-His recombinant protein. Following the primary immunization (50 μg / mouse, emulsified in complete Freund's adjuvant, intraperitoneal injection), booster immunizations were administered every other week (25 μg / mouse, emulsified in incomplete Freund's adjuvant, intraperitoneal injection). Blood was collected after the second booster immunization, and serum antibody titers and specificity against the immunogen were determined using ELISA and FACS.

[0374] 2.2 Cell fusion

[0375] Four days after the final booster immunization (unemulsified, intraperitoneal injection, 50 μg / mouse), mouse spleens were removed and triturated in DPBS. The spleen trituration fluid was collected in a 50 ml centrifuge tube (Corning), and the erythrocytes were lysed to obtain a splenocyte suspension. Splenocytes were fused with mouse myeloma Sp2 / 0 cells using conventional electroporation. The fusion product was adjusted to an appropriate density and cultured in DMEM (Gibco) supplemented with 20% fetal bovine serum (Gibco) and 1× HAT (hypoxanthine, methotrexate, and thymidine) in an incubator with 5% CO2 and 37°C. A half-medium change was performed on the seventh day of culture. On the tenth day, hybridoma supernatants were screened by ELISA to identify clones that positively bound to human FGFR2b-His and cyno FGFR2b-His, but negatively bound to human FGFR2c-His. After obtaining the target clone, flow cytometry was performed to screen out clones that bind positively to CHO-K1-huFGFR2b cells (prepared by the inventor, Met1-Thr822, Accession#P21802-3) and negatively to CHO-K1-huFGFR2c cells (prepared by the inventor, Met1-Thr821, Accession#P21802-1). In addition, flow cytometry was used to screen out clones with FGF10 (Cat. No.: Kactus-FGF-HE010B) / FGFR2b blocking activity. Next, the positive clones that met the above screening conditions were subcloned by limiting dilution and cultured in DMEM (Gibco) medium containing 10% fetal bovine serum. ELISA was used on the 10th day after subcloning, and the positive clones were selected and expanded to 24-well plates for further culture. Three days later, cell binding activity was verified by FACS. The best clone was selected based on the test results, cultured in DMEM (Gibco) medium containing 10% fetal bovine serum in a 5% CO2, 37°C incubator, and frozen in liquid nitrogen for subsequent sequencing.

[0376] Example 3: Screening of mouse hybridoma cells

[0377] Based on titer testing of blood samples collected after immunization, mice with high titers were selected for seven rounds of fusion. From 33,600 different polyclonal hybridoma cell lines, 58 hybridoma cells that specifically bound FGFR2b and exhibited ligand-blocking activity were selected according to the methods and screening criteria described in Example 2.2 for antibody light and heavy chain variable region sequencing.

[0378] Example 4: Determination of variable region sequences of murine antibodies (according to Kabat or IMGT)

[0379] Positive clones were transferred from 24-well plates (Corning) to T75 culture flasks (Corning). After the cells expanded to 70-90% confluence, they were frozen and then stored on dry ice and sent to GenScript for mouse antibody variable region sequencing. When using NGS sequencing, RNA is first extracted and purified, reverse transcribed into cDNA, and two rounds of PCR amplification using the cDNA as a template yield heavy and light chain positive bands. After sequencing, the paired antibody heavy and light chain sequencing results were analyzed to ultimately obtain the mouse antibody variable region sequence.

[0380] Example 5: Construction of chimeric antibodies

[0381] After sequencing the mouse antibody light and heavy chain variable regions, we commissioned GenScript to synthesize the gene fragments and clone the heavy and light chain variable region fragments into plasmids containing the human heavy chain constant region Fc or light chain constant region κ sequences, respectively. Sequencing confirmed correct cloning. GenScript was commissioned to perform transient transfection and purification of the antibodies, obtaining chimeric antibodies with a purity greater than 90% as determined by SDS-PAGE and SEC for subsequent activity testing.

[0382] Example 6: Screening of chimeric antibodies

[0383] 6.1 Binding activity of chimeric antibodies to CHO-K1-huFGFR2b / huFGFR2c cells

[0384] CHO-K1-huFGFR2b (CHO-K1 cells overexpressing human FGFR2b, constructed by the inventors) or CHO-K1-huFGFR2c (CHO-K1 cells overexpressing human FGFR2c, constructed by the inventors) cells were digested and counted in a T75 culture flask (Corning). The cell density was adjusted to 1E6 / ml and 100 μl was added to each well of a 96-well U-shaped plate (Corning). The anti-FGFR2b chimeric antibody constructed in Example 5, positive controls (Bemarituzumab / Bema and Aprutumab / Apru), and negative control (anti-KLH IgG1) were diluted in FACS buffer (2% FBS / PBS) (starting from 20 μg / ml or 10 μg / ml, with a 3-fold serial dilution, for a total of 12 or 8 dilutions). The cells were resuspended in 100 μl per well and incubated at 4°C for 1 hour. Wash cells three times with FACS buffer, dilute the secondary antibody Alexa fluor 488 F(ab′)2Fragment Goat Anti-Human IgG, Fcγ fragment specific (Cat. No.: Jackson immunoresearch-109-546-098) at 1:1000, resuspend cells in 100 μl per well, and incubate at 4°C in the dark for 30 minutes. After washing cells twice with FACS buffer, resuspend cells in 50 μl per well with FACS buffer, and use Intellicyt iQue3 (Satorius) to detect the mean fluorescence intensity (MFI) of cells. Fitting analysis of cell binding activity curves was performed on GraphPad Prism9 software. The EC curve 50 The plateau values ​​are shown in Table 3 (CHO-K1-huFGFR2b) and Table 4 (CHO-K1-huFGFR2c). Bemarituzumab binds to FGFR2b with high affinity and specificity, while aprutumab binds to both FGFR2b and FGFR2c, but binds weakly to FGFR2b. The results in Table 3 show that Chi-004, Chi-013, Chi-019, Chi-038, Chi-044, Chi-045, Chi-046, Chi-047, Chi-048, Chi-049, Chi-051, Chi-052, Chi-053, Chi-054, Chi-055, Chi-056, Chi-057, and Chi-058 bind relatively weakly to CHO-K1-huFGFR2b; the results in Table 4 show that Chi-027 binds weakly to CHO-K1-huFGFR2c and will not be studied further.

[0385] Table 3. Binding activity of chimeric antibodies to CHO-K1-huFGFR2b cells

[0386] Table 4. Binding activity of chimeric antibodies to CHO-K1-huFGFR2c cells

[0387] 6.2 Binding activity of chimeric antibodies to huFGFR1b-Fc, huFGFR3b-his, and huFGFR4-his

[0388] Antigens huFGFR1b-Fc (Cat. No.: Sino Biological-16482-H02H), huFGFR3b-His (Cat. No.: Sino Biological-16486-H08H), and huFGFR4-His (Cat. No.: Sino Biological-10538-H08H) were diluted to 2 μg / ml in DPBS (Gibco). 50 μl was added to each well of a 96-well high-adsorption ELISA plate (Corning) and incubated overnight at 4°C. The next day, the plate was washed three times with washing buffer (0.05% Tween 20 / DPBS) and blocked with blocking buffer (PBS containing 5% nonfat dry milk) at room temperature for 1 hour. After washing three times with wash buffer, anti-FGFR2b chimeric antibody, positive control (bemarituzumab), and negative control (anti-KLH IgG1) were diluted in ELISA buffer (PBS containing 1% nonfat dry milk) (starting at 20 μg / ml or 60 μg / ml, with a three-fold serial dilution sequence of eight steps). 100 μl was added to each well of a 96-well plate and incubated at room temperature for 1 hour. After washing three times with wash buffer, 50 μl of a 1:5000 dilution of the secondary antibody Goat anti-human IgG (Fab) HRP (Sigma-A0293) was added to each well and incubated at room temperature for 30 minutes. After washing three times, 50 μl of TMB substrate was added to each well and incubated at room temperature for 2-3 minutes. 50 μl of 1N HCl stop solution was added to each well. OD450 values ​​were measured using a multi-function microplate reader (Thermo Scientific, Multiskan FC). The results of chimeric antibody binding activity to FGFR1b are shown in Table 5, and those to FGFR3b and FGFR4 are shown in Table 6. Compared to IgG, bemarituzumab weakly bound to FGFR1b and did not bind to FGFR3b or FGFR4. Chi-006, Chi-018, and Chi-032 weakly bound to FGFR1b but not to FGFR3b or FGFR4. Chi-042 weakly bound to FGFR1b and FGFR3b at high concentrations but did not bind to FGFR4.

[0389] Table 5. Chimeric Antibodies Binding to FGFR1b ELISA

[0390] Table 6. Chimeric Antibodies Binding to FGFR3b and FGFR4 ELISA

[0391] 6.3 Binding activity of chimeric antibodies to huFGFR1c-His and huFGFR3c-His

[0392] Antigens huFGFR1c-His (Cat. No.: Sino Biological-10616-H08H) and huFGFR3c-His (Cat. No.: Sino Biological-16044-H08H) were diluted to 2 μg / ml in DPBS (Gibco). 50 μl was added to each well of a 96-well high-adsorption ELISA plate (Corning) and incubated overnight at 4°C. Binding activity of anti-FGFR2b chimeric antibodies (Chi-006, Chi-018, Chi-032, Chi-042), a positive control (Bemarituzumab), and a negative control (anti-KLH IgG1) to huFGFR1c-his and huFGFR3c-his was measured according to the experimental protocol described in 6.2. The experimental results are shown in Figure 1 . Chi-006, Chi-018, Chi-032, Chi-042, and Bemarituzumab did not bind to huFGFR1c-His and huFGFR3c-His proteins.

[0393] 6.4 Binding activity of chimeric antibodies to cyno FGFR2b-His and mouse FGFR2b-His

[0394] Antigens cyno FGFR2b-His (Cat. No. Kactus-FGF-CM1BB) and mouse FGFR2b-His (Cat. No. Acro-FGB-M52H5) were diluted to 2 μg / ml in DPBS (Gibco). 50 μl was added to each well of a 96-well high-adsorption ELISA plate (Corning) and incubated overnight at 4°C. The anti-FGFR2b chimeric antibodies (Chi-006, Chi-018, Chi-032, and Chi-042) were tested for species cross-binding activity according to the protocol described in 6.2. Bemarituzumab (Bema) was used as a positive control. The results are shown in Figures 2 and 3. Chi-006, Chi-018, Chi-032, Chi-042, and Bema all showed strong cross-binding activity against both cyno FGFR2b and mouse FGFR2b.

[0395] 6.5 Ligand-blocking activity of chimeric antibodies

[0396] CHO-K1-huFGFR2b(S252W) or CHO-K1-huFGFR2b were digested and counted from a T75 culture flask (Corning). The cell density was adjusted to 1E6 / ml and 100 μl was added to each well of a 96-well U-shaped plate (Corning). Anti-FGFR2b chimeric antibodies (Chi-006, Chi-018, Chi-032, Chi-042), a positive control (Bema), and a negative control (anti-KLH IgG1) were diluted in FACS buffer (starting at 10 μg / ml or 20 μg / ml, with a 3-fold serial dilution). The cells were resuspended in 100 μl per well and incubated at 4°C for 1 hour. Cells were washed three times with FACS buffer (2% FBS / PBS), and the ligands 0.2 μg / ml FGF7-biotin (Cat. No.: Kactus-FGF-HE101B) or 0.1 μg / ml FGF10-biotin (Cat. No.: Kactus-FGF-HE010B) were added and incubated at 4°C in the dark for 1 hour. Cells were washed three times with FACS buffer, and a 1:1000 dilution of SA-PE (Cat. No.: BD-554061) was added and incubated at 4°C in the dark for 30 minutes. Cells were washed twice with FACS buffer and resuspended in 50 μl of FACS buffer. The mean fluorescence intensity (MFI) of the cells was measured using Intellicyt iQue3 (Satorius), and curve fitting analysis was performed using GraphPad Prism 9 software. As shown in Figures 4 and 5, the candidate antibodies effectively blocked the interaction between the ligands FGF7 or FGF10 and the receptor FGFR2b.

[0397] 6.6 ADCC activity of chimeric antibodies

[0398] PBMCs were resuspended and adjusted to a density of 2E6-4E6 / ml using RPMI 1640 medium (Gibco, Cat#A1049101). The cells were then placed in a T75 cell culture flask and incubated overnight at 37°C in a 5% CO2 incubator. Target KATO3 cells were harvested, washed once with PBS, and labeled with Cell Trace Far Red (Invitrogen, Cat#C34564) according to the manufacturer's instructions. The cells were stained at 37°C for 20 min. Five volumes of RPMI 1640 were added and the cells were incubated at 37°C for 5 min to terminate the staining. 3E5 PBMCs (50 μl) and 1.5E4 KATO3 cells (50 μl) were added to each well of a 96-well U-bottom plate (Corning). The candidate antibody was diluted in culture medium (starting at 60 μg / ml, with a 5-fold serial dilution). 20 μl / well of the antibody was added to the cells and incubated at 37°C for 4 hours. 80 μl / well of buffer (PBS + 2% FBS) was added, and the cells were centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded. The cells were washed once with 200 μl / well of buffer and incubated with 100 μl / well of PI (Sigma, Cat# P4864) at 4°C for 5 minutes. Target cell viability was measured using a Fortessa flow cytometer. The experimental results are shown in Figure 6, showing that all candidate chimeric antibodies exhibited strong ADCC activity, similar to that of the control antibody Bema-Ref. The ADCC activity of the other control antibody, Apru-Ref, was weaker.

[0399] 6.7 Chimeric Antibody Cell Proliferation Inhibitory Activity

[0400] FGFR2b / BaF3 cells (overexpressing FGFR2b in Baf3 cells, constructed by GenScript, cultured in RPMI 1640 + 10% FBS + 10 ng / ml IL3 (Sino biological, Cat#51066-MNAH)) in the logarithmic growth phase were washed three times with RPMI 1640 medium without IL3 to remove the IL3 in the original culture medium. The cells were adjusted to an appropriate density and 3,000 cells were plated at 100 μl / well in a 96-well black, clear-bottom flat plate (Corning, Cat#3340). 50 μl of a mixture of 100 ng / ml FGF7 (Peprotech, Cat#100-19-10 ug) and 40 μg / ml Heparin (Selleck, Cat#S1346) was added to each well. The candidate antibody was diluted with culture medium (starting concentration was 40 μg / ml, 3-fold dilution), 50 μl / well was added to a 96-well plate, and cultured in a 37°C incubator for 5 days. After five days, the cells in the 96-well plate were mixed, 70 μl / well of cells were transferred to a new 96-well plate, 70 μl / well of CellTiter-Glo detection reagent (Promega, Cat# G7572) was added, and the chemiluminescent signal was detected using a multifunctional microplate reader (Molecular Devices, SpectraMax M5). The parameter regression curve was fitted by GraphPadprism software to calculate the EC 50 As shown in Figure 7, the candidate chimeric antibodies can effectively inhibit the proliferation of FGFR2b / BaF3 cells in vitro, and the inhibitory activity is similar to that of the control antibody Bemarituzumab (Bema-Ref), while another control antibody Aprutumab (Apru-Ref) has no cell proliferation inhibitory activity.

[0401] 6.8 Chimeric Antibodies Inhibit ERK Phosphorylation Activity

[0402] Following antibody treatment, the Advanced phospho-ERK (ThR202 / TyR204) cellular kit (Perkin Elmer, 64AERPEG) was used to detect phosphorylated ERK protein levels in the FGFR2b downstream signaling pathway. FGFR2b / BaF3 cells were washed three times with RPMI 1640 blank medium, resuspended to 2E5 / ml, placed in a T75 cell culture flask, and incubated overnight at 37°C in a 5% CO2 incubator. The next day, cells were harvested and resuspended to 4E6 / ml in RPMI 1640, and 50 μl / well was added to a 96-well U-bottom plate. The candidate antibody was serially diluted (starting at 50 μg / ml, followed by 3-fold dilutions). 12.5 μl of each concentration of antibody was added to each well of the 96-well plate and incubated at 37°C for 30 minutes. Add 12.5 μl of a mixture of FGF7 (final concentration 100 ng / ml) and Heparin (final concentration 10 μg / ml) to each well of a 96-well plate and incubate in a 37°C incubator for 15 min. Add 25 μl of 4x lysis buffer to each well of a 96-well plate and incubate at room temperature on a shaker at 350 rpm for 40 min. Transfer 16 μl of the above lysis supernatant to a White Opaque 96-well Microplate (Perkin Elmer, Cat# 6005680) and add 4 μl of a pre-mixed mixture of Phospho-ERK1 / 2 d2 antibody and Phospho-ERK1 / 2Eu Cryptate antibody. Incubate at room temperature for 4 h. Read the fluorescence value using Envision (Perkin Elmer). Calculate the EC using GraphPad Prism software to fit the parametric regression curve. 50 The experimental results are shown in Figure 8, and the candidate chimeric antibody inhibits the phosphorylation of ERK protein in the FGF7 / FGFR2b signaling pathway in a concentration-dependent manner.

[0403] 6.9 Affinity

[0404] The affinity of candidate chimeric antibodies and control antibodies for human FGFR2b protein was determined using an Octet RED96e molecular interaction instrument. The candidate chimeric antibodies and control antibodies were diluted to 1 μg / mL in PBST (0.05% Tween 20) buffer and captured onto a pre-wetted Protein A biosensor at a capture capacity of 1.3 nm. They were then bound to a gradient-diluted PBST solution of human FGFR2b protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.563 nM) for 180 seconds. Subsequently, the Protein A biosensor was immersed in PBST buffer for dissociation, with a dissociation time of 360 seconds. The acquired data were fitted to a 1:1 binding model using Octet RED96e Data Analysis version 12.0 software. The affinity results are shown in Table 7. The results show that Chi-018, Chi-032, Chi-042 and Bemarituzumab have similar affinities, around 3-5 nM, while Chi-006 has a relatively higher affinity.

[0405] Table 7. Chimeric Antibody Affinity

[0406] Example 7: Humanization of antibody variable regions

[0407] To humanize the antibody variable region, the human immunoglobulin gene database at NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) is first searched for human germline IgG genes homologous to the murine antibody cDNA sequence. The amino acid sequence and precise boundaries of the variable region CDRs are then defined using the Kabat or IMGT numbering systems. In principle, a human IGHV with high homology to the murine antibody is selected as the humanization template, and humanization of the antibody variable region is performed via CDR grafting.

[0408] Chi-042 and Chi-032 were humanized using the CDR grafting approach. Chi-042 humanization generated 5 heavy chains and 9 light chains, resulting in 20 humanized molecules; Chi-032 humanization generated 3 heavy chains and 3 light chains, resulting in 9 humanized molecules. Specific combinations of heavy and light chains for the humanized antibodies are shown in Tables 1 & 2.

[0409] Example 8: Screening of humanized antibodies

[0410] 8.1 Binding of Humanized Antibodies to CHO-K1-huFGFR2b Cells

[0411] CHO-K1-huFGFR2b cells were digested and counted in a T75 culture flask (Corning). The cell density was adjusted to 1E6 / ml and 100 μl was added to each well of a 96-well U-shaped plate (Corning). The anti-FGFR2b humanized antibody constructed in Example 7, a positive reference (Bema), and a negative reference (anti-KLH IgG1) were diluted in FACS buffer (starting at 20 μg / ml or 10 μg / ml, with a 3-fold or 4-fold serial dilution, for a total of 8 dilutions). The cells were resuspended in 100 μl per well and incubated at 4°C for 1 hour. Cells were washed three times with FACS buffer (2% FBS / PBS) and incubated with secondary antibodies (Alexa fluor 488 F(ab')2 Fragment Goat Anti-Human IgG, Fcγ fragment specific (Cat. No. Jackson immunoresearch-109-546-098) or PE F(ab')2 Goat anti-human IgG Fcγ Antibody (Cat. No. Biolegend-398004)) at a dilution of 1:1000 at 4°C in the dark for 30 min. Cells were washed twice with FACS buffer and resuspended in 50 μl of FACS buffer. Mean fluorescence intensity (MFI) was measured using IntellicytiQue3 (Satorius), and curve fitting analysis was performed using GraphPad Prism 9 software. Figure 9 shows that the binding activity of the humanized antibody to CHO-K1-huFGFR2b cells is similar to that of the chimeric antibody.

[0412] 8.2 ADCC activity of humanized antibodies

[0413] The ADCC activity of different versions of the Chi-042 and Chi-032 chimeric antibodies, hu-042 and hu-032, was tested using the experimental method described in Example 6.6. The results are shown in Figure 10 , indicating that the ADCC activity of the humanized molecules was similar to that of the chimeric parent antibody.

[0414] 8.3 Humanized Antibody Cell Proliferation Inhibitory Activity

[0415] The cell proliferation inhibitory activity of different versions of the Chi-042 and Chi-032 chimeric antibodies, hu-042 and hu-032, was tested using the experimental method described in Example 6.7. As shown in Figure 11, the humanized molecules had similar cell proliferation inhibitory activity as the chimeric parent antibody.

[0416] 8.4 Humanized Antibodies Inhibit ERK Phosphorylation Activity

[0417] Following the experimental protocol described in Example 6.8, the inhibitory activity of hu-042 and hu-032, the humanized versions of the Chi-042 and Chi-032 chimeric antibodies, was tested for ERK phosphorylation. The results, shown in Figure 12, demonstrate that the humanized hu-042 molecule exhibited concentration-dependent inhibition of ERK phosphorylation, with comparable inhibitory activity to the parent chimeric antibody.

[0418] 8.5 Humanized Antibody Affinity

[0419] The affinity of humanized antibodies and control antibodies for human FGFR2b protein was determined using an Octet RED96e molecular interaction instrument. Humanized antibodies and control antibodies were diluted to 1 μg / mL in PBST (0.05% Tween 20) buffer and captured onto pre-wetted Protein A biosensors at a capture capacity of 1.3-1.5 nm. They were then bound to a gradient-diluted PBST solution of human FGFR2b protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM; or 100 nM, 25 nM, 5 nM) for 180 seconds. Subsequently, the Protein A biosensor was immersed in PBST buffer for dissociation, with a dissociation time of 360 seconds. The acquired data were fitted to a 1:1 binding model using Octet RED96e Data Analysis version 12.0 software. The affinity results are shown in Tables 8 and 9, where the affinity of the humanized antibody hu-042 is comparable to that of the chimeric antibody Chi-042, and the affinity of the humanized antibody hu-032 is slightly lower than that of the chimeric antibody Chi-032.

[0420] Table 8. Affinity of humanized antibody hu-042

[0421] Table 9. Affinity of humanized antibody hu-032

[0422] Example 9: Preparation of anti-FGFR2b antibody drug conjugates

[0423] 9.1 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin vc-MMAE to Prepare Conjugate

[0424] Antibodies used: Reference antibody (Bemarituzumab), hu-032-005, hu-042-012, hu-042-015, hu-042-016.

[0425] Drugs and their sources: vc-MMAE was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0426] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin vc-MMAE is as follows:

[0427] first step:

[0428] Step 2:

[0429] Preparation of ADC

[0430] ADC-1: Bema

[0431] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 2.86 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB), and the mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and vc-MMAE (8.0 eq.) were added sequentially (total DMA ratio was 10%) to the solution. The solution was incubated at 25°C for 1 hour. After completion of the reaction, desalting and purification were performed, and the reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5). Filtering was performed to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0432] ADC-2:hu-032-005

[0433] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 2.86 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB). The mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and vc-MMAE (8.0 eq.) were added sequentially (total DMA ratio: 10%). The solution was incubated at 25°C for 1 hour. After completion of the reaction, desalting and purification were performed. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0434] ADC-3:hu-042-012

[0435] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 2.45 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB), and the mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and vc-MMAE (8.0 eq.) were added sequentially (total DMA ratio was 10%) to the solution. The solution was incubated at 25°C for 1 hour. After completion of the reaction, desalting and purification were performed, and the reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5). Filtering was performed to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0436] ADC-4:hu-042-015

[0437] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 2.35 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB). The mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and vc-MMAE (8.0 eq.) were added sequentially (total DMA ratio: 10%). The solution was incubated at 25°C for 1 hour. After the reaction, desalting and purification were performed. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0438] ADC-5:hu-042-016

[0439] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 2.45 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB). The mixed solution was incubated at 37°C for 3.5 h to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and vc-MMAE (8.0 eq.) were added sequentially (total DMA ratio: 10%). The solution was incubated at 25°C for 1 h. After the reaction, desalting and purification were performed. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography. The physicochemical properties of ADCs-1 to -5 are summarized in Table 10.

[0440] Table 10. Physicochemical properties of ADC-1 to ADC-5

[0441] FD:free drug ratio

[0442] 9.2 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin Deruxtecan to Prepare Conjugate

[0443] Antibodies: Bemarituzumab, hu-032-005, hu-042-012, hu-042-015, hu-042-016, Isotype IgG1

[0444] Drugs and their sources: Deruxtecan was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0445] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin is as follows:

[0446] first step:

[0447] Step 2:

[0448] Preparation of ADC

[0449] ADC-6: Bema

[0450] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 8.0 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB), and the mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and Deruxtecan (14.0 eq.) were added sequentially (total DMA ratio: 10%) to the solution. The solution was incubated at 25°C for 1 hour. After completion of the reaction, ultrafiltration was used for purification. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0451] ADC-7:hu-032-005

[0452] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 8.0 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB), and the mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and Deruxtecan (14.0 eq.) were added sequentially (total DMA ratio: 10%) to the solution. The solution was incubated at 25°C for 1 hour. After completion of the reaction, ultrafiltration was used for purification. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0453] ADC-8:hu-042-012

[0454] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 8.0 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB). The mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and Deruxtecan (14.0 eq.) were added sequentially (total DMA ratio: 10%). The solution was incubated at 25°C for 1 hour. After completion of the reaction, ultrafiltration was used for purification. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0455] ADC-9:hu-042-015

[0456] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 8.0 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB), and the mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and Deruxtecan (14.0 eq.) were added sequentially (total DMA ratio: 10%) to the solution. The solution was incubated at 25°C for 1 hour. After completion of the reaction, ultrafiltration was used for purification. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0457] ADC-10:hu-042-016

[0458] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 8.0 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB), and the mixed solution was incubated at 37°C for 3.5 hours to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and Deruxtecan (14.0 eq.) were added sequentially (total DMA ratio: 10%) to the solution. The solution was incubated at 25°C for 1 hour. After completion of the reaction, ultrafiltration was used for purification. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography.

[0459] ADC-11: Isotype IgG1

[0460] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 8.0 eq.) was added to an antibody solution (buffer pH 7.0, 40 mM PB). The mixed solution was incubated at 37°C for 3.5 h to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and Deruxtecan (14.0 eq.) were added sequentially (total DMA ratio: 10%). The solution was incubated at 25°C for 1 h. After completion of the reaction, ultrafiltration was used for purification. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography. A summary of the physicochemical properties of ADCs-6 to -11 is shown in Table 11.

[0461] Table 11. Physicochemical properties of ADC-6 to -11

[0462] 9.3 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin CPD2 to Prepare Conjugate

[0463] Antibody: hu-042-016

[0464] Drug and its source: CPD2, commissioned to Shanghai WuXi AppTec Co., Ltd.

[0465] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin CPD2 is as follows:

[0466] first step:

[0467] Step 2:

[0468] Preparation of ADC-12

[0469] Step 1: TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) was added to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5. The reaction was stirred at 25°C for 4 hours to obtain a reduced antibody solution. Step 2: DMA (N,N-dimethylacetamide) and CPD2 (14.0 eq.) (total DMA content: 20%) were added to the reduced antibody solution. The reaction was maintained at 25°C for 1 hour. The reaction solution was replaced with a buffer solution (20 mM L-histidine, pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC was stored at -60 to -90°C. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The physical and chemical properties of ADC-12 are shown in Table 12.

[0470] Table 12. Physicochemical properties of ADC-12

[0471] 9.4 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin CPD4 to Prepare Conjugate

[0472] Antibody: Bemarituzumab, hu-042-016

[0473] Drug and its source: CPD4, commissioned to Shanghai WuXi AppTec Co., Ltd.

[0474] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin CPD4 is as follows:

[0475] first step:

[0476] Step 2:

[0477] Preparation of ADC-13 and ADC-14

[0478] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5. The reaction was stirred at 25°C for 4 hours to obtain a reduced antibody solution. Step 2: Add DMA (N,N-dimethylacetamide) and CPD4 (14.0 eq.) (total DMA content: 20%) to the reduced antibody solution. The reaction was maintained at 25°C for 1 hour. The reaction solution was replaced with a buffer solution (20 mM L-histidine, pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC was stored at -60 to -90°C. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The physical and chemical properties of ADC-13 and ADC-14 are shown in Table 13.

[0479] Table 13. Physicochemical properties of ADC-13 to -14

[0480] 9.5 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin CPD5 to Prepare Conjugate

[0481] Antibody: Bemarituzumab

[0482] Drug and its source: CPD5, commissioned by Shanghai WuXi AppTec Co., Ltd.

[0483] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin CPD5 is as follows:

[0484] first step:

[0485] Step 2:

[0486] Preparation of ADC-15

[0487] Step 1: TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) was added to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5. The reaction was stirred at 25°C for 4 hours to obtain a reduced antibody solution. Step 2: DMA (N,N-dimethylacetamide) and CPD5 (14.0 eq.) (total DMA content: 20%) were added to the reduced antibody solution. The reaction was maintained at 25°C for 1 hour. The reaction solution was replaced with a buffer solution (20 mM L-histidine, pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC was stored at -60 to -90°C. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The physical and chemical properties of ADC-15 are shown in Table 14.

[0488] Table 14. Physicochemical properties of ADC-15

[0489] 9.6 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin CPD6 to Prepare Conjugate

[0490] Antibody: Bemarituzumab, hu-042-016

[0491] Drug and its source: CPD6, commissioned to Shanghai WuXi AppTec Co., Ltd.

[0492] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin CPD6 is as follows:

[0493] first step:

[0494] Step 2:

[0495] Preparation of ADC-16 and ADC-17

[0496] Step 1: TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) was added to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5. The reaction was stirred at 25°C for 4 hours to obtain a reduced antibody solution. Step 2: DMA (N,N-dimethylacetamide) and CPD6 (14.0 eq.) (total DMA content: 20%) were added to the reduced antibody solution. The reaction was maintained at 25°C for 1 hour. The reaction solution was replaced with a buffer solution (20 mM L-histidine, pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC was stored at -60 to -90°C. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The physical and chemical properties of ADC-16 and -17 are shown in Table 15.

[0497] Table 15. Physicochemical properties of ADC-16 to -17

[0498] 9.7 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin SN38-Comp5 to Prepare Conjugate

[0499] Antibody: hu-042-016

[0500] Drug and its source: SN38-Comp5, synthesized by Suzhou Junmeng Biopharmaceutical Technology Co., Ltd.

[0501] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin SN38-Comp5 is as follows:

[0502] first step:

[0503] Step 2:

[0504] Preparation of ADC-18

[0505] Step 1: TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) was added to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5. The reaction was stirred at 25°C for 3.5 hours to obtain a reduced antibody solution. Step 2: DMA (N,N-dimethylacetamide), Comp5 (16.0 eq.) (total DMA content: 20%) was added to the reduced antibody solution. The reaction was maintained at 25°C for 1 hour. The reaction solution was replaced with a buffer solution (50 mM citric acid, pH 6.0) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC was stored at -60 to -90°C. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by PLRP chromatography. Purity was determined by size-exclusion chromatography. The physical and chemical properties of ADC-18 are shown in Table 16.

[0506] Table 16. Physicochemical properties of ADC-18

[0507] 9.8 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin Deruxtecan to Prepare Conjugate (DAR4)

[0508] Antibody: hu-042-016

[0509] Drugs and their sources: Deruxtecan was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0510] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin is as follows:

[0511] first step:

[0512] Step 2:

[0513] Preparation of ADC-19

[0514] Reduction: 2 mM TCEP (tris(2-carbonylethyl)phosphine, 2.38 eq.) was added to an antibody solution (buffer: pH 6.5, 50 mM PBS / 2 mM EDTA). The mixed solution was incubated at 37°C for 3 h to obtain a reduced antibody solution. Conjugation: The reduced antibody solution was placed on ice, and DMA (N,N-dimethylacetamide) and Deruxtecan (7 eq.) were added sequentially (total DMA ratio: 15%). The solution was incubated at 25°C for 1 h. After completion of the reaction, ultrafiltration was used to purify the reaction solution. The reaction solution was exchanged into a buffer solution (20 mM L-His / pH 5.5) and filtered to obtain the antibody-drug conjugate (ADC) of the present invention. The ADC product was stored at -60 to -90°C. Free linker-toxin and other small molecules were detected by reverse-phase chromatography, and DAR was detected by HIC chromatography. Purity was determined by size exclusion chromatography. The physical and chemical properties of ADC-19 are shown in Table 17.

[0515] Table 17. Physicochemical properties of ADC-19

[0516] 9.9 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin CPD2 to Prepare Conjugate (DAR4)

[0517] Antibody: hu-042-016

[0518] Drug and its source: CPD2, commissioned to Shanghai WuXi AppTec Co., Ltd.

[0519] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin CPD2 is as follows:

[0520] first step:

[0521] Step 2:

[0522] Preparation of ADC-20

[0523] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.2 eq.) to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5. The reaction was stirred at 37°C for 3 hours to obtain a reduced antibody solution. Step 2: Add DMA (N,N-dimethylacetamide) and CPD2 (6.0 eq.) (total DMA ratio: 15%) to the reduced antibody solution. The reaction was maintained at 25°C for 1 hour. The reaction solution was replaced with a buffer solution (20 mM L-histidine / pH 5.5), filtered to obtain the antibody-drug conjugate (ADC) of the present invention, and stored at -60 to -90°C. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The physical and chemical properties of ADC-20 are shown in Table 18.

[0524] Table 18. Physicochemical properties of ADC-20

[0525] 9.10 Anti-FGFR2b Antibody Conjugated to Linker-Cytotoxin CPD6 to Prepare Conjugate (DAR4)

[0526] Antibody: hu-042-016

[0527] Drug and its source: CPD6, commissioned to Shanghai WuXi AppTec Co., Ltd.

[0528] The preparation method of the conjugate prepared by coupling the anti-FGFR2b antibody to the linker-cytotoxin CPD6 is as follows:

[0529] first step:

[0530] Step 2:

[0531] Preparation of ADC-21

[0532] Step 1: TCEP (tris(2-carbonylethyl)phosphine, 2.2 eq.) was added to the antibody in a buffer solution of 50 mM PBS / 2 mM EDTA, pH 6.5. The reaction was stirred at 37°C for 3 hours to obtain a reduced antibody solution. Step 2: DMA (N,N-dimethylacetamide) and CPD6 (6.0 eq.) (total DMA ratio of 15%) were added to the reduced antibody solution. The reaction was controlled at 25°C for 1 hour. The reaction solution was replaced with a buffer solution (20 mM L-histidine / pH 5.5), filtered to obtain the antibody-drug conjugate ADC of the present invention, and stored at -60 to -90°C. Free small molecules were detected by reverse-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The physical and chemical properties of ADC-21 are shown in Table 19.

[0533] Table 19. Physicochemical properties of ADC-21

[0534] Example 10: Endocytic activity of anti-FGFR2b antibody drug conjugates

[0535] Follow Zenon TM pHrodo TM Each ADC sample prepared in Example 9 was labeled with iFL IgG Labeling Reagent (Invitrogen, Cat# Z25612) according to the instructions. The sample was serially diluted with culture medium and added to an equal volume of SNU16 cells at 50 μl / well. The cells were incubated at 37°C for 4 hours, and the fluorescence value in the PE channel was measured by flow cytometry to reflect the number of internalized molecules. As shown in Figure 13, hu-042-MMAE and hu-032-MMAE exhibited superior target-dependent endocytosis activity compared to the negative control, KLH-MMAE.

[0536] Example 11: In vitro killing experiment of anti-FGFR2b antibody drug conjugate

[0537] After 6 days of co-culture of each ADC sample with FGFR2b-expressing SNU16 cells at a concentration gradient, the viable cell count was determined using a cell counting lite (Vazyme #DD1102-01). The in vitro cytotoxicity of the candidate molecules was determined by calculating the proliferation inhibition rate (Proliferation inhibition rate (%) = (viable cell count in untreated group - viable cell count in treated group) / (viable cell count in untreated group - viable cell count in solution control group)). Figure 14 shows that bemarituzumab and aprutumab did not affect the proliferation activity of SNU16 cells, but ADC-1, -2, and -3 exhibited significant cytotoxicity. Figure 15 shows that ADC samples constructed with different linkers (ADC-13, -15, and -16) exhibited significant and similar cytotoxicity on SNU16 cells, comparable to that of ADC-6. Table 20 summarizes the in vitro cytotoxicity EC values ​​of ADC-13, -15, and -16 compared to ADC-6. 50 The relative killing activities (%) of ADC-13, -15, and -16 were calculated based on ADC-6.

[0538] Table 20: Anti-FGFR2b ADC's ability to inhibit the proliferation of SNU16 cells N / A: Not applicable.

[0539] In addition, Figure 16 compares the cytotoxicity of hu-042-016 ADC samples constructed with different linkers on SNU16 cells. The results show that each hu-042-016-CPD ADC sample (ADC-12, -14, and -17) can significantly inhibit the in vitro proliferation of SNU16 cells, with similar activity and slightly higher than that of hu-042-016-DXD ADC (ADC-10).

[0540] Example 12: In vitro bystander assay of anti-FGFR2b antibody drug conjugates

[0541] Refer to CellTrace TM Violet (Cat. No.: Invitrogen TM -C34557) instructions for pre-labeling N87 cells, staining with PBS at 37°C for 20 minutes, adding 5 times the volume of complete medium (RPMI 1640 + 10% FBS), and incubating at 37°C for 5 minutes to terminate staining. 5000 SNU16 cells (100 μl) and 15,000 N87 cells (100 μl) were added to each well of a 24-well plate (Corning). The candidate antibody was diluted to 100nM with complete medium, added to each well of the 24-well plate at 50 μl, and each well was filled with complete medium to 500 μl. The cells were gently mixed back and forth and incubated in a 37°C incubator for 5 days. After 5 days, the supernatant was gently shaken, and the cells in the supernatant were collected into a flow tube. The culture medium was then washed with 300 μl PBS per well, and the PBS was recovered into the corresponding flow tube. 150 μl TrypLE was added. TM (Cat. No.: Gibco-12604021) Digest in a 37°C incubator for 5 minutes, then terminate with the same volume of complete medium. Recover the digested cells into corresponding flow cytometry tubes. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 300 μl of buffer to each tube, including 250 μl FACS buffer (PBS + 2% FBS + 0.1% PI) and 50 μl CountBright TM Absolute Counting Beads (Cat. No.: Invitrogen-C36950). Vortex and mix thoroughly. Analyze on a Fortessa flow cytometer. Collect 5000 beads per well as the stopping gate. Analyze SNU16 and N87 PI-negative live cells using FlowJo. TM Absolute Counting Beads was used to count viable SNU16 and N87 PI-negative cells in a complete volume of buffer. GraphPad Prism was used to plot the data.

[0542] The results, as shown in Figure 17, show that when SNU16 and N87 cells were co-cultured, hu-042-016-Dxd-D4, hu-042-016-CPD6-D4, and hu-042-016-CPD6-D8 significantly killed FGFR2b-positive SNU16 cells and exhibited strong bystander-killing activity against FGFR2b-negative N87 cells. Due to the poor membrane-penetrating ability of MMAF, the anti-FGFR2b MMAF sample lacked bystander-killing activity and only inhibited SNU16 cell proliferation without affecting N87 cell viability. Anti-KLH-CPD6-D8 and blank medium groups showed no effective killing effect on either cell type and served as negative controls. When SNU16 cells were cultured alone, all anti-FGFR2b ADCs effectively killed SNU16 cells; however, when N87 cells were cultured alone, none of the anti-FGFR2b ADCs affected N87 cell proliferation.

[0543] Example 13: In vivo efficacy study of anti-FGFR2b antibody-drug conjugates

[0544] Experimental animals: 48 female NDG mice, 6-8 weeks old, weighing approximately 18-22 g, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0545] Antibody drugs:

[0546] hu-042-016-DXD, lot number: 20230704-R1; DAR: 7.28;

[0547] hu-042-016-CPD2, lot number: 20230626-R1; DAR: 7.66;

[0548] hu-042-016-CPD4, lot number: 20230616-R1; DAR: 7.88;

[0549] hu-042-016-CPD6, lot number: 20230616-R2; DAR: 7.82;

[0550] KLH-CPD6, batch number: 20230713-R1; DAR: 7.84.

[0551] Test process:

[0552] SNU16 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS). SNU16 cells in the logarithmic growth phase were collected and resuspended in PBS to an appropriate concentration for inoculation of NDG mice. SNU16 cells were collected and inoculated at a density of 3.5×10 6 / 100ul, inoculated subcutaneously on the right side of NDG mice.3 Afterwards, the mice were randomly divided into groups and administered according to their tumor volumes. The day of grouping was designated as day 0. Detailed administration methods, dosages, and routes of administration are shown in Table 21 below.

[0553] Table 21: Dosage, dosage and route of administration Note: N: number of animals used; iv: intravenous injection; administration volume: the administration volume was adjusted according to the weight of the tumor-bearing mice (0.1 mL / 10 g).

[0554] After the start of the experiment, the tumor volume and mouse body weight were measured twice a week. The experimental results are shown in Table 22 and Figure 18.

[0555] Table 22: Average tumor volume of mice in each group (Mean ± SEM) Note: p<0.05 is considered to be a significant difference; the anti-tumor efficacy of the test substance is evaluated by the tumor growth inhibition rate TGI (%), which reflects the tumor growth inhibition rate. TGI (%) = [(1-(TV Treatment _DayN-TV Treatment_ Day0)) / (TV vehicle _DayN-TV Vehicle _Day0)]×100%. TV Treatment _DayN、TV Treatment _Day0、TV Vehicle _DayN and TV Vehicle _Day0 is the average tumor volume at the treatment day (Day N) and the initial time (Day 0) in the treatment group, and at the vehicle control group. T / C (%) is the relative tumor growth rate, T / C (%) = (1-TGI) × 100%.

[0556] At the end of the experiment (28 days after administration), compared with the control group, the anti-FGFR2b antibody-drug conjugate (ADC) hu-042-016-CPD2, hu-042-016-CPD4, hu-042-016-CPD6 and hu-042-016-DXD at a dose level of 2 mg / kg significantly inhibited tumor growth, with tumor growth inhibition rates of 79.68% (p < 0.0001), 92.49% (p < 0.0001), 77.02% (p < 0.0001), and 67.92% (p < 0.0001), respectively.

[0557] Example 14: In vivo efficacy study of anti-FGFR2b antibody drug conjugate (DAR4)

[0558] Experimental animals: 40 female NDG mice, 6-8 weeks old, weighing approximately 18-22 g, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0559] Antibody drugs:

[0560] hu-042-016-DXD DAR4, lot number: 20231001-R12; DAR: 4.05;

[0561] hu-042-016-CPD2 DAR4, lot number: 20231001-R1; DAR: 4.14;

[0562] hu-042-016-CPD1 DAR4, lot number: 20231017-L1; DAR: 4.10;

[0563] hu-042-016-CPD6 DAR4, lot number: 20231011-R1; DAR: 4.10;

[0564] hu-042-016-CPD6 DAR8, lot number: 20231011-R2; DAR: 7.90;

[0565] KLH-CPD6 DAR4, lot number: 20231107-R1; DAR: 4.23.

[0566] Test process:

[0567] SNU16 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS). SNU16 cells in the logarithmic growth phase were collected, resuspended in PBS to an appropriate concentration, and plated at 3.5×10 6 100ul was inoculated subcutaneously on the right side of NDG mice. When the tumor grew to about 150mm 3 The grouping started, with the day of grouping being day 0. The mice were randomly divided into groups and dosed according to their tumor volumes. The detailed administration method, dosage and route of administration are shown in Table 23.

[0568] Table 23: Dosage regimen, dosage and route of administration Note: N: number of animals used; iv: intravenous injection; administration volume: the administration volume was adjusted according to the weight of the tumor-bearing mice (0.1 mL / 10 g).

[0569] After the start of the experiment, the tumor volume and mouse body weight were measured twice a week. The experimental results are shown in Table 24 and Figure 19.

[0570] Table 24. Average tumor volume of mice in each group (Mean ± SEM) Note: p<0.05 is considered to be a significant difference; the anti-tumor efficacy of the test substance is evaluated by the tumor growth inhibition rate TGI (%), which reflects the tumor growth inhibition rate. TGI (%) = [(1-(TV Treatment _DayN-TV Treatment _Day0)) / (TV Vehicle _DayN-TV Vehicle _Day0)]×100%. TV Treatment _DayN、TV Treatment _Day0、TV Vehicle _DayN and TV Vehicle _Day0 is the average tumor volume at the treatment day (Day N) and the initial time (Day 0) in the treatment group, and at the vehicle control group. T / C (%) is the relative tumor growth rate, T / C (%) = (1-TGI) × 100%.

[0571] At the end of the experiment (after 20 days of dosing), the anti-FGFR2b antibody-drug conjugates (ADCs) hu-042-016-CPD6 DAR4, hu-042-016-CPD2 DAR4, hu-042-016-CPD1 DAR4, and hu-042-016-DXd DAR4 significantly inhibited tumor growth at a dose of 2 mg / kg compared to the control group, with TGIs of 84.89% (p < 0.0001), 115.51% (p < 0.0001), 107.06% (p < 0.0001), and 92.88% (p < 0.0001), respectively. In addition, hu-042-016-CPD6 DAR8 showed the greatest tumor inhibition effect, with a TGI of 123.63% (p < 0.0001).

[0572] Example 15: In vivo pharmacodynamic study of anti-FGFR2b antibody-drug conjugates on human gastric cancer subcutaneous transplanted tumors

[0573] Experimental Procedure: Successfully revived FP4+2 generation LD1-0017-200652 human gastric cancer xenografts were cut into small pieces approximately 3mm x 3mm x 3mm in size. These pieces were then inoculated subcutaneously on the right side of the back of NU / NU mice (generation FP4+3 for efficacy experiments). Approximately 30-45mg of tumor tissue was inoculated per animal, along with 10μL of Matrigel. Mice were observed and tumor growth monitored after inoculation. On day 18 of inoculation, the average tumor volume in tumor-bearing mice was 163.61mm. 3 The grouping and dosing were performed at 1:00 pm, and the day of grouping and dosing was defined as day 0. The grouping and dosing information is shown in Table 25 below.

[0574] Table 25. Dosage regimen, dosage, and route of administration Note: N: number of animals used; BIW*4W: administration twice a week for 4 consecutive weeks; IV: intravenous injection.

[0575] After the start of the experiment, the tumor volume was measured twice a week. The experimental results are shown in Table 26 and Figure 20.

[0576] Table 26. Evaluation of the antitumor efficacy of the test drugs in a human gastric cancer subcutaneous xenograft model

[0577] The experimental results showed that compared with the Vehicle group, all dose groups of hu-042-016-CPD6 DAR4 could inhibit tumor growth, and the inhibitory activity was significantly higher than the positive control Bemarituzumab (TGI was only 10.95%); among them, the inhibitory effects of the 3mg / kg group, 6mg / kg group and 10mg / kg group were more significant, with TGI reaching 106.57%, 108.76% and 108.76%, respectively.

[0578] Example 16: In vivo pharmacodynamic study of anti-FGFR2b antibody drug conjugates on subcutaneous human breast cancer xenografts

[0579] Experimental Procedure: Successfully revived FP3+3 generation LD1-2009-361825 human breast cancer xenografts were cut into small pieces approximately 3mm x 3mm x 3mm in size. These pieces were then inoculated subcutaneously on the right side of the back of NCG mice (generation FP3+4 for efficacy studies). Approximately 50-90mg of tumor tissue was inoculated per animal, along with 30μL of Matrigel. Mice were observed and tumor growth monitored after inoculation. On day 28 after inoculation, the average tumor volume in tumor-bearing mice was 149.22mm. 3 The day of group administration was defined as day 0. The specific group information is shown in Table 26.

[0580] Table 26. Dosage regimen, dosage, and route of administration Note: N: number of animals used; BIW*4W: administration twice a week for 4 consecutive weeks; IV: intravenous injection.

[0581] After the start of the experiment, the tumor volume was measured twice a week. The experimental results are shown in Table 27 and Figure 21.

[0582] Table 27. Evaluation of the antitumor efficacy of the test drugs in a human breast cancer subcutaneous xenograft model

[0583] The experimental results showed that compared with the Vehicle group, hu-042-016-CPD6 DAR4 in each dose group could significantly inhibit tumor growth (TGI was 85.49%, 129.04% and 146.02%, respectively). The inhibitory activity was positively correlated with the dose and was better than Bemarituzumab (TGI was 47.76%).

[0584] Example 17: In vivo pharmacodynamic study of anti-FGFR2b antibody-drug conjugates on subcutaneous human lung cancer xenografts

[0585] Successfully revived FP2+1 generation LD1-0025-200783 human lung cancer xenografts were cut into small pieces approximately 3 mm × 3 mm × 3 mm in size. These pieces were inoculated subcutaneously on the right side of the back of NU / NU mice (generation FP2+2 for efficacy experiments). Each animal was inoculated with approximately 40-75 mg of tumor tissue and 15 μL of Matrigel. Mice were observed and tumor growth was monitored. On day 21 after inoculation, the average tumor volume of the tumor-bearing mice was 149.12 mm. 3 The day of group administration was defined as Day 0. The specific group information is shown in Table 28 below.

[0586] Table 28. Dosage regimen, dosage, and route of administration Note: N: number of animals; IV: tail vein injection; QW: administration once a week; BIW*4W: administration twice a week for 4 consecutive weeks.

[0587] After the start of the experiment, the tumor volume was measured twice a week. The experimental results are shown in Table 29 and Figure 22.

[0588] Table 29. Evaluation of the antitumor efficacy of the test drugs in a human lung cancer subcutaneous xenograft tumor model

[0589] The experimental results showed that compared with the vehicle group, Bemarituzumab had almost no anti-tumor activity, with a TGI of -0.33%; hu-042-016-CPD6 DAR4 in each dose group could significantly inhibit tumor growth, with TGIs of 47.82%, 74.07% and 93.74, respectively, and the inhibitory activity was positively correlated with the dose.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to FGFR2b, wherein the antibody or antigen-binding fragment thereof comprises: An HCDR1 having an amino acid sequence as shown in SEQ ID NO: 25, 1, 9 or 17, or having 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 25, 1, 9 or 17; An HCDR2 having an amino acid sequence as shown in SEQ ID NO: 45, 2, 10, 18, 26, 33, 43 or 44, or having 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 45, 2, 10, 18, 26, 33, 43 or 44; An HCDR3 having an amino acid sequence as shown in SEQ ID NO: 27, 3, 11 or 19, or having 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 27, 3, 11 or 19; An LCDR1 having an amino acid sequence as shown in SEQ ID NO: 51, 5, 13, 21, 29, 37 or 38, or having 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 51, 5, 13, 21, 29, 37 or 38; An LCDR2 having an amino acid sequence as shown in SEQ ID NO: 53, 6, 14, 22, 30, 39 or 52, or having 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 53, 6, 18, 22, 30, 39 or 52; An LCDR3 having an amino acid sequence as shown in SEQ ID NO: 31, 7, 15 or 23, or having 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO: 31, 7, 15 or 23.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region: The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 45 and SEQ ID NO: 27, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 45 and SEQ ID NO: 27; or HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; or HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; or HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; or HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19; or HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 43, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 43, and SEQ ID NO: 27; or HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27; The light chain variable region comprises: LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 31, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 31; LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; or LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; or LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; or LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31; or LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23; or LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23; or LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23; or LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31; Preferably, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 45, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 31, respectively; or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 33, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 21, SEQ ID NO: 39, and SEQ ID NO: 23, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 43, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 44, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 31, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 45, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 31, respectively; or A heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 43, and SEQ ID NO: 27, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 51, SEQ ID NO: 53, and SEQ ID NO: 31, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and wherein: The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 49, 4, 12, 20, 28, 34, 35, 36, 46, 47, 48, or 50, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 49, 4, 12, 20, 28, 34, 35, 36, 46, 47, 48, or 50; The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, 8, 16, 24, 32, 40, 41, 42, 54, 55, 56, 57, 58, 59, 60, or 61, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 62, 8, 16, 24, 32, 40, 41, 42, 54, 55, 56, 57, 58, 59, 60, or 61; Preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 49, 34, 35, 36, 46, 47, 48, or 49; the light chain variable region comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 62, 40, 41, 42, 54, 55, 56, 57, 58, 58, 60, or 61; More preferably, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 49, 34, 35, 36, 46, 47, 48, or 50; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, 40, 41, 42, 54, 55, 56, 57, 58, 58, 60, or 61.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 49; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 62; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 20, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 34; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 40, 41 or 42, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 35; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 40, 41 or 42, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 36; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 40, 41 or 42, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, 47 or 48, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 46, 47 or 48; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 54; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46, 47 or 48, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 46, 47 or 48; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 55, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 55; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 48 or 49; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 56; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 48 or 49; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 57; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48 or 49, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 48 or 49; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 58; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49 or 50, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 49 or 50; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 59; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49 or 50, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 49 or 50; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 60; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49 or 50, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 49 or 50; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 61; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 50, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 50; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

62.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 49 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 62; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 34 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 35 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 46, 47 or 48 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 54; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 46, 47 or 48 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 55; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 48 or 49 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 48 or 49 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 57; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 48 or 49 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 58; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 49 or 50 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 59; or A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 49 or 50 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 60; or A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 49 or 50 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 61; or A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 50 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

62.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody comprises: A heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 63 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 63; and A light chain comprising an amino acid sequence as shown in SEQ ID NO: 64, 65 or 66 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 64, 65 or 66; Preferably, the antibody comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 63 and a light chain having an amino acid sequence as shown in SEQ ID NO: 64, or a heavy chain having an amino acid sequence as shown in SEQ ID NO: 63 and a light chain having an amino acid sequence as shown in SEQ ID NO: 65, or a heavy chain having an amino acid sequence as shown in SEQ ID NO: 63 and a light chain having an amino acid sequence as shown in SEQ ID NO:

66.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody is selected from murine antibodies, chimeric antibodies, humanized antibodies or fully human antibodies, and the antigen-binding fragment is selected from Fab, Fab', F(ab′)2, Fv, scFv or sdAb.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the antibody or antigen-binding fragment is any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably the IgG1 subtype.

9. An isolated anti-FGFR2b antibody or antigen-binding fragment thereof having at least one of the following characteristics: (I) The epitope of the human FGFR2b protein bound is the same, completely overlapping or partially overlapping with the antibody or antigen-binding fragment thereof according to any one of claims 1-8; (II) It competes with the antibody or antigen-binding fragment thereof according to any one of claims 1-8 for binding to the epitope of the human FGFR2b protein.

10. A polynucleotide molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-9.

11. An expression vector comprising the polynucleotide molecule according to claim 10; preferably, the vector is a eukaryotic expression vector.

12. A host cell comprising the polynucleotide molecule according to claim 10 or the expression vector according to claim 11; preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

13. A method for preparing an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9, wherein, The method includes expressing the antibody or its antigen-binding fragment in the host cell according to claim 12 under conditions suitable for the expression of the antibody or its antigen-binding fragment, and recovering the expressed antibody or its antigen-binding fragment from the host cell.

14. An immunoconjugate comprising the antibody or its antigen-binding fragment according to any one of claims 1-9 and a conjugate moiety.

15. The immunoconjugate according to claim 14, wherein, The conjugate moiety is selected from: an anti-tumor agent, an immunomodulator, a cytotoxic drug, a fluorescent substance, a luminescent substance, an enzyme, a small nucleic acid molecule, and any combination thereof; preferably, the small nucleic acid is siRNA or an antisense oligonucleotide (ASO).

16. The immunoconjugate according to claim 14, wherein, The immunoconjugate is an antibody-drug conjugate having a structure of Ab-(L-D)m, wherein Ab is the antibody or its antigen-binding fragment that binds to FGFR2b according to any one of claims 1-9; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient; m represents the average number of L-D units conjugated to Ab, and the range of m is 1-8, preferably 4.

17. The antibody-drug conjugate according to claim 16, wherein, The L structure is as shown in formula (A): Wherein: L1 represents a group or bond formed after a functional group capable of reacting with a thiol reacts with the thiol; L2 and Y are each independently selected from -(CH2) p -, -(OCH2CH2) p -, and -(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; R6 is selected from H, C1-C6 alkoxy, -(OCH2CH2) p O-C1-C6 alkyl and -(CH2CH2O) p -C1-C6 alkyl; X is selected from -NH-, -NH(CH2) n C(=O)-, -C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; o is selected from 0 and 1; n is selected from 0, 1, and 2; p is an integer selected from 1 to 10.

18. The immunoconjugate according to claim 17, wherein, The functional group capable of reacting with a thiol of L1 is selected from maleimide, halogen, halogen-substituted functional groups (such as halogen-substituted aldehyde groups, halogen-substituted -S(O)2-), aldehyde groups, alkenyl groups, alkynyl groups, alkanones, sulfonyl groups, silanes, isocyanate groups, and norbornenyl groups; Preferably, the functional group capable of reacting with a mercapto group described in L1 is selected from: and their derivatives; Wherein: A is a halogen; The wavy line indicates the position where L1 is connected to L2; Preferably, the functional group of L1 is:

19. The immunoconjugate according to any one of claims 17-18, wherein: L2 is -(CH2) p -, and p is an integer from 1 to 4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-; and / or X is -NH- or -NH(CH2) n C(=O)-; preferably, X is -NH- or -NHC(=O)-; and / or Y is -(CH2) p -, p is 1, 2 or 3; preferably, Y is methylene; and / or Z is C.

20. The immunoconjugate according to any one of claims 17-19, wherein, L3 is selected from monosaccharides, disaccharides, and five- or six-membered saturated heterocycles containing 1-2 nitrogen atoms and divalent hydrophilic groups having two monovalent group centers generated by removing two hydrogen atoms from them and their derivatives; Preferably, the monosaccharide is selected from triose, tetrose, pentose, hexose, and heptose; Preferably, the disaccharide is selected from maltose, sucrose, and lactose; Preferably, the five- or six-membered saturated heterocycle containing 1-2 nitrogen atoms or its derivative is selected from piperazinyl, piperidinyl, and pyrrolidinyl; Preferably, L3 is selected from divalent hydrophilic groups having two monovalent group centers generated by removing two hydrogen atoms from glucose, galactose, mannose, glucuronic acid, galacturonic acid, mannuronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, and N-acetylmuramic acid; More preferably, L3 has the structure represented by formula L3-1: In the formula: R1 is selected from -O(CH2) p -, and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4; R2, R3, and R4 are independently selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group, and phosphoric acid group; t is an integer from 1 to 20; The wavy line indicates the position where L3 is connected to X and Y.

21. The immunoconjugate according to any one of claims 17-20, wherein: L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine and tyrosine; the amino acid residues are optionally substituted by one or more -OR 20 wherein, R 20 is selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group and phosphoric acid group, wherein t is an integer from 1 to 20; or L4 is selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine-glycine, glycine-glycine-phenylalanine-glycine-glycine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine, and lysine-phenylalanine, preferably selected from the following peptide segments: valine-citrulline (VC), valine-alanine (VA), and glycine-glycine-phenylalanine-glycine (GGFG); each of the peptide segments is optionally substituted with one or more substituents selected from the group consisting of -OR20 and -(OCH2CH2) p O-C1-C6 alkyl, wherein R20 is selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group, and phosphoric acid group, t is an integer from 1 to 20, and p is an integer from 1 to 6.

22. The immunoconjugate according to claim 21, wherein, L4 is selected from: In the formula, the wavy line indicates the position where L4 is connected to Z and NH.

23. The immunoconjugate according to any one of claims 14-22, wherein, R6 is H, C1-C3 alkoxy or -(OCH2CH2) p O-C1-C3 alkyl, wherein p is an integer from 1 to 6; preferably, R6 is H, methoxy or -(OCH2CH2) p OCH3, wherein p is an integer from 1 to 6 or from 1 to 4.

24. The immunoconjugate according to claim 16, wherein, The structure of the joint L is shown as any of the following structures: In each formula, R2, R3 and R4 are as described in claim 20, R6 is as described in claim 17 or 22, and R” is H or C1-C6 alkyl.

25. The immunoconjugate according to any one of claims 14-24, characterized in that, The pharmaceutical moiety D includes tubulin inhibitors, DNA damaging agents, vitamin A precursors, and folic acid; Preferably, the tubulin inhibitors include dolastatin and auristatin, maytansine; the DNA damaging agents include calicheamicin, duocarmycin, anthramycin derivatives PBD (pyrrolobenzodiazepine), topoisomerase I inhibitors; More preferably, the auristatin drugs include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF) or their derivatives, the maytansine drugs include DM1, DM3, DM4 or their derivatives; the topoisomerase I inhibitors include irinotecan, topotecan, irinotecan, 9-nitro camptothecin, camptothecin derivative SN-38.

26. The immunoconjugate according to any one of claims 14-25, wherein The linker-drug (L-D) moiety has the structure shown by formula VCMMAE, CPD2, CPD4, CPD5, CPD6 or SN-38Comp5 as follows:

27. The immunoconjugate according to any one of claims 14-26, wherein The immunoconjugate is selected from the structures shown by ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI or ADC-VII of the formula: In the formula, m represents the average number of linker-drug (L-D) units coupled to the Ab, and the range of m is 1-8, preferably m is 4.

28. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, the polynucleotide molecule of claim 10, the expression vector of claim 11, the host cell of claim 12, or the immunoconjugate of any one of claims 14-27, and a pharmaceutically acceptable carrier or excipient.

29. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, the polynucleotide molecule of claim 10, the expression vector of claim 11, the host cell of claim 12, the immunoconjugate of any one of claims 14-27, or the pharmaceutical composition of claim 28 in the preparation of a drug for treating and / or preventing FGFR2b-mediated diseases or disorders, preferably the disease or disorder is cancer; more preferably, the cancer is selected from endometrial cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, bladder cancer, and cholangiocarcinoma.

30. An article of manufacture comprising a first container containing a composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, the polynucleotide molecule of claim 10, the expression vector of claim 11, the host cell of claim 12, the immunoconjugate of any one of claims 14-27, or the pharmaceutical composition of claim 28.

31. The article of manufacture as claimed in claim 31, wherein: The article further includes a second container, which contains a pharmaceutically acceptable buffer; or The article further includes a container containing a diluent, a buffer, or a control antibody for detection.

32. A method for detecting the presence of FGFR2b in a sample using the antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a detection composition comprising the antibody or antigen-binding fragment thereof, comprising the step of contacting the antibody or antigen-binding fragment thereof or the detection composition comprising the antibody or antigen-binding fragment thereof with the sample.

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