Anti-human FGFR2b antibody and use thereof

Abstract

The present invention belongs to the field of biomedicine. Provided are an anti-human FGFR2b antibody and the use thereof. Further provided are a nucleic acid molecule encoding the antibody, a vector, a host cell and a composition, and the use thereof for treating a disease.

Description

Anti-human FGFR2b antibody and its uses

[0001] This invention claims priority to Chinese patent application filed on October 14, 2024 (application number: CN202411436447.3, invention title: Anti-human FGFR2b antibody and its use) and Chinese patent application filed on November 6, 2024 (application number: CN202411585627.8, invention title: Anti-human FGFR2b antibody and its use), the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine and relates to anti-human FGFR2b antibodies and their applications, as well as nucleic acid molecules, vectors, host cells, compositions encoding the antibody and their applications in disease treatment. Technical Background

[0003] Tumors are new growths formed by the proliferation of local tissue cells under the influence of various tumorigenic factors. Growth factors are closely related to tumor formation and proliferation, and growth factor receptors on the tumor surface have always been popular targets for tumor-targeted drugs. Among them, the most representative targets are the epidermal growth factor receptor (EGFR) family, and marketed antibody drugs targeting EGFR include Cetuximab and Panitumumab. Fibroblast growth factor receptors (FGFRs) involve multiple signaling pathways with different functions downstream, and abnormalities in the FGFR signaling pathway are an important cause of the development and progression of various tumors. FGFRs are receptor tyrosine kinases composed of an extracellular ligand domain and an intracellular tyrosine kinase domain, including four subtypes: FGFR1, FGFR2, FGFR3, and FGFR4. Under normal conditions, FGFR participates in physiological processes including embryonic development, metabolic homeostasis, tissue repair and regeneration; however, once the FGFR gene undergoes amplification, fusion or activating mutation, it will lead to excessive activation of FGFR signaling, thereby promoting cell proliferation, survival, tumor drug resistance, angiogenesis and, more importantly, immune escape.

[0004] Based on the extracellular IgIII domain classification of FGFR2, FGFR2 can be divided into FGFR2-III b (FGFR2b) and FGFR2-III c (FGFR2c). FGFR2-III c is mainly expressed in stromal cells and can bind FGF1 and FGF2 with high affinity, while FGFR2b is mainly expressed in epithelial cells and can bind FGF1 and KGF family cell growth factors (FGF7, FGF10, FGF22) with high affinity. FGFR2b is the only receptor for KGF family cell growth factors and is also known as KGFR. KGF family cell growth factors and FGFR2b are highly expressed in various solid tumors, and tumor cell proliferation, survival, and development are mainly driven by FGFR2 gene amplification and hyperactivation of this signaling pathway, making them markers of poor prognosis in many tumors. It has been reported that approximately 30% of HER2-negative gastroesophageal cancer patients overexpress FGFR2b, making it a highly promising therapeutic target. Currently, the FGFR2-targeting antibody Bemarituzumab has entered phase 3 clinical trials. In phase 2 clinical trials, its efficacy in combination with chemotherapy drugs showed better treatment effects in advanced gastric cancer and gastroesophageal junction cancer with FGFR2 overexpression or amplification. Compared with the chemotherapy control group, the use of Bemarituzumab in combination with chemotherapy increased the median progression-free survival (PFS) of patients with advanced gastric cancer from 7.4 months to 9.5 months. More importantly, the overall survival (OS) of patients (including gastric cancer and gastroesophageal junction (GEJ) cancer) was also significantly improved, confirming the effectiveness of FGFR2b as a new therapeutic target for gastric cancer.

[0005] However, because FGFR2 plays a crucial role in maintaining cell development and differentiation, it is expressed at low levels in various normal human tissues. While high-affinity antibody drugs targeting FGFR2 effectively target tumor tissues, there is a certain probability that they may also bind to normal tissues. Currently, the main mechanism of FGFR2-targeted antibody therapy for tumors relies on the Fc-mediated killing of target cells by immune cells. Therefore, these antibody drugs can cause a certain degree of specific targeting of non-tumor tissues, leading to side effects.

[0006] Therefore, new anti-human FGFR2b antibodies are still in urgent need. Summary of the Invention

[0007] The inventors of this application have screened for FGFR2b-specific binding antibodies using hybridoma technology, which exhibit good binding activity in vitro. In some embodiments, the antibodies of this invention can specifically kill FGFR2b-expressing cells via ADCC; in some embodiments, the antibodies of this invention maintain their in vitro biological activity after humanization; in some embodiments, a pH-dependent anti-human FGFR2b antibody is constructed using antibody engineering, exhibiting low affinity for the FGFR2b antigen under neutral pH conditions (e.g., pH 7.4) and high affinity for FGFR2 under acidic pH conditions (e.g., pH 6.0). The pH-dependent anti-human FGFR2b antibody of this invention can more specifically target target cells in the tumor microenvironment, reducing binding to FGFR2b-expressing cells in normal tissues, thereby reducing potential side effects and providing more and better options for clinical treatment of this type of target-abnormal solid tumors.

[0008] In a first aspect, the present invention provides an anti-human FGFR2b antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to HCDR1-3 in the VH having at least 80% sequence identity with those shown in or having at least 80% sequence identity with those in SEQ ID NO:53, 23, 25, 11-15, 1, or 9; and,

[0009] The LCDR1, LCDR2, and LCDR3 of the light chain variable regions are identical to LCDR1-3 in the VL shown in SEQ ID NO:54, 24, 26, 16-18, 2, or 10, or have at least 80% sequence identity with SEQ ID NO:54, 24, 26, 16-18, 2, or 10.

[0010] In addition, the present invention provides an anti-human FGFR2b antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region is as shown in SEQ ID NO:53, 23, 25, 11-15, 1 or 9, or has at least 80% sequence identity with SEQ ID NO:53, 23, 25, 11-15, 1 or 9, and the light chain variable region is as shown in SEQ ID NO:54, 24, 26, 16-18, 2 or 10, or has at least 80% sequence identity with SEQ ID NO:54, 24, 26, 16-18, 2 or 10.

[0011] In a second aspect, the present invention provides an anti-human FGFR2b antibody, said antibody having a pH-dependent binding function to the human FGFR2b antigen.

[0012] In addition, the present invention provides an anti-human FGFR2b antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence as shown in SEQ ID NO:56 or having at least 80% sequence identity with SEQ ID NO:56, and / or the light chain comprises a sequence as shown in SEQ ID NO:57 or having at least 80% sequence identity with SEQ ID NO:57; the heavy chain comprises a sequence as shown in SEQ ID NO:49 or having at least 80% sequence identity with SEQ ID NO:49, and / or the light chain comprises a sequence as shown in SEQ ID NO:50 or having at least 80% sequence identity with SEQ ID NO:50; or the heavy chain comprises a sequence as shown in SEQ ID NO:51 or having at least 80% sequence identity with SEQ ID NO:51, and / or the light chain comprises a sequence as shown in SEQ ID NO:52 or having at least 80% sequence identity with SEQ ID NO:52.

[0013] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-human FGFR2b antibody described in any of the preceding claims.

[0014] In a fourth aspect, the present invention provides a carrier comprising the nucleic acid described in any of the preceding claims.

[0015] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid or vector described in any of the preceding claims.

[0016] In a sixth aspect, the present invention provides a method for preparing an antibody that binds to human FGFR2b, comprising culturing a host cell as described in any of the preceding claims under conditions suitable for expressing the antibody, and recovering the antibody from the cultured host cell culture.

[0017] In a seventh aspect, the present invention provides a multispecific molecule comprising the anti-human FGFR2b antibody as described in any of the preceding claims.

[0018] In an eighth aspect, the present invention provides a conjugate comprising the anti-human FGFR2b antibody as described in any of the preceding claims, and a conjugation portion.

[0019] In a ninth aspect, the present invention provides a pharmaceutical composition comprising an anti-human FGFR2b antibody, a multispecific molecule, a conjugate, a nucleic acid molecule, a carrier or cell, and a pharmaceutically acceptable carrier as described in any of the preceding claims.

[0020] In a tenth aspect, the present invention provides a method for treating tumors, the method comprising administering to a subject in need a therapeutically effective amount of any of the preceding anti-human FGFR2b antibodies, multispecific molecules, conjugates, nucleic acid molecules, carriers, cells, or pharmaceutical compositions.

[0021] In an eleventh aspect, the present invention provides the use of the anti-human FGFR2b antibody, multispecific molecule, conjugate, nucleic acid molecule, carrier, cell or pharmaceutical composition described in any of the preceding claims in the preparation of a medicament.

[0022] In a twelfth aspect, the present invention provides an anti-human FGFR2b antibody, multispecific molecule, conjugate, nucleic acid molecule, carrier, cell, or pharmaceutical composition as described in any of the preceding claims, for use as a medicament. Attached Figure Description

[0023] Figure 1: Results of enzyme-linked immunosorbent assay (ELISA) for detecting the binding of FGFR2b antibody to human FGFR2b protein. The absorbance at 450 nm (OD450) is used as the Y-axis. The results are expressed as the mean ± SD of two replicates. The isotype control (IgG1 isotype control unrelated to FGFR2b antigen) is a negative control.

[0024] Figure 2: Results of enzyme-linked immunosorbent assay (ELISA) for detecting the binding of FGFR2b antibody to monkey FGFR2b protein. The absorbance at 450 nm (OD450) is used as the Y-axis. The results are expressed as the mean ± SD of two replicates. The isotype control (IgG1 isotype control unrelated to FGFR2b antigen) is a negative control.

[0025] Figure 3: Results of enzyme-linked immunosorbent assay (ELISA) to detect the binding of FGFR2b antibody to mouse FGFR2b protein. The absorbance at 450 nm (OD450) is used as the Y-axis. The results are expressed as the mean ± SD of two replicates. The isotype control (IgG1 isotype control unrelated to FGFR2b antigen) is a negative control.

[0026] Figure 4: Results of enzyme-linked immunosorbent assay (ELISA) to detect the binding of FGFR2b antibody to human FGFR2c protein. The natural ligand of FGFR2, FGF1, was used as a positive control. The absorbance at 450 nm (OD450) was used as the Y-axis. The results are expressed as the mean ± SD of two replicates.

[0027] Figure 5 shows the results of flow cytometry assays to determine the binding of FGFR2b antibody to 293T-hFGFR2b cells, with the Isotype control (IgG1 isotype control unrelated to FGFR2b antigen) serving as the negative control.

[0028] Figure 6 shows the results of flow cytometry assays to determine the binding of FGFR2b antibody to SNU-16 cells, with the Isotype control (IgG1 isotype control unrelated to FGFR2b antigen) serving as the negative control.

[0029] Figure 7: The results of flow cytometry assay for the binding of FGF1 ligand to 293T-hFGFR2b cells by FGFR2b antibody blocking FGF1 ligand were measured. Bemarituzumab antibody, which is known to have FGF1 blocking activity, was used as a positive control. The amount of FGF1 on the cells was measured by flow cytometry and expressed as average fluorescence intensity (Y-axis).

[0030] Figure 8 shows the results of flow cytometry analysis of the FGFR2b antibody blocking the binding of FGF7 ligand to 293T-hFGFR2b cells. Bemarituzumab antibody, which is known to have FGF7 blocking activity, was used as a positive control. The amount of FGF7 on the cells was measured by flow cytometry and expressed as average fluorescence intensity (Y-axis).

[0031] Figure 9: Results of ADCC activity assay for FGFR2b antibody using luciferase reporter gene assay. Effector cells overexpressing CD16a-NFAT reporter gene (Jurkat-CD16a-NFAT-Luc) and target cells overexpressing human FGFR2b protein (293T-hFGFR2b) were co-cultured. Different concentrations of chimeric antibody were added to the culture system, and after co-incubation, luciferase assay reagent was added. The chemiluminescence intensity (Y-axis) was recorded to reflect the activation intensity of CD16a under these conditions, thus representing the ADCC activity of the antibody. The isotype control (IgG1 isotype control unrelated to FGFR2b antigen) served as the negative control.

[0032] Figure 10: Determination of the ADC killing activity of FGFR2b antibody against target cells using an indirect method. Using 293T-hFGFR2b cells as the target cells, different concentrations of chimeric antibody and a fixed concentration of anti-human IgG Fc secondary antibody conjugated with MMAE were added to the culture system. The isotype control (IgG1 isotype control unrelated to the FGFR2b antigen) served as the negative control, and the background control was the addition of only MMAE-anti-human IgG Fc secondary antibody. After co-incubation, the number of viable cells in each well was detected using a luciferase cell viability assay kit, expressed as chemiluminescence intensity (Y-axis).

[0033] Figure 11 shows the computer-predicted structure of the complex of FGFR2b chimeric antibody and FGFR2b antigen, and the amino acid sites that can be used for pH-dependent modification.

[0034] Figure 12: The results of enzyme-linked immunosorbent assay (ELISA) for detecting the binding of FGFR2b antibodies with different pH-dependent designs and mutations to human FGFR2b protein are shown. The absorbance at 450 nm (OD450) is used as the Y-axis, and the results are expressed as the mean ± SD of two replicates.

[0035] Figure 13: The results of flow cytometry assays of the binding of humanized and pH-dependent hybrid mutant FGFR2b molecules to 293T-hFGFR2b cells. The amount of antibody on the cells was measured by flow cytometry and is expressed as average fluorescence intensity (Y-axis).

[0036] Figure 14 shows the binding kinetics and affinity of humanized and pH-dependent combined mutant FGFR2b molecules to hFGFR2b protein as determined by SPR. Figures 14A and 14B are the fitting curves of M15-cu08-B008 at pH 7.4 and pH 6.0, respectively; Figures 14C and 14D are the fitting curves of M15-cu23-B008 at pH 7.4 and pH 6.0, respectively. In Figures 14B and 14D, the light-colored lines represent the actual detected signal lines, and the dark, smooth lines represent the fitted curves. The upper line represents the curve with an antibody concentration of 20 nM, and the lower line represents the curve with an antibody concentration of 4 nM. In Figures 14A and 14C, due to weak binding signals, fitting is not possible, therefore there are no fitted curves, only two light-colored lines representing the actual detected signals (the upper line represents the actual detected signal line with an antibody concentration of 100 nM, and the lower line represents the actual detected signal line with an antibody concentration of 25 nM).

[0037] Figure 15 shows the results of flow cytometry assays of the binding of anti-human FGFR2b antibody to 293T-hFGFR2b cells. The amount of antibody on the cells was measured by flow cytometry and expressed as average fluorescence intensity (Y-axis).

[0038] Figure 16 shows the results of flow cytometry assays of the binding of anti-human FGFR2b antibody to KATO-III cells. The amount of antibody on the cells was measured by flow cytometry and expressed as average fluorescence intensity (Y-axis).

[0039] Figure 17 shows the results of flow cytometry assays to determine the binding of anti-human FGFR2b antibody to SNU-16 cells. The amount of antibody on the cells was measured by flow cytometry and expressed as average fluorescence intensity (Y-axis).

[0040] Figure 18 shows the results of flow cytometry analysis of the endocytosis of anti-human FGFR2b antibody by 293T-hFGFR2b cells at 37°C. The amount of remaining antibody on the cell surface was measured by flow cytometry as the incubation time increased (X-axis), and the average fluorescence intensity (Y-axis) was used to reflect the endocytosis effect of the cells.

[0041] Figure 19 shows the results of flow cytometry analysis of the endocytosis of anti-human FGFR2b antibody by KATO-III cells at 37°C. The amount of remaining antibody on the cell surface was measured by flow cytometry as the incubation time increased (X-axis), and the average fluorescence intensity (Y-axis) was used to reflect the endocytosis effect of the cells.

[0042] Figure 20 shows the results of flow cytometry analysis of the endocytosis of anti-human FGFR2b antibody by SNU-16 cells at 37°C. The amount of remaining antibody on the cell surface was measured by flow cytometry as the incubation time increased (X-axis), and the average fluorescence intensity (Y-axis) was used to reflect the endocytosis effect of the cells.

[0043] Figure 21 shows the results of flow cytometry assays to determine the binding of ADC molecules to 293T-hFGFR2b cells. The amount of antibody on the cells was measured by flow cytometry and is expressed as average fluorescence intensity (Y-axis). Figures 21A and 21B show the results under pH 7.4 and pH 6.0 conditions, respectively. The isotype control (IgG1 isotype control unrelated to FGFR2b antigen) serves as a negative control.

[0044] Figure 22 shows the results of flow cytometry assays to determine the binding of ADC molecules to SNU-16 cells. The amount of antibody on the cells was measured by flow cytometry and expressed as average fluorescence intensity (Y-axis). Figures 22A and 22B show the results under pH 7.4 and pH 6.0 conditions, respectively. The isotype control (IgG1 isotype control unrelated to FGFR2b antigen) serves as the negative control.

[0045] Figure 23: Results of flow cytometry assays to determine the binding of ADC molecules to FGF7 ligands in 293T-hFGFR2b cells. Bemarituzumab antibody, known to have FGF7 blocking activity and no significant pH-dependent binding, was used as a positive control. The amount of FGF7 on the cells was measured by flow cytometry and expressed as average fluorescence intensity (Y-axis). Figures 23A and 23B show results under pH 7.4 and pH 6.0 conditions, respectively. The isotype control (IgG1 isotype control unrelated to the FGFR2b antigen) served as a negative control.

[0046] Figure 24: Results of flow cytometry assay for the binding of ADC molecules to FGF10 ligand in 293T-hFGFR2b cells. Bemarituzumab antibody, known to have FGF10 blocking activity, was used as a positive control. The amount of FGF10 on the cells was measured by flow cytometry and expressed as mean fluorescence intensity (Y-axis). The isotype control (IgG1 isotype control unrelated to the FGFR2b antigen) served as a negative control.

[0047] Figure 25: This shows the curves of how ADC molecules inhibit tumor growth in a SNU-16 gastric cancer-bearing mouse model.

[0048] Figure 26: Body weight curve of ADC molecule in SNU-16 gastric cancer-bearing mouse model. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. However, this description should not be construed as limiting the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions according to the product instructions are followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products obtained through commercial purchase. Unless expressly defined in this disclosure, the terminology used herein has the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0050] The terms “a / an” and “an” include plural references unless the context clearly indicates otherwise. For example, “an antibody” means one or more antibodies. The terms “first” and “second” are used for descriptive purposes only and should not be construed as implying relative importance or the number of technical features indicated. The term “multiple” means at least two, such as 2, 3, etc., unless it is explicitly stated in the text that this is not the case.

[0051] The terms “include” or “have” are understood to mean “include” rather than “exclusive” or “exhaustive”; that is, “includes but is not limited to”. For example, “includes A” means “includes A, but is not limited to A”.

[0052] The term "and / or" has both the meanings of "and" and "or," referring to each or a combination of specific characteristics. For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0053] The terms “optional,” “optionally,” “optionally,” and “optionally” refer to the occurrence or non-occurrence of the event or condition described following the word, that is, including both the occurrence and non-occurrence of the event or condition. For example, “optionally, the antibody contains a constant region” means that the antibody may or may not contain a constant region.

[0054] The term "about" refers to a range of values ​​that includes a specific value and that a person skilled in the art would reasonably consider similar to that specific value. In some embodiments, the term "about" refers to within the standard error of a measurement generally accepted in the art. For example, in some embodiments, "about" refers to + / - 10% of a specific value; in other embodiments, "about" refers to + / - 5% of a specific value.

[0055] The range “X to XX” in this document should be considered to specifically disclose all possible subranges and the individual values ​​within that range. For example, the description “1 to 4” should be considered to explicitly disclose the subranges: 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, 3 to 4, etc., and the individual numbers within that range: 1, 2, 3, or 4.

[0056] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 ((1968)).

[0057] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0058] The term "amino acid mutation" refers to amino acid substitution (or replacement), deletion, insertion, and modification. Those skilled in the art can substitute, delete, insert, and / or modify amino acids as needed to obtain new constructs, provided the construct has the desired function. Amino acid deletions and insertions can be performed at the amino terminus (N-terminus), middle, and / or carboxyl terminus (C-terminus) of the amino acid sequence. In some embodiments, the amino acid mutation is an amino acid substitution; in some embodiments, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having a different structure and / or chemical properties. In some embodiments, the amino acid mutation is a conserved amino acid substitution, i.e., replacing one amino acid with another amino acid having similar structure and / or chemical properties. Amino acid substitutions can be performed using non-naturally occurring amino acids or derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be performed using genetic or chemical methods known in the art; for example, genetic methods include site-directed mutagenesis, PCR, gene synthesis, etc., and methods other than genetic engineering that alter the side chain groups of amino acids include, for example, chemical modification.

[0059] The term "antibody" refers to a protein molecule capable of specifically binding to an antigen. The term "antibody" is used in the broadest sense in this disclosure and encompasses a variety of antibody structures, including but not limited to: monoclonal / polyclonal antibodies, monospecific / multispecific antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies), murine / chimeric / humanized / human antibodies, full-length antibodies / antigen-binding fragments (also called antigen-binding moieties), etc., provided they exhibit the desired antigen-binding activity.

[0060] "Natural antibodies" are naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N to the C-terminus, each heavy chain of a natural IgG antibody has a heavy chain variable region (VH), followed by a heavy chain constant region (CH), which includes three constant domains (CH1, CH2, and CH3); similarly, from the N to the C-terminus, each light chain has a light chain variable region (VL), followed by a light chain constant region (CL). Based on whether an antibody contains α, δ, ε, γ, and μ heavy chains, antibodies can be classified into five isotypes: IgA, IgD, IgE, IgG, and IgM. Isotype antibodies can be further divided into different subtypes. For example, the IgG isotype includes four subtypes: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), and IgG4 (γ4 heavy chain). The IgA isotype is divided into two subtypes: IgA1 (α1 heavy chain) and IgA2 (α2 heavy chain).

[0061] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this disclosure to refer to antibodies that have a structure similar to that of natural antibodies or that have an Fc domain.

[0062] The term "isolated antibody" refers to an antibody that has been separated from its native components. In some embodiments, the antibody is purified to a purity of at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), which can be purified and determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0063] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in antibody binding to the antigen. Natural antibodies include a heavy chain variable region (VH) and a light chain variable region (VL), each of which contains four frame regions (FR) and three hypervariable regions (also known as complementarity-determining regions, HVR, or CDR).

[0064] The terms "complementarity-determining region," "hypervariant region," or "CDR" refer to the main region within the variable region that facilitates binding to the antigen; "framework," "frame region," or "FR" refers to the structural domains within the antibody variable region other than the CDR residues. Each VH and VL typically consists of three CDRs and four FRs arranged in the following order (from the amino terminus to the carboxyl terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable regions of both the heavy and light chains contain three CDRs (CDR1, CDR2, and CDR3), with the three CDRs of the heavy chain labeled HCDR1, HCDR2, and HCDR3, and the three CDRs of the light chain labeled LCDR1, LCDR2, and LCDR3. The boundaries of CDRs can be defined according to known numbering systems in the field, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the Abm numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272), and the Contact numbering system (MacCallum, RM, Martin, ACR, & Thornton, JM (1996) Antibody-antigen Interactions: Contact Analysis and Binding). Site Topography. Journal of Molecular Biology, 262(5), 732-745.) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody variable region, those skilled in the art can readily determine the CDR according to the numbering system.Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0065] The term "constant region" or "constant domain" refers to the carboxyl-terminal portion of the antibody's light and heavy chains, which does not directly participate in antibody-antigen binding. Compared to VH and VL, the constant region has a relatively conserved amino acid sequence. While the constant domain typically does not directly participate in antibody-antigen binding, it exhibits various effector functions.

[0066] The term "light chain" includes the variable region (VL) and the constant region (CL) of the light chain. The VL is located at the amino terminus of the light chain, and the CL is located at the carboxyl terminus of the light chain. The light chain can be a κ chain or a λ chain, etc.

[0067] The term "heavy chain" includes the variable region (VH) and the constant region (CH). The variable region is located at the amino terminus of the heavy chain, and the constant region is located at the carboxyl terminus. The constant region of the IgG antibody heavy chain includes three constant region domains: CH1, CH2, and CH3. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgD, IgM, and IgE, etc.

[0068] The term "antigen-binding fragment" refers to a portion containing a complete antibody (which is not the complete antibody itself) that specifically binds to the antigen bound by the complete antibody. Examples of antigen-binding fragments include, but are not limited to: Fv, Fab, Fab', Fab'-SH, F(ab')2, dsFv, (dsFv)2, single-chain Fab (scFab), single-chain antibody (scFv), diabody, and multispecific antibodies formed from antigen-binding fragments.Among them, "Fab" is an antibody fragment composed of VL, VH, CL and CH1 domains; "Fv" is an antibody fragment composed of the VL and VH domains of a single arm of the antibody; "Fab'" is a Fab fragment containing part of the hinge region; it is usually obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and a heavy chain Fd fragment (composed of VH and CH1 domains); "F(ab')2" is a bivalent fragment containing two Fab' fragments connected by disulfide bonds in the hinge region; "scFab" is a polypeptide composed of VH, CH1, VL, CL, and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; "scFv" is a single-chain protein containing a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are linked by a peptide linker and can be expressed as a single-chain multi-chain protein. Peptides, scFvs, typically retain the specificity of the intact antibody (parent antibody) from which they originate. The N-terminus to C-terminus of an scFv may contain: a) VL-linker-VH or b) VH-linker-VL. In some embodiments, a disulfide bond may also exist between the VH and VL of the scFv. In some implementations, scFvs can form di-scFvs (i.e., two or more individual scFvs linked together to form an antibody). In some implementations, scFvs can form (scFv)2 (i.e., two or more individual scFvs linked in parallel to form an antibody). (The antibody formed); "dsFv" is a disulfide bond-stabilized Fv fragment; (dsFv)2 is a dimerized dsFv; "Fab'-SH" is a cysteine ​​residue in the hinge region of the Fab' fragment carrying a free thiol group; "diabody" refers to the expression of VH and VL domains on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0069] The term "antibody effector function" refers to those biological activities attributable to the Fc region of an antibody. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0070] The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling the cytotoxic effector cells to specifically bind to target cells carrying the antigen and kill the target cells with cytotoxins. Antibodies “arm” the cytotoxic cells, thereby exerting their cytotoxic effect. NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Further FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet's *Annu. Rev. Immunol.* 9:457-92 (1991). To assess the ADCC activity of a target molecule (e.g., an antibody), it can be evaluated via in vitro ADCC assays (e.g., as described in U.S. Patent Nos. 5,500,362 or 5,821,337), using effector cells such as peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. The ADCC activity of a target molecule (e.g., an antibody) can also be assessed in vivo, for example, in animal models (such as those disclosed in Clynes et al., (USA) 95:652-656 (1998)).

[0071] The term “antibody-dependent phagocytosis” or (“ADCP”) refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0072] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors on leukocytes (e.g., CR3).

[0073] The term "mouse antibody" refers to an antibody whose variable region and constant region are both derived from immunoglobulins of a mouse lineage (e.g., mouse or rat).

[0074] The term "chimeric" refers to antibodies in which a portion of the heavy and / or light chains is derived from a specific species, while the remainder of the heavy and / or light chains is derived from other species. For example, a human-mouse chimeric antibody can be constructed by combining the variable region of a mouse antibody with the constant region of a human antibody.

[0075] The term "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulins. For example, a humanized antibody can be constructed by retaining the CDR region of a non-human antibody and replacing the rest of the antibody with the framework and constant regions of a human antibody. In some embodiments, in a humanized antibody, some, most, or all of the amino acids outside the CDR domain have been replaced by amino acids derived from human immunoglobulins, while some, most, or all of the amino acids in one or more CDR regions remain unchanged. Generally, minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted, as long as they preserve the antibody's ability to bind to a specific antigen.

[0076] The terms “affinity” and “binding strength” refer to the overall strength of the non-covalent interaction between the antibody binding site and the antigen. Unless otherwise specified, “affinity” as used in this disclosure refers to internal binding affinity, which reflects a 1:1 interaction between the antibody and the antigen. Affinity is typically expressed as a dissociation constant (KD). Affinity can be measured using conventional methods known in the art. The terms “kassoc” or “ka” refer to the association rate of the antibody-antigen interaction, and the terms “kdis” or “kd” refer to the dissociation rate of the antibody-antigen interaction. The term “KD” refers to the dissociation constant, which is the ratio of kd to ka (i.e., kd / ka), and is typically expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. Methods for determining antibody KD include using a biosensing system, such as a system measuring surface plasmon resonance, or by solution equilibrium titration (SET).

[0077] The term "affinity-matured antibody" refers to an antibody that has one or more amino acid residue changes in one or more CDRs, resulting in improved affinity for the antigen compared to the parent antibody. In some embodiments, affinity-matured antibodies have nanomolar or picomolar affinity for the target antigen. Affinity-matured antibodies can be generated by methods known in the art (Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation through VH and VL domain shuffling; the following literature describes random mutagenesis of CDR and / or framework residues: Barbas et al., PNAS, 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995) and Hawkins et al., J. Mol. Biol. 226:889-896 (1992)).

[0078] The term "monoclonal antibody" refers to a substantially homogeneous group of antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence (except for the possible small number of naturally occurring mutations). In contrast, polyclonal antibodies typically contain different antibodies with variable domains having different amino acid sequences, and they are usually specific to different epitopes and / or different antigens. "Monoclonal" indicates an antibody obtained from a substantially homogeneous group of antibodies. Monoclonal antibodies can be produced by methods known in the art, for example, by hybridoma methods (Kohler et al., (1975) Nature 256:495), by recombinant DNA methods (see, for example, US Patent No. 4,816,567), by isolation methods from phage antibody libraries (Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597), and also by using transgenic animals containing all or part of the human immunoglobulin loci (see Presta (2005) J. Allergy Clin. Immunol. 116:731).

[0079] The term "antigen" refers to a protein that can selectively bind to an antibody. An antigen may have one or more epitopes that interact with different antibodies.

[0080] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody. Epitopes are typically antigenic determinants consisting of specific chemical groups with a defined composition and structure. Epitopes can be formed from consecutive amino acid residues (linear epitopes) or from discontinuous amino acid residues (conformal epitopes), such as spatially close discontinuous amino acid residues formed due to antigen folding. In some embodiments, an epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acid residues in a unique spatial conformation. Epitopes can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural assays (e.g., nuclear magnetic resonance spectroscopy). Antibodies that bind to specific epitopes (i.e., those that bind to the same epitope) can be obtained using methods known in the art, including but not limited to, alanine scanning, Western blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496) and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0081] When “competition” is used to describe the competition between antigen-binding proteins (e.g., antibodies) for the same epitope, it means that antigen-binding proteins competitively bind to a common antigen, which can usually be determined by measuring that the antigen-binding protein to be detected (e.g., antibody) inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., reference antibody) to the antigen. Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including but not limited to: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeled sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIA of I-125 (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15), etc. In some embodiments, the binding of the reference antibody to the antigen is inhibited by at least 40% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 90%, 95%, 97%, or 98% or more) by an antibody that competitively binds to it. In some embodiments, to determine whether the test antibody binds to the same epitope as the reference antibody, the ability of the reference antibody to bind to the antigen can be detected under saturation conditions. For example, after removing excess reference antibody, the ability of the test antibody to bind to the antigen can be evaluated. If the test antibody is able to bind to the antigen after saturation binding of the reference antibody, then the test antibody binds to a different epitope than the reference antibody; however, if the test antibody is unable to bind to the antigen after saturation binding of the reference antibody, then the test antibody may bind to the same epitope as the reference antibody.

[0082] The term "specific binding" or "specifically bound" refers to a non-random binding between two molecules. For example, an antibody binds to the antigen or epitope it corresponds to with a higher affinity than it binds to other antigens or epitopes. Typically, antibodies bind with an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or an epitope within the antigen. In some embodiments, the KD of antibody binding to an antigen is 10%, 1%, or less of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). The KD value can be measured using methods known in the art, such as by... Surface plasmon resonance assay. Antibodies that specifically bind to antigens or epitopes within antigens may exhibit cross-reactivity with other related antigens, for example, cross-reactivity with corresponding antigens from other species (such as humans or monkeys, such as cynomolgus (Cyno) and chimpanzee (Chimp)) or common marmoset (Callithrix jacchus).

[0083] The terms "anti-human FGFR2b antibody" and "antibody binding to human FGFR2b" refer to antibodies capable of binding to human FGFR2b with sufficient affinity. In one embodiment, the degree of binding to an unrelated, non-human FGFR2b protein is less than at least 10%, 1%, or less of the antibody's binding to human FGFR2b, said binding can be achieved through... Measured by surface plasmon resonance assay. In some embodiments, the antibody binding to human FGFR2b has the following dissociation constant (KD): < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10). -8 M or smaller, such as 10 -8 M to 10 -12 M, for example, 10 -9 M to 10 -10 M). In some embodiments, anti-human FGFR2b antibodies bind to conserved antigenic epitopes in FGFR2b from different species.

[0084] As used in this article, "pH-dependent binding" refers to the ability of a protein (e.g., an antibody) to bind with a higher binding affinity to its ligand (e.g., the human FGFR2b antigen protein) at an acidic pH than at a neutral pH.

[0085] As used herein, “acidic pH” means a pH of about 6.6 or lower (e.g., pH 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5 or lower, or any value in between); “neutral pH” means a pH of about 7.0 to about 7.6 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or any value in between).

[0086] The terms "multispecific antibody" and "multispecific molecule" refer to antibody molecules that can bind to multiple (two or more) different antigenic epitopes of the same antigen or multiple (two or more) different antigens.

[0087] The term "conjugate" refers to an antibody chemically or biologically linked to another pharmaceutical agent (e.g., including but not limited to: protein tags, detectable labels, or therapeutic agents). Examples of conjugates include, for example, conjugates constructed by linking the anti-human FGFR2b antibody of the present invention with a cytotoxic drug via a linker. Pharmaceutical agents may be, for example, antitumor compounds, chemotherapeutic agents, molecularly targeted drugs, immune activators, immunosuppressants, toxins, photosensitizing substances, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, antibiotics, hormones, etc. Protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select appropriate protein tags (e.g., purification tags, detection tags, or tracer tags) according to the desired purpose. The conjugated portion is selected from detectable labels, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acrid esters), and magnetic beads (e.g., The label may include pyrometric markers such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.) and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, such labels are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).

[0088] In some embodiments, the coupling portion is selected from therapeutic agents, such as antitumor drugs or immunosuppressants.

[0089] In some embodiments, the conjugate is an antibody-drug conjugate (ADC) comprising the anti-human FGFR2b antibody and a conjugation moiety consisting of a cytotoxic drug, the conjugation moiety being linked to the antibody via a linker.

[0090] The terms "connector unit," "connector," and "linker" refer to a chemical structural fragment or bond that is linked at one end to an antibody and at the other end to a drug. A connector can be a cleavable connector (which can break within the target cell to release the cytotoxic drug) or an incleavable connector. Cleavable connectors can be, for example, acid-labile connectors (e.g., hydrazone-containing connectors), protease-sensitive connectors (e.g., peptidase-sensitive connectors), photostable connectors, dimethyl connectors, or disulfide-containing connectors (Chari et al., Cancer Research 52:127-131 (1992); US Patent US5208020). In some embodiments, the connector is an incleavable connector (e.g., SMCC). In some embodiments, the connector is a disulfide connector, a hydrazone connector, or a protease-cleavable connector. In some embodiments, the protease-cleavable adapter is selected from cathepsin B substrate adapters (e.g., adapters containing Val-Cit, Val-Ala, or Gly-Gly-Phe-Gly), β-glucuronidase substrate adapters, β-galactosidase substrate adapters, or sulfatase substrate adapters. In some embodiments, the linker may have a reactive group capable of reacting with certain amino acid residues before being attached to the antibody, thereby enabling attachment to the antibody; for example, the linker may have a functional group capable of reacting with free cysteine ​​present on the antibody to form a covalent bond, exemplary reactive functional groups include maleimide, haloacetamide, α-haloacetyl, reactive esters such as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydride, acyl chloride, sulfonyl chloride, isocyanate and isothiocyanate (see, for example, Klussman et al. (2004), Bioconjugate Chemistry 15(4):765-7730); or the linker may have a functional group capable of reacting with electrophilic groups present on the antibody, exemplary electrophilic groups including but not limited to aldehyde and ketone carbonyl groups including but not limited to acyl hydrazides, oximes, amino groups, hydrazine, thiourea, hydrazide carboxylates and aryl hydrazides, etc. In some embodiments, the connector unit comprises one or more of the following structural units: the connector unit L is a linker comprising one or more of the following structures: maleimide hexanoyl (MC), valine-citrulline (Val-Cit), p-aminobenzyloxycarbonyl (PAB), glycine-glycine-phenylalanine-glycine (GGFG), maleimide, carbonyl, amino, amide, aminoacyl, -(PEG)n- (where n is independently selected from an integer from 1 to 20), C 1-20 Alkylene, C 1-20Heteroalkyl, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimide propionyl (MP), methylsulfonylpyrimidinyl, valine-alanine (Val-Ala), N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), dimethylethylenediamine (DMED), N-succinimide-4-(2-pyridinylthio) The options include valerate (SPP), N-succinimide-4-(N-maleimide-methyl)-cyclohexane-1-carboxylate, N-succinimide-(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-3-(pyridin-2-yldithio)-propionate (SPDP), or acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC). In some embodiments, the linker may include: an extension unit (attaching the antibody to the linker portion, such as maleimide, MC, MP, etc.), a protease cleavage unit (e.g., an amino acid that imparts protease cleavage activity to the linker, such as Val-Cit, Val-Ala), and a spacer unit (attaching the antibody to the drug portion directly or via the extension unit or protease cleavage unit, such as PAB or peptide bond, etc.). The connector can be synthesized by methods known in the art, such as those described in US20050238649A1 or US10973924B2.

[0091] The term "alkyl" refers to a straight-chain or branched monovalent saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1 to 12, for example, 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) carbon atoms. For example, as used herein, the term "C 1-6 "Alkyl" refers to an alkyl group having 1 to 6 carbon atoms, for example: "C 2-6 Alkyl", C 2-5 Alkyl", C 1-4 "alkyl" and "C" 1-3 Alkyl group. "C" 1-6 Examples of "alkyl" include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl. The term "C" 1-4 "Alkyl" refers to an alkyl group having 1 to 4 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl). The term "C"... 1-3 "Alkyl" refers to an alkyl group having 1 to 3 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl).

[0092] The term "alkylene" refers to a divalent group obtained by further losing one hydrogen atom from an "alkyl" group as defined above. In some embodiments, it typically has 1 to 20 carbon atoms (e.g., 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2). The term "C..." 1-20 "Alkylene" refers to an alkylene containing 1 to 20 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2- or -CH2CH(CH3)-), butylene (-CH2CH2CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-), etc.

[0093] The term "heteroalkylene" refers to an alkylene chain in which at least one, but not all, of the CH2 atoms are independently replaced by the same or different heteroatom groups. Non-limiting examples of heteroatom groups include O, S, NR, etc.; in NR, R is a suitable substituent, and in some embodiments, R is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyl, cyano, cycloalkyl, heterocyclic, aryl, and heteroaryl; optionally, the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are each independently replaced by a substituent selected from halogen, hydroxyl, cyano, amino, haloalkyl, and hydroxyalkyl. As used herein, the prefix "x-membered" or "x to y-membered" associated with a heteroalkylene group indicates the total number of C atoms and heteroatom members in the heteroalkyl skeletal chain. Non-limiting examples of heteroalkyl groups include, but are not limited to, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -CH2OCH2-, -OCH2O-, -OCH2CH2O-, -OCH2OCH2CH2-, -SCH2-, -SCH2CH2-, -SCH2CH2CH2-, -CH2SCH2-, -SCH2CH2S-, -SCH2SCH2CH2-, -NHCH2-, -NHCH2CH2-, -NHCH2CH2CH2-, -CH2NHCH2-, -N(CH3)CH2-, -CH2N(CH3)-, -OCH2NH-, -OCH2CH2NH-, -OCH2NHCH2CH2-, -OCH2N(CH3)CH2-, etc.

[0094] The term "cytotoxic drug" refers to substances that inhibit or prevent cell function and / or cause cell damage. Examples of cytotoxic drugs include: paclitaxel, tubulysins, duostatins, cytochalasin B, bacitracin D, ethidium bromide, emetine, etoposide, tenoposide, camptothecin or its analogues or derivatives, vincristine, vinblastine, colchicine, dihydroxydiketone anthrax, maytansin or its analogues or derivatives, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, chachiin or its analogues or derivatives, and antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil). Pyrimidines, dacarbamate, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, cisplatin and other platinum derivatives (e.g., carboplatin), docamycin A, docamycin SA, CC-1065 (also known as rachelmycin) or analogs or derivatives of CC-1065), dolalastatin, auristatin, pyrrolo[2,1-c][1,4]benzodiazepines Drug class (PDB), indolebenzodiazepine (IGN) or its analogues, antibiotics (e.g., bleomycin, daunorubicin, doxorubicin, idarubicin, scintillan, mitomycin, mitoxantrone, procainamide, amycin (AMC)), antimitotic agents (e.g., tubulin targets), diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules), ricin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, shiga Toxins, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alarin, saponins, Capsula root toxin, gelanin, Abrus precatorius toxin A chain, Capsula root toxin A chain, α-Acriflavin, tung oil protein, caryophyllin protein, Phytolacca acinosa protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, Jatropha curcas toxin, croton toxin, sphagnum moss inhibitor, white tree toxin, mitogellin, localized trachomatis, phenolic toxin, antimicrobial / cleavage peptides (e.g., CLIP, Magaiin 2, bee venom peptide, silkworm antimicrobial peptide, and P18), ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, and Pseudomonas endotoxin. For example, cytotoxic drugs can be one or more of the following: aurestatin compounds (e.g., MMAE, MMAF, etc., see US patent US5635483, international patents WO2004010957, WO2002088172, etc.), camptothecin compounds (e.g., DXd, camptothecin, hydroxycamptothecin, SN-38, ixotecan, irinotecan, etc., see US patent US10973924B2, US5658920A, etc.), or maytansine alkaloids (DM1, DM3, or DM4, etc., see US patent US5208020, US6441163, etc.). For example, cytotoxic drugs can be MMAE (monomethyl aurestatin E) or DXd.

[0095] The term "DAR," or "drug-antibody ratio," refers to the average amount of drug conjugated to each antibody in a conjugate composition. DAR values ​​can be decimals or integers; for example, DAR can be any decimal or integer between 1 and 10. The amount of drug conjugated to the antibody (drug loading) can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, HIC, and RP-HPLC.

[0096] The terms "nucleic acid," "polynucleotide," and "nucleic acid molecule" are used interchangeably to refer to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. Nucleic acids include those containing known nucleotide analogs or modified backbone residues or links. Nucleic acids can be synthetic, naturally occurring, or non-natural, such as non-natural nucleic acids that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of a reference nucleotide. This includes, but is not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleotide (PNA) modified nucleic acids.

[0097] The term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules, but which are located extrachromosomally or at a chromosomal location different from their natural chromosomal location. "Nucleic acid encoding anti-human FGFR2b antibody" refers to one or more nucleic acid molecules encoding the antibody heavy chain and / or light chain (or fragments thereof), including one or more nucleic acid molecules in a single or separate vector, and one or more nucleic acid molecules present at one or more locations in the host cell. In some embodiments, the nucleic acid sequence also includes conserved modified variants (e.g., degenerate codon substitutions) and complementary sequences. For example, degenerate codon substitutions can be obtained by producing sequences in which the third position of one or more codons is substituted with a mixture of bases 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).

[0098] The term "vector" is a delivery vehicle capable of transporting a genetic element (e.g., nucleic acid) linked to it. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Exemplarily, vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. In some embodiments, the vector is a "plasmid," which is a circular double-stranded DNA loop in which additional DNA segments can be linked. In some embodiments, the vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which DNA segments can be linked to a viral genome. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain replication initiation sites and may include components that facilitate their entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. Vectors can be expression vectors. In some embodiments, the vector (e.g., an expression vector) contains the nucleic acid sequence of the antibody encoded by this disclosure, a promoter (e.g., SV40, CMV, EF-1α), and may also contain at least one selection marker.

[0099] The term "expression vector" or "expression construct" is a vector containing a nucleic acid sequence suitable for transformation of host cells and which directs and / or controls (together with the host cell) the expression of one or more coding regions operatively linked to it. Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, affect the splicing of coding regions operatively linked to them.

[0100] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells (including their progeny) infused with exogenous nucleic acids. Host cells include "transformers" and "transformed cells," encompassing primary transformed cells and their derived progeny cells, regardless of passage number. Progeny cells may not be identical to parental cells in their nucleic acid contents and may contain mutations. In some embodiments, host cells include mutant progeny cells that have the same function or biological activity as the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Exemplary host cells include: Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei.

[0101] The terms "identity" and "sequence identity" refer to the degree (percentage) to which the amino acids (amino acid sequence identity) or nucleic acids (nucleic acid sequence identity) of two sequences are identical at equivalent positions when two sequences are optimally aligned. To achieve optimal alignment, gaps may be introduced to obtain the maximum percentage of sequence identity. For example, when two sequences are optimally aligned, if a position in the first sequence is occupied by the corresponding amino acid residue or nucleotide in the second sequence, the sequences are identical at that position. In some embodiments, the percentage identity between two sequences is determined by the number of identity positions shared by the sequences: Percentage identity = Number of identity positions / Total number of positions × 100%. The percentage of amino acid sequence identity can be determined using various methods known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0102] A polypeptide sequence having at least 80% identity with its parent sequence includes polypeptide variants with altered amino acid sequences (e.g., polypeptide variants obtained through conserved substitution), and polypeptide variants that have been modified (e.g., by covalently linking the molecule to the polypeptide) (e.g., through glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc.). Polypeptide variants can be generated through chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. In some embodiments, the polypeptide variant has a function similar to, identical to, or improved upon that of the polypeptide from which it is derived.

[0103] The terms "conservative substitution," "conservative replacement," or "conservative mutation" refer to amino acid substitutions that do not adversely affect or alter the intended properties of a polypeptide containing an amino acid sequence. Amino acids in a sequence can be replaced with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, skeletal structure, and rigidity) without altering the protein's biological activity. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0104] The term "pharmaceutical composition" refers to a mixture containing one or more antibodies described in this disclosure and other components, such as physiological / pharmaceutical carriers and excipients.

[0105] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is distinct from the active ingredient and non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Those skilled in the art will understand that other drug carriers can be used in this invention. If desired, the pharmaceutical composition may be contained in a box, vial, or dispenser, which may, for example, contain one or more unit doses of antibody. The box, vial, or dispenser may be accompanied by instructions for use.

[0106] The terms "subject" or "individual" include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless specifically indicated, the terms "patient" or "subject" are used interchangeably in this disclosure, unless explicitly defined herein. In some embodiments, the subject is a human. In some embodiments, the subject has a solid tumor, hematologic malignancy, or autoimmune disease.

[0107] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between exogenous drugs, therapeutic agents, diagnostic agents, or compositions and animals, humans, subjects, cells, tissues, organs, or biological fluids.

[0108] The term "treatment" refers to a clinical intervention that attempts to alter the natural processes of the individual being treated, and can be implemented during prevention or in the course of clinicopathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis.

[0109] The term "effective dose" refers to an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, or improve damage associated with a disease state (e.g., lung disease). In some embodiments, an effective dose may be a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. A complete therapeutic or preventative effect may not occur after a single dose is administered, but may occur after a series of doses. Thus, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the therapeutic agent to elicit the desired response in the individual.

[0110] The inventors of this application obtained a human FGFR2b-specific binding antibody through hybridoma screening, which exhibits good binding activity in vitro. Furthermore, various anti-human FGFR2b antibodies were constructed using antibody engineering, and their excellent antigen-binding ability and anti-tumor function were verified through in vitro and in vivo experiments.

[0111] In a first aspect, the present invention provides an anti-human FGFR2b antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to HCDR1-3 in the VH shown in or corresponding to SEQ ID NO:53, 23, 25, 11-15, 1, or 9, or have at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity; and LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to HCDR1-3 in the VH shown in or corresponding to SEQ ID NO:53, 23, 25, 11-15, 1, or 9; and LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to HCDR1-3 in the VH shown in or corresponding to SEQ ID NO:53, 23, 25, 11-15, 1, or 9, having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity. LCDR1-3 in VLs that are shown in or have at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 100%) sequence identity with SEQ ID NO:54, 24, 26, 16-18, 2, or 10.

[0112] In some implementations, the aforementioned anti-human FGFR2b antibody, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat, IMGT, Abm, Contact, or Chothia numbering system.

[0113] In some implementations, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system.

[0114] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments, wherein:

[0115] A) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as HCDR1-3 in SEQ ID NO:53; and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as LCDR1-3 in SEQ ID NO:54;

[0116] B) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as HCDR1-3 in SEQ ID NO:23; and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as LCDR1-3 in SEQ ID NO:24;

[0117] C) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as HCDR1-3 in SEQ ID NO:25; and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as LCDR1-3 in SEQ ID NO:26;

[0118] D) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as HCDR1-3 in SEQ ID NO:11, 12, 13, 14 or 15, respectively; and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as LCDR1-3 in SEQ ID NO:16, 17 or 18, respectively.

[0119] E) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable regions are identical to HCDR1-3 in SEQ ID NO:1; and the LCDR1, LCDR2, and LCDR3 of the light chain variable regions are identical to LCDR1-3 in SEQ ID NO:2; or

[0120] F) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as HCDR1-3 in SEQ ID NO:9; and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are the same as LCDR1-3 in SEQ ID NO:10.

[0121] In some implementations, the anti-human FGFR2b antibody described in any of the preceding claims, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system.

[0122] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments, wherein:

[0123] a) The HCDR1 of the heavy chain variable region includes DX 20 X1MN (SEQ ID NO:32), where X1 is Y or H, X 20 For Y or H; HCDR2 includes X2X3X4NKAX5IYTTX6YX 18 X 19SVKG (SEQ ID NO:33), where X2 is F or H, X3 is I or H, X4 is R or H, X5 is N or H, X6 is E or H, X 18 For S or A, X 19 It is either A or D; and HCDR3 includes X7X8X. 21 X9X 10 X 11 AMDY (SEQ ID NO:34), where X7 is R or H, X8 is L or H, X9 is Y or H, X 10 For A or H, X 11 For F or H, X 21 For L or H; and

[0124] b) The LCDR1 of the light chain variable region includes KASQNVX 22 TX 12 VA (SEQ ID NO:35), where X 12 For A or H, X 22 For G or R; LCDR2 includes X 13 ASX 14 RYT(SEQ ID NO:36), where X 13 For S or H, X 14 For N, S, or H; and LCDR3 includes QQX 15 X 16 TX 17 PMYT(SEQ ID NO:37), where X 15 For Y or H, X 16 For S or H, X 17 It can be Y or H.

[0125] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims, wherein X1 to X 17 X 20 and X 21 At least one of them is H.

[0126] In some implementations, the anti-human FGFR2b antibody described in any of the preceding embodiments,

[0127] A) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:55, LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:42;

[0128] B) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:27, LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0129] C) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:27, LCDR2 includes the amino acid sequence shown in SEQ ID NO:29, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0130] D) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, 20 or 21, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7 or 22, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0131] E) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:20, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0132] F) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:21, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0133] G) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:22, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0134] H) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:20, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:22, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0135] I) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:39;

[0136] J) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:39;

[0137] K) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:44, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:45; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0138] L) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:19, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0139] M) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:46; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0140] N) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0141] O) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:40;

[0142] P) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:42;

[0143] Q) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:47; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0144] R) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:48, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0145] S) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:44, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; or

[0146] T) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8.

[0147] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims is selected from any one or more of groups A) to T) above (e.g., any group 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 therein).

[0148] In some implementations, the anti-human FGFR2b antibody described in any of the preceding embodiments,

[0149] i) The heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, 4 and 5 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:55, 28 and 42 respectively.

[0150] ii) The heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:27, 28, and 8, respectively; or

[0151] iii) The heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, 4 and 5 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:27, 29 and 8 respectively.

[0152] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments is a murine antibody, a chimeric antibody, or a humanized antibody.

[0153] In some implementations, the antibody is a humanized antibody.

[0154] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments, wherein:

[0155] The heavy chain variable region comprises an amino acid sequence as shown in or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO: 53, 23, 25, 11-15, 1, or 9; and / or

[0156] The light chain variable region comprises an amino acid sequence as shown in or having at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 100%) sequence identity with SEQ ID NO: 54, 24, 26, 16-18, 2, or 10.

[0157] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments comprises:

[0158] A) The heavy chain variable region as shown in SEQ ID NO:53, and the light chain variable region as shown in SEQ ID NO:54;

[0159] B) The heavy chain variable region as shown in SEQ ID NO:23, and the light chain variable region as shown in SEQ ID NO:24;

[0160] C) The heavy chain variable region as shown in SEQ ID NO:25, and the light chain variable region as shown in SEQ ID NO:26;

[0161] D) Heavy chain variable regions as shown in SEQ ID NO:11, 12, 13, 14 or 15, and light chain variable regions as shown in SEQ ID NO:16, 17 or 18;

[0162] E) The heavy chain variable region as shown in SEQ ID NO:1, and the light chain variable region as shown in SEQ ID NO:2; or

[0163] F) The heavy chain variable region as shown in SEQ ID NO:9, and the light chain variable region as shown in SEQ ID NO:10.

[0164] In some embodiments, the anti-human FGFR2b antibody is selected from any one or more of groups A) to F) above (e.g., any 2, 3, 4, 5 or 6 of them).

[0165] In some embodiments, the present invention also provides an anti-human FGFR2b antibody, comprising a heavy chain variable region and a light chain variable region, wherein:

[0166] The heavy chain variable region comprises an amino acid sequence as shown in or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO: 53, 23, 25, 11-15, 1, or 9; and / or

[0167] The light chain variable region comprises an amino acid sequence as shown in or having at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 100%) sequence identity with SEQ ID NO: 54, 24, 26, 16-18, 2, or 10.

[0168] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments comprises:

[0169] A) The heavy chain variable region as shown in SEQ ID NO:53, and the light chain variable region as shown in SEQ ID NO:54;

[0170] B) The heavy chain variable region as shown in SEQ ID NO:23, and the light chain variable region as shown in SEQ ID NO:24;

[0171] C) The heavy chain variable region as shown in SEQ ID NO:25, and the light chain variable region as shown in SEQ ID NO:26;

[0172] D) Heavy chain variable regions as shown in SEQ ID NO:11, 12, 13, 14 or 15, and light chain variable regions as shown in SEQ ID NO:16, 17 or 18;

[0173] E) The heavy chain variable region as shown in SEQ ID NO:1, and the light chain variable region as shown in SEQ ID NO:2; or

[0174] F) The heavy chain variable region as shown in SEQ ID NO:9, and the light chain variable region as shown in SEQ ID NO:10.

[0175] In some embodiments, the anti-human FGFR2b antibody is selected from any one or more of groups A) to F) above (e.g., any 2, 3, 4, 5 or 6 of them).

[0176] Secondly, the present invention provides an anti-human FGFR2b antibody, wherein the antibody has a pH-dependent binding function to the human FGFR2b antigen.

[0177] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments has weaker binding activity to the FGFR2b antigen under neutral pH (e.g., 7.4) conditions than under acidic pH (e.g., 6.0) conditions.

[0178] In some embodiments, the ratio of the EC50 value of the anti-human FGFR2b antibody described in any of the preceding claims to the EC50 value of the antibody binding to the human FGFR2b antigen at pH 7.4 to the EC50 value of the antibody binding to the human FGFR2b antigen at pH 6.0 is greater than 1 (e.g., greater than 1.2, greater than 1.5, greater than 2, greater than 3, greater than 10, greater than 20, greater than 50, greater than 100, greater than 120, greater than 200, greater than 300, or greater).

[0179] In some implementations, the EC50 value is detected by an ELISA method.

[0180] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims has a heavy chain variable region containing at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) histidine (H) at amino acid positions 32, 33, 50, 51, 52, 53, 58, 95, 96, 97, 98, 99, and / or 100 corresponding to the amino acid sequence shown in SEQ ID NO:1; and / or the light chain variable region of the antibody has at least one (e.g., 1, 2, 3, 4, 5, or 6) histidine at amino acid positions 32, 50, 53, 91, 92, and / or 94 corresponding to the amino acid sequence shown in SEQ ID NO:2; the amino acid positions are the corresponding positions numbered according to the Kabat numbering system.

[0181] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments has a light chain variable region having at least one (e.g., 1, 2, 3, or 4) histidine residues at amino acid positions 32, 50, 91, and / or 92.

[0182] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims has amino acids 32, 50, 91, and 92 of its light chain variable region being histidine, or amino acids 32 and 50 being histidine, or amino acids 50 and 92 being histidine.

[0183] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments has at least one (e.g., 1, 2, 3, or 4) histidine residues in the heavy chain variable region at amino acid positions 33, 51, 96, and / or 98.

[0184] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments has amino acids at positions 33 and 51 of the heavy chain variable region being histidine, or amino acids at positions 33, 51, 96, and 98 being histidine.

[0185] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims has a light chain variable region in which at least one (e.g., 1, 2, 3, or 4) of the amino acids at positions 32, 50, 91, and / or 92 corresponding to the amino acid sequence shown in SEQ ID NO:2 is a histidine.

[0186] In some embodiments, the anti-human FGFR2b antibody of any of the preceding claims has a light chain variable region containing histidine at amino acids 32, 50, 91, and 92 corresponding to the amino acid sequence shown in SEQ ID NO:2, or containing histidine at amino acids 32 and 50 corresponding to the amino acid sequence shown in SEQ ID NO:2, or containing histidine at amino acids 50 and 92 corresponding to the amino acid sequence shown in SEQ ID NO:2.

[0187] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims has a heavy chain variable region in which at least one (e.g., 1, 2, 3, or 4) of the amino acids at positions 33, 51, 96, and / or 98 corresponding to the amino acid sequence shown in SEQ ID NO:1 is a histidine.

[0188] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims has histidine at positions 33 and 51 corresponding to the amino acid sequence shown in SEQ ID NO:1, or histidine at positions 33, 51, 96, and 98 corresponding to the amino acid sequence shown in SEQ ID NO:1.

[0189] In some embodiments, the amino acid position of the anti-human FGFR2b antibody described in any of the preceding embodiments is the corresponding position numbered according to the Kabat numbering system.

[0190] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims, wherein the heavy chain variable region of the antibody has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) histidine (H) in the amino acid sequence corresponding to Y32, Y33, F50, I51, R52, N56, E61, R101, L102, L103, Y104, A105, and F106 (Note: these positions are natural ordinal numbers relative to the sequence of SEQ ID NO:1, such as Y32, Y33, F50, I51, R52, N53, E58, R95, L96, L97, Y98, A99, and F100) amino acids as defined by the Kabat numbering system; and / or the light chain variable region of the antibody has at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) histidine (H) in the amino acid sequence corresponding to SEQ ID NO:1; and / or the heavy ...); and / or the heavy chain variable region of the antibody has at least one (e.g., 1 At least one (e.g., 1, 2, 3, 4, 5, or 6) of the amino acid positions A32, S50, N53, Y91, S92, and Y94 (Note: these positions are natural sequence numbers relative to the sequence of SEQ ID NO:2, such as A32, S50, N53, Y91, S92, and Y94 according to the Kabat numbering system) of the amino acid sequence shown in NO:2 is a histidine (H).

[0191] In some implementations, the anti-human FGFR2b antibody described in any of the preceding embodiments,

[0192] a) The HCDR1 of the heavy chain variable region includes DX 20 X1MN (SEQ ID NO:32), where X1 is Y or H, X 20 For Y or H; HCDR2 includes X2X3X4NKAX5IYTTX6YX 18 X 19 SVKG (SEQ ID NO:33), where X2 is F or H, X3 is I or H, X4 is R or H, X5 is N or H, X6 is E or H, X 18 For S or A, X 19 It is either A or D; and HCDR3 includes X7X8X. 21 X9X 10 X 11 AMDY (SEQ ID NO:34), where X7 is R or H, X8 is L or H, X9 is Y or H, X 10 For A or H, X 11 For F or H, X 21 For L or H; and b) the LCDR1 of the light chain variable region includes KASQNVX 22 TX 12 VA (SEQ ID NO:35), where X 12For A or H, X 22 For G or R; LCDR2 includes X 13 ASX 14 RYT(SEQ ID NO:36), where X 13 For S or H, X 14 For N, S, or H; and LCDR3 includes QQX 15 X 16 TX 17 PMYT(SEQ ID NO:37), where X 15 For Y or H, X 16 For S or H, X 17 For Y or H; where X1 to X 17 X 20 and X 21 At least one of them (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19) is H.

[0193] In some implementations, the anti-human FGFR2b antibody described in any of the preceding embodiments, wherein,

[0194] a) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:55, LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:42;

[0195] b) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:27, LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0196] c) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:27, LCDR2 includes the amino acid sequence shown in SEQ ID NO:29, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8;

[0197] d) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:39;

[0198] e) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; or

[0199] f) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:42.

[0200] In some embodiments, the anti-human FGFR2b antibody is selected from any one or more of groups a) to f) above (e.g., any 2, 3, 4, 5 or 6 of them).

[0201] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims comprises a heavy chain variable region as shown in SEQ ID NO:53 and a light chain variable region as shown in SEQ ID NO:54; or comprises a heavy chain variable region as shown in SEQ ID NO:23 and a light chain variable region as shown in SEQ ID NO:24; or comprises a heavy chain variable region as shown in SEQ ID NO:25 and a light chain variable region as shown in SEQ ID NO:26.

[0202] In some implementations, the anti-human FGFR2b antibody described in any of the preceding embodiments includes an antibody constant region.

[0203] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments has a heavy chain constant region selected from the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4, and a light chain constant region selected from the κ or λ chain constant region.

[0204] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims, wherein the heavy chain constant region comprises a sequence as shown in SEQ ID NO:30 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:30, and / or the light chain constant region comprises a sequence as shown in SEQ ID NO:31 ...99%, or 100%) sequence identity with SEQ ID NO:30, and / or the light chain constant region comprises a sequence as shown in SEQ ID NO:31 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 99%, or 100%) sequence identity with SEQ ID NO:30, and / or the light chain constant region comprises a sequence as shown in SEQ ID NO:31, and / or the light chain constant region comprises a sequence as shown in SEQ ID NO:31, and / or the light chain constant region comprises a sequence as shown in SEQ ID NO:31, and / or the light chain constant region comprises a sequence as shown in SEQ ID NO:31, and / or the light chain constant region comprises a sequence NO:31 is a sequence that has at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%) sequence identity.

[0205] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments comprises a heavy chain and a light chain, wherein

[0206] i) The heavy chain comprises a sequence as shown in SEQ ID NO:56 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:56, and / or the light chain comprises a sequence as shown in SEQ ID NO:57 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:57; or

[0207] ii) The heavy chain comprises a sequence as shown in SEQ ID NO:49 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:49, and / or the light chain comprises a sequence as shown in SEQ ID NO:50 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:50; or

[0208] iii) The heavy chain comprises a sequence as shown in SEQ ID NO:51 or having at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%) sequence identity with SEQ ID NO:52, and / or the light chain comprises a sequence as shown in SEQ ID NO:52 or having at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%) sequence identity with SEQ ID NO:52.

[0209] In some implementations, the anti-human FGFR2b antibody described in any of the preceding embodiments,

[0210] The antibody comprises a heavy chain as in SEQ ID NO:56 and a light chain as in SEQ ID NO:57; or

[0211] The antibody comprises a heavy chain, such as SEQ ID NO:49, and a light chain, such as SEQ ID NO:50; or

[0212] The antibody comprises a heavy chain as in SEQ ID NO:51 and a light chain as in SEQ ID NO:52.

[0213] In some embodiments, the present invention provides an anti-human FGFR2b antibody comprising a heavy chain and a light chain, wherein i) the heavy chain comprises a sequence as shown in SEQ ID NO:56 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID NO:56, and / or the light chain comprises a sequence as shown in SEQ ID NO:57 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, or 100%) sequence identity with SEQ ID NO:56, and / or the light chain comprises a sequence as shown in SEQ ID NO:57 or having at least 80% (e.g., more than 80%, 85%, 86%, 87%, 98%, 9 ..., and / or the light chain comprises a sequence as shown in SEQ ID NO:57, and / or the light chain comprises a sequence as shown in SEQ ID NO:57, and / or the light chain comprises a sequence as shown in SEQ ID NO:57, and / or the light chain comprises a sequence as shown in SEQ ID NO:57, and / or the light chain comprises a sequence as shown in SEQ ID NO:57, and / or the light chain comprises a sequence as shown in SEQ ID NO NO:57 has a sequence identity of at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%); ii) the heavy chain comprises a sequence as shown in SEQ ID NO:49 or having a sequence identity of at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%), and / or the light chain comprises a sequence as shown in SEQ ID NO:50 ... or 100%). NO:50 has a sequence identity of at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%); or iii) the heavy chain comprises a sequence as shown in SEQ ID NO:51 or having a sequence identity of at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%), and / or the light chain comprises a sequence as shown in SEQ ID NO:52 ... or 100%). NO:52 is a sequence that has at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%) sequence identity.

[0214] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding claims comprises a heavy chain as in SEQ ID NO:56 and a light chain as in SEQ ID NO:57; or the antibody comprises a heavy chain as in SEQ ID NO:49 and a light chain as in SEQ ID NO:50; or the antibody comprises a heavy chain as in SEQ ID NO:51 and a light chain as in SEQ ID NO:52.

[0215] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments is a full-length antibody or an antigen-binding fragment selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, scFab, and (dsFv)2.

[0216] In some embodiments, an anti-human FGFR2b antibody that competes with the antibody described in any of the preceding claims for binding to human FGFR2b or that binds to the same antigenic epitope as the antibody described in any of the preceding claims is also disclosed.

[0217] In some embodiments, the anti-human FGFR2b antibody described in any of the preceding embodiments, wherein the antibody has at least one of the following functions (1) to (6):

[0218] (1) The antibody is capable of specifically binding to the human FGFR2b antigen; in some embodiments, the antibody is capable of binding to the human FGFR2b protein with an EC50 value of less than 10 nM (e.g., less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, or less), the EC50 value being detected by an ELISA method; optionally, the antibody is capable of binding to the human FGFR2b protein with a KD value of less than 10 E-09M (e.g., less than 8 E-09M, less than 6 E-09M, less than 5 E-09M, less than 4 E-09M, less than 3 E-09M, less than 2 E-09M, less than 1 E-09M, or less), the KD value being detected by a surface plasmon resonance assay.

[0219] (2) The antibody is capable of specifically binding to the cynomolgus monkey and / or mouse FGFR2b antigen; in some embodiments, the antibody is capable of binding to monkey and / or mouse FGFR2b protein with an EC50 value of less than 100 nM (e.g., less than 100 nM, less than 50 nM, less than 30 nM, less than 20 nM or less), the EC50 value being detected by an ELISA method;

[0220] (3) The antibody is capable of pH-dependent binding to the human FGFR2b antigen; in some embodiments, the antibody has a higher binding activity to the FGFR2b antigen under neutral pH conditions (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or any value between them, e.g., pH = 7.4) than under acidic pH conditions (e.g., pH = 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5 or lower, or any value between them, e.g., pH = 6). Under certain conditions, the antibody exhibits weak binding activity to the FGFR2b antigen. In some embodiments, the ratio of the EC50 value of the antibody binding to the human FGFR2b antigen at pH 7.4 to the EC50 value of the antibody binding to the human FGFR2b antigen at pH 6.0 is greater than 1 (e.g., greater than 1.2, greater than 1.5, greater than 2, greater than 3, greater than 10, greater than 20, greater than 50, greater than 100, greater than 120, greater than 200, greater than 300, or greater). In some embodiments, the EC50 value is detected by an ELISA method.

[0221] (4) The antibody is capable of binding to cells expressing human FGFR2b protein; in some embodiments, the cells are SNU-16 cells, 293T cells, or KATO-III cells expressing FGFR2b; in some embodiments, the antibody is capable of binding to cells expressing human FGFR2b protein with an EC50 value of less than 10 nM (e.g., less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, or less), the EC50 value being detected by an ELISA method; optionally, the antibody is capable of pH-dependent binding to cells expressing FGFR2b; in some embodiments In some embodiments, the antibody exhibits weaker binding activity to FGFR2b-expressing cells at neutral pH (e.g., 7.4) than at acidic pH (e.g., 6.0); in some embodiments, the ratio of the EC50 value of the antibody binding to FGFR2b-expressing cells at pH 7.4 to the EC50 value of the antibody binding to FGFR2b-expressing cells at pH 6.0 is greater than 1 (e.g., greater than 1.2, greater than 1.5, greater than 2, greater than 3, greater than 10, greater than 20, greater than 50, greater than 100, greater than 120, greater than 200, greater than 300, or greater).

[0222] (5) The antibody can block the binding of FGF1 / 7 to FGFR2b; or

[0223] (6) The antibody has ADCC activity.

[0224] Thirdly, the present invention provides a nucleic acid molecule that encodes the anti-human FGFR2b antibody described in any of the preceding claims.

[0225] Fourthly, the present invention provides a carrier comprising the nucleic acid described in any of the preceding claims.

[0226] Fifthly, the present invention provides a host cell comprising the nucleic acid or vector described in any of the preceding claims.

[0227] In a sixth aspect, the present invention provides a method for preparing an antibody that binds to human FGFR2b, comprising culturing the host cells described in any of the preceding claims under conditions suitable for expressing the antibody, and recovering the antibody from the cultured host cell culture.

[0228] In a seventh aspect, the present invention provides a multispecific molecule comprising the anti-human FGFR2b antibody as described in any of the preceding claims.

[0229] In some embodiments, the multispecific molecule specifically binds to human FGFR2b and specifically binds to one or more other antigens.

[0230] In an eighth aspect, the present invention provides a conjugate comprising the anti-human FGFR2b antibody as described in any of the preceding claims, and a conjugation portion.

[0231] In some implementations, the coupling portion is selected from protein tags, detectable markers, or therapeutic agents.

[0232] In some embodiments, the conjugation portion is a cytotoxic drug, and the conjugation portion is linked to an antibody described in any of the preceding embodiments via a linker.

[0233] In some embodiments, the conjugate has the structure shown in formula a:

[0234] Formula a: Ab-(LD)m

[0235] In formula a, Ab is the anti-human FGFR2b antibody described in any of the preceding terms; L is the linker unit connecting Ab and D; D is the cytotoxic drug; and m is any integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0236] In some embodiments, the conjugate, the anti-human FGFR2b antibody, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to HCDR1-3 in the VH shown in or corresponding to SEQ ID NO:53, 23, 25, 11-15, 1, or 9, or have at least 80% (e.g., more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to SEQ ID NO:53, 23, 25, 11-15, 1, or 9. LCDR1-3 in VLs that are shown in or have at least 80% (e.g., more than 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 100%) sequence identity with SEQ ID NO:54, 24, 26, 16-18, 2, or 10.

[0237] In some embodiments, the conjugate contains an anti-human FGFR2b antibody comprising a heavy chain variable region and a light chain variable region, wherein a) the HCDR1 of the heavy chain variable region comprises DX 20 X1MN (SEQ ID NO:32), where X1 is Y or H, X 20 For Y or H; HCDR2 includes X2X3X4NKAX5IYTTX6YX 18 X 19 SVKG (SEQ ID NO:33), where X2 is F or H, X3 is I or H, X4 is R or H, X5 is N or H, X6 is E or H, X 18 For S or A, X 19 It is either A or D; and HCDR3 includes X7X8X. 21 X9X 10 X 11 AMDY (SEQ ID NO:34), where X7 is R or H, X8 is L or H, X9 is Y or H, X 10 For A or H, X 11 For F or H, X 21 For L or H; and b) the LCDR1 of the light chain variable region includes KASQNVX 22 TX 12VA (SEQ ID NO:35), where X 12 For A or H, X 22 For G or R; LCDR2 includes X 13 ASX 14 RYT(SEQ ID NO:36), where X 13 For S or H, X 14 For N, S, or H; and LCDR3 includes QQX 15 X 16 TX 17 PMYT(SEQ ID NO:37), where X 15 For Y or H, X 16 For S or H, X 17 For Y or H; where X1 to X 17 X 20 and X 21 At least one of them is H.

[0238] In some embodiments, the conjugate comprises a heavy chain variable region of the anti-human FGFR2b antibody including HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, respectively, and a light chain variable region including LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:55, 28, and 42, respectively; or the heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:27, 28, and 8, respectively.

[0239] In some embodiments, the conjugate contains an amino acid sequence as shown in SEQ ID NO:53 in the heavy chain variable region and an amino acid sequence as shown in SEQ ID NO:54 in the light chain variable region; or the heavy chain variable region of the anti-human FGFR2b antibody contains an amino acid sequence as shown in SEQ ID NO:23 and an amino acid sequence as shown in SEQ ID NO:24 in the light chain variable region.

[0240] In some embodiments, the conjugate comprises an amino acid sequence as shown in SEQ ID NO:56 in the heavy chain and an amino acid sequence as shown in SEQ ID NO:57 in the light chain; or the heavy chain of the anti-human FGFR2b antibody comprises an amino acid sequence as shown in SEQ ID NO:49 and an amino acid sequence as shown in SEQ ID NO:50 in the light chain.

[0241] In some embodiments, the connector unit L in the conjugate comprises one or more of the following structures: maleimide hexanoyl (MC), valine-citrulline (Val-Cit), p-aminobenzyloxycarbonyl (PAB), glycine-glycine-phenylalanine-glycine (GGFG), maleimide, carbonyl, amino, amide, aminoacyl, -(PEG)n- (wherein n is independently selected from an integer from 1 to 20), C 1-20 Alkylene, C 1-20 Heteroalkyl, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimide propionyl (MP), methylsulfonylpyrimidinyl, valine-alanine (Val-Ala), N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), dimethylethylenediamine (DMED), N-succinimide-4-(2-pyridinylthio) ) Valerate (SPP), N-succinimide-4-(N-maleimide-methyl)-cyclohexane-1-carboxylic acid ester, N-succinimide-(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimide-4-(2-pyridyl dithio)butyrate (SPDB), N-succinimide-3-(pyridin-2-yl dithio)-propionate (SPDP), or acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC).

[0242] In some embodiments, the adapter unit L comprises: an extension unit for attaching an antibody to other portions of the adapter, a protease cleavage unit (to give the adapter protease cleavage activity), and a spacer unit for attaching a drug to other portions of the adapter; the extension unit may be selected from: maleimide hexanoyl, maleimide propionyl, or maleimide; the protease cleavage unit may be selected from: valine-citrulline, valine-alanine, alanine-phenylalanine, phenylalanine-lysine, or glycine-glycine-phenylalanine-glycine; the spacer unit may be selected from aminobenzyloxycarbonyl or a peptide bond.

[0243] In some embodiments, the connector unit L comprises maleimide hexanoyl, valine-citrulline, and p-aminobenzyloxycarbonyl, or maleimide hexanoyl and glycine-glycine-phenylalanine-glycine.

[0244] In some implementations, the connector unit L is MC-Val-Cit-PAB or MC-GGFG.

[0245] In some embodiments, the cytotoxic drug D in the conjugate is one or more selected from auratestatin compounds, camptothecin compounds, or maytansine alkaloids.

[0246] In some embodiments, the cytotoxic drug D is MMAE or Dxd.

[0247] In some embodiments, the cytotoxic drug D has the following structural formula D1:

[0248] In some embodiments, the cytotoxic drug D has the following structural formula D2:

[0249] In some embodiments, the conjugate, wherein the (LD) portion is derived from the structure shown in formula L-D1 or L-D2:

[0250] In some embodiments, the conjugate has a structure represented by formula Ab-L-D1 or formula Ab-L-D2:

[0251] In formulas Ab-L-D1 and Ab-L-D2, Ab is the anti-human FGFR2b antibody of any of the preceding terms; n is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0252] In some embodiments, the conjugate (e.g., Ab-L-D1, Ab-L-D2), the heavy chain of the anti-human FGFR2b antibody is as shown in SEQ ID NO:56, and the light chain is as shown in SEQ ID NO:57; or the heavy chain of the anti-human FGFR2b antibody is as shown in SEQ ID NO:49, and the light chain is as shown in SEQ ID NO:50.

[0253] In some embodiments, the conjugate structure is as shown in formula Ab-L-D1 or Ab-L-D2, and the heavy chain of the anti-human FGFR2b antibody is as shown in SEQ ID NO:56, and the light chain is as shown in SEQ ID NO:57.

[0254] In some embodiments, the present invention provides a conjugate having the structure shown in formula Ab-L-D1, wherein the heavy chain of the anti-human FGFR2b antibody is shown in SEQ ID NO:56, and the light chain is shown in SEQ ID NO:57.

[0255] In some embodiments, the present invention provides a conjugate having the structure shown in formula Ab-L-D2, wherein the heavy chain of the anti-human FGFR2b antibody is shown in SEQ ID NO:56, and the light chain is shown in SEQ ID NO:57.

[0256] In some embodiments, the DAR value in the composition of the conjugate is a decimal or integer among 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.

[0257] In some embodiments, the DAR value of the conjugate composition is 3.69, 3.76, or 8.00.

[0258] In a ninth aspect, the present invention provides a pharmaceutical composition comprising an anti-human FGFR2b antibody, a multispecific molecule, a conjugate, a nucleic acid molecule, a carrier or cell, and a pharmaceutically acceptable carrier as described in any of the preceding claims.

[0259] In some embodiments, the pharmaceutical composition may further comprise additional pharmaceutical agents.

[0260] In a tenth aspect, the present invention provides a method for treating tumors, the method comprising administering to a subject in need a therapeutically effective amount of any of the preceding claims an anti-human FGFR2b antibody, a multispecific molecule, a conjugate, a nucleic acid molecule, a carrier, a cell, or a pharmaceutical composition thereof;

[0261] In some implementations, the tumor is a disease associated with high expression of FGFR2b.

[0262] In some implementations, the subject is a human being.

[0263] In some implementations, the tumor is selected from skin cancer, non-Hodgkin's lymphoma, renal cell carcinoma, lung cancer, glioma, gastric cancer, head and neck cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma, etc.

[0264] In one aspect, the present invention provides the use of the anti-human FGFR2b antibody, multispecific molecule, conjugate, nucleic acid molecule, carrier, cell or pharmaceutical composition described in any of the preceding claims in the preparation of a medicament.

[0265] In some implementations, the drug is used to treat tumors.

[0266] In some implementations, the anti-human FGFR2b antibody, multispecific molecule, conjugate, nucleic acid molecule, carrier, cell, or pharmaceutical composition described in any of the preceding embodiments may be used alone or in combination with other agents.

[0267] The tumor is associated with FGFR2b high expression.

[0268] In some implementations, the tumor is selected from skin cancer, non-Hodgkin's lymphoma, renal cell carcinoma, lung cancer, glioma, gastric cancer, head and neck cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma, etc.

[0269] In a twelfth aspect, the present invention provides a pharmaceutical composition of any of the preceding claims, including an anti-human FGFR2b antibody, a multispecific molecule, a conjugate, a nucleic acid molecule, a carrier, a cell, or the like, for use as a medicament.

[0270] In some implementations, the drug is used to treat tumors.

[0271] In some implementations, the anti-human FGFR2b antibody, multispecific molecule, conjugate, nucleic acid molecule, carrier, cell, or pharmaceutical composition described in any of the preceding embodiments may be used alone or in combination with other agents.

[0272] The tumor is associated with FGFR2b high expression.

[0273] In some implementations, the tumor is selected from skin cancer, non-Hodgkin's lymphoma, renal cell carcinoma, lung cancer, glioma, gastric cancer, head and neck cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma, etc.

[0274] Furthermore, those skilled in the art will foresee that any of the preceding technical solutions can be freely combined with one or more other technical solutions of the present invention to form new technical solutions (without mutual exclusion). These will not be listed one by one here, but they are all within the scope of protection claimed by the present invention.

[0275] The present invention is described in the following non-limiting embodiments. Those skilled in the art will understand that the embodiments are described by way of example and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0276] Example 1: Screening and preparation of monoclonal hybridomas

[0277] 1. Immunization and fusion

[0278] Immunization was performed using 6-8 week old Balb / c mice (purchased from Shanghai Slack). Mice were housed under SPF conditions after receipt. A human FGFR2b extracellular domain fusion protein (huFGFR2b-Fc, purchased from Beijing Baipusaisi, catalog number FGB-H5256) with a C-terminal human IgG Fc tag was used as the immunogen. For the primary immunization, huFGFR2b-Fc protein was emulsified with Freund's complete adjuvant, and each mouse was intraperitoneally injected with 0.20 mL of 50 μg protein. For the booster immunization, huFGFR2b-Fc was emulsified with Freund's incomplete adjuvant, and each mouse was intraperitoneally injected with 0.20 mL of 25 μg protein. Each immunization was spaced 2 weeks apart. Blood samples were collected 7 days after each booster immunization, and serum antibody titers and specificities were detected by ELISA. After 2-3 booster immunizations, mice with the highest titers were selected for the final titer shock. 50 μg huFGFR2b-Fc was injected intraperitoneally into selected mice. Five days later, the mice were euthanized by cervical dislocation, and spleen cells were collected. These cells were then fused with the mouse myeloma cell line SP2 / 0 (ATCC) using an electrofusion method and cultured in a diluted form.

[0279] 2. Screening of hybridoma clones

[0280] ELISA was performed using a human FGFR2b extracellular domain fusion protein (huFGFR2b-His, purchased from Beijing Baipusaisi, catalog number FGB-H5223) with a C-terminal human His tag as the antigen. FACS was performed using 293T cells (293T-hFGFR2b, purchased from Kangyuan Bochuang, catalog number KC-2051) overexpressing the extracellular and transmembrane regions of human FGFR2b. Positive clones with OD 450nm > 1.0 in ELISA and MFI values ​​10 times greater than the negative control in FACS were amplified into 24-well plates. The supernatant was collected after 3 days to test the inhibitory activity of the hybridoma. Hybridoma clones with an FGF1 receptor inhibition rate of 50% or higher were subcloned using limiting dilution to ensure monoclonal replication. Monoclonal positive hybridoma clones were inoculated into serum-free SFM medium (Invitrogen, 12045076) containing 2% (w / w) FBS and cultured at 37°C under 5% (v / v) CO2 conditions. Selected positive hybridoma clones were then cryopreserved in liquid nitrogen. Positive clones were cultured and then detected by agarose gel electrophoresis. Positive samples were sequenced to obtain the heavy chain variable region and light chain variable region sequences of the anti-human FGFR2b antibody M15 (see Table 1).

[0281] Table 1: Amino acid sequence of antibody M15

[0282] Note: The CDR is defined according to the Kabat numbering system.

[0283] Example 2: Preparation of anti-FGFR2b chimeric antibody

[0284] Based on the variable region of antibody M15 obtained in Example 1, the heavy chain variable region was directionally cloned into an expression vector (Invitrogen, catalog number V79020) containing a signal peptide and a constant region of the human antibody heavy chain IgG1 (amino acid sequence as shown in SEQ ID NO: 30), and the light chain variable region was directionally cloned into an expression vector (Invitrogen, catalog number V79020) containing a signal peptide and a constant region of the human antibody light chain kappa (amino acid sequence as shown in SEQ ID NO: 31). The recombinant vector plasmid was subjected to ExpiFectamine... TM The 293 transfection kit (Invitrogen, A14525) was used to transiently transfect Expi293F cells (Gibco). Expi293 was then used... TM Expression Medium (Gibco) was cultured at 37°C, and feed was added after 18-22 hours. After 5-7 days, the cell culture medium was collected, cells were removed by filtration, and the supernatant was filtered through a 0.45 μm filter until clear. The supernatant was then subjected to MabSelect PrismA (Cytiva, 17-5498-02) affinity chromatography. The MabSelect PrismA column was regenerated with 0.1M NaOH, washed with pure water, and equilibrated with PBS. After binding the supernatant, the column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with 0.1M acetate buffer at pH 3.5 and neutralized with 1M Tris-HCl. The eluted sample was appropriately concentrated and further purified using a PBS-equilibrated Superdex 200 gel chromatography system (Cytiva, 28-9893-35). The collected protein samples were then concentrated to an appropriate concentration. The purified anti-human FGFR2b chimeric antibody (M15xi) was sterile filtered through a 0.22-micron filter and analyzed for protein concentration (A280 / 1.4), purity, and endotoxin (Rhinogen recombinant factor C endotoxin assay kit). The results are shown in Table 2.

[0285] Table 2: Expression and purification of anti-human FGFR2b chimeric antibody M15xi

[0286] The amino acid sequence of the constant region of the human antibody heavy chain IgG1 (SEQ ID NO:30):

[0287] The amino acid sequence of the constant region of the human antibody light chain kappa (SEQ ID NO:31):

[0288] Example 3: Determination of the affinity constant of anti-FGFR2b antibody

[0289] The affinity of the antibody for human FGFR2b protein (purchased from Beijing Baipusaisi, catalog number FGB-H5223) was determined using surface plasmon resonance (SPR) on a Biacore 8K Plus (GE Healthcare) instrument. The candidate antibody was captured at a concentration of 1 μg / mL using a ProteinA chip (Cytiva, 29127555). Serially diluted hFGFR2b-His (starting concentration 25 nM, 2-fold dilution, 5 concentration points) was flowed through the sensor chip at a flow rate of 10 μL / min. The running buffer was 1×PBS-P+ (Cytiva, 50-105-5354), and the detection conditions were: capture time 60 s ("s" refers to seconds); antigen binding time 120 s; dissociation time 600 s; and regeneration conditions were Gly-HCl (pH 1.5) for 30 s. After double subtraction (control channel and zero concentration), the association and dissociation curves were fitted to the "1:1 binding" model in Biacore 8K Plus insight evaluation software.

[0290] Some experimental results are shown in Table 3. The results show that the anti-human FGFR2b antibody of the present invention can bind to human FGFR2b.

[0291] Table 3: Affinity constant determination of anti-human FGFR2b antibody

[0292] Example 4: Enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of anti-human FGFR2b antibody to human / monkey / mouse FGFR2b antigen protein.

[0293] The binding reaction of anti-human FGFR2b antibody with FGFR2b protein was detected. Human FGFR2b (purchased from Beijing Baipusaisi, catalog number FGB-H5223), human FGFR2c (purchased from Beijing Baipusaisi, catalog number FGC-H5225), monkey FGFR2b (purchased from Beijing Baipusaisi, catalog number FGB-C52H6), and mouse FGFR2b (purchased from Beijing Baipusaisi, catalog number FGB-M52H5) proteins were diluted to a final concentration of 1 μg / mL with PBS. Then, 100 μL / well was added to each well of a 96-well high-binding-capacity ELISA plate (BEAVER, 40301), sealed with plastic film, and incubated overnight at 4°C. The next day, the plates were washed twice with PBST (PBS + 0.05% Tween 20), and then blocked with PBST + 1% (w / w) BSA at room temperature for 2 hours. The blocking buffer was then discarded, and the plate was washed twice with PBST. 100 μL of the purified, serially diluted anti-human FGFR2b chimeric antibody obtained in Example 2 was added to each well (initial concentration 100 nM, 10-fold dilution for detecting binding activity with human FGFR2b and FGFR2c; initial concentration 2 μM, 5-fold dilution for detecting binding activity with monkey and mouse FGFR2b). For detecting binding activity with human FGFR2c protein, FGF1 protein (hFGF1, purchased from Beijing Yiqiao Shenzhou, catalog number 10013-HNAE) was used as a positive control, with an initial concentration of 1 μM, 3-fold dilution. After incubation at 37°C for 1 hour, the plate was washed three times with PBST, and 1:5000 dilution of HRP (horseradish peroxidase)-labeled anti-human Kappa light chain secondary antibody (Sigma, A7164) was added. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. 100 μL of TMB substrate (SURMODICS, TMBC-1000-01) was added to each well, and the plate was incubated at room temperature for 15 minutes. Then, 50 μL of stop solution (Solarbio, C1058) was added to each well to terminate the reaction. OD450 values ​​were read using an ELISA reader (SpectraMax 384 plus, Molecular Device). A four-parameter logistic model was used to fit the dose-response data and OD450 readings using GraphPad Prism.

[0294] Some experimental results are shown in Figures 1-4 and Table 4. The experimental results show that the anti-FGFR2b antibody of the present invention can bind to human FGFR2b recombinant protein in a dose-dependent manner under ELISA detection conditions. At the same time, the anti-human FGFR2b antibody of the present invention has cross-binding activity with monkey and mouse FGFR2b proteins. In addition, the chimeric antibody M15xi cannot bind to FGFR2c protein under ELISA detection conditions, while the natural ligand FGF1 of FGFR2 can bind to FGFR2c, indicating that the chimeric antibody specifically binds to the FGFR2b isotype protein.

[0295] Table 4: Results of M15xi binding experiments with human, monkey, and mouse FGFR2b proteins

[0296] Note: OD450 refers to the absorbance at a wavelength of 450nm.

[0297] Example 5: Detection of the binding activity of anti-human FGFR2b antibody to FGFR2b-expressing cells.

[0298] 293T cells (293T-hFGFR2b, purchased from Kangyuan Bochuang, catalog number KC-2051) that overexpress the extracellular and transmembrane regions of human FGFR2b, or SNU-16 human gastric cancer cell line that highly expresses FGFR2 membrane protein (purchased from Nanjing Kebai Biotechnology Co., Ltd.), were cultured in T-75 cell culture flasks. 293T-hFGFR2b cells were cultured in DMEM medium (Gibco, 11995065) containing 1 μg / mL puromycin (Gibco, A1113803) and 10% (v / v) fetal bovine serum (Biological Industries, 04-002-1A) at 37°C and incubated at 5% (v / v) CO2 until 90% confluence. The medium was discarded, and the cells were washed twice with PBS buffer. Then, the cells were digested into single cells with Trypsin-EDTA (Gibco, 25200-072). The digestion reaction was terminated with medium, and the cell suspension was collected. SNU-16 cells were semi-adherent and cultured in RPMI-1640 medium (Gibco, 22400089) containing 10% (v / v) fetal bovine serum (Biological Industries, 04-002-1A) at 37°C in a 5% (v / v) CO2 incubator. Single-cell suspensions were collected by pipetting the cells from the bottom of the culture flask. After centrifugation, the supernatant was discarded, and the cells were collected and washed twice with PBS buffer. The collected cells were then resuspended in FACS buffer (2% FBS PBS, percentage by volume) to a concentration of 2 × 10⁶ cells / mL. 6 Cells / mL, 50 μL per well, were added to a 96-well V plate. An equal volume of the test sample (serially diluted anti-human FGFR2b chimeric antibody obtained in Example 2, starting at 200 nM, 6-fold diluted in 7 spots, with one blank control) was added. Incubate on ice for 1 hour, wash twice with FACS buffer, and add 100 μL of 1:1000 diluted goat anti-human-IgG(H+L)-AF488 secondary antibody (Invitrogen, A11013) to each well. Incubate on ice for 30 min ("min" refers to minutes). Wash twice with FACS buffer, and finally resuspend the cells in 100 μL of FACS buffer. Analyze the cells using flow cytometry. iQue3 (SARTORIUS) detection and analysis results. Dose response data were fitted to fluorescence signals (MFI) using a four-parameter logistic model via GraphPad Prism.

[0299] Some experimental results are shown in Figures 5-6 and Table 5. The experimental results show that the anti-human FGFR2b antibody of the present invention can specifically bind to 293T-FGFR2b cells and SNU-16 cells.

[0300] Table 5: FACS detection of binding activity of anti-human FGFR2b chimeric antibody (M15xi) to 293T-FGFR2b and SNU-16

[0301] Note: MFI refers to average fluorescence intensity.

[0302] Example 6: Experiment on the blocking of FGF1 / 7 and FGFR2b binding by anti-human FGFR2b antibody

[0303] The 293T-hFGFR2b cell line was cultured and collected according to the method described in Example 5. The collected cells were resuspended in FACS buffer (2% FBS PBS, the percentage is volume percentage) to 4 × 10⁻⁶ cells / mL. 6 Cells / mL, 25 μL per well, were added to a 96-well V plate. An equal volume of the test sample (the serially diluted anti-human FGFR2b chimeric antibody obtained in Example 2, starting at 200 nM, 6-fold diluted to 7 spots, with one blank control) or the positive control Bemarituzumab (an anti-human FGFR2b antibody whose structure is listed in the Tabs database (Therapeutic Antibody)) was added. The database (https: / / tabs.craic.com / ) was used to search for "Bemarituzumab" in the Tabs database to obtain the light and heavy chain sequences of Bemarituzumab. After incubating on ice for 30 min, 50 μL of FGF1 protein (with a C-terminal His tag, purchased from MCE, catalog number: HY-P700053AF) or FGF7 protein (with a C-terminal His tag, purchased from Beijing Baipusaisi, catalog number: FG7-H52H5) was added to each well, bringing the final concentrations to 0.4 nM and 69 nM, respectively. After incubating on ice for another 60 min, the cells were washed twice by centrifugation with FACS buffer. 100 μL of 1:500 diluted anti-His-AF488 secondary antibody (Nanjing GenScript, catalog number A01800) was added to each well, and the cells were incubated on ice for 30 min. After washing twice by centrifugation with FACS buffer, the cells were resuspended in 100 μL of FACS buffer and analyzed by flow cytometry. iQue3 (SARTORIUS) detection and analysis results. A four-parameter logical model was used to fit the dose-response data and fluorescence signals using GraphPad Prism.

[0304] Some experimental results are shown in Figures 7-8. The experimental results show that the anti-human FGFR2b antibody of the present invention can block the binding of FGF7 ligand to FGFR2b protein, but cannot block the binding of FGF1 ligand to FGFR2b protein.

[0305] Example 7: Detection of ADCC activity of anti-human FGFR2b antibody using reporter gene assay

[0306] Using 293T-hFGFR2b cells as target cells and Jurkat cells (Jurkat-CD16a-NFAT-Luc cells, purchased from Beijing Baipusaisi, catalog number SCJUR-STF067) overexpressing the full-length CD16a gene, NFAT response element promoter, and luciferase expression gene as effector cells, the ability of anti-human FGFR2b antibody to activate the CD16a signaling pathway was detected to evaluate its ADCC activity. After culturing and collecting 293T-hFGFR2b cells as described in Example 5, they were resuspended in culture medium to a concentration of 2 × 10⁻⁶ cells / mL. 5 100 μL / well of cells / mL was added to a 96-well black clear-bottom cell culture plate (Beyotime, FCP965) and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the test antibody (the serially diluted anti-human FGFR2b chimeric antibody obtained in Example 2, starting at 200 nM, 6-fold dilution in 5 spots, with one blank control) was diluted with experimental medium (RPMI 1640 medium containing 2% (v / v) FBS). The medium in the culture plate containing 293T-hFGFR2b cells was aspirated, and 50 μL / well of the diluted antibody was added to the cell culture plate. The plate was incubated at room temperature for 5 min. The Jurkat-CD16a-NFAT-Luc cell suspension was collected by centrifugation and resuspended in experimental medium to a density of 2 × 10⁻⁶ cells / well. 6 Cells / mL, 50 μL / well added to culture plates, incubated at 37°C, 5% CO2 for 4 h. After incubation, the cell culture plates were removed from the incubator and placed for 15 minutes. The activity of firefly luciferase in the cells was detected using a luciferase reporter gene assay kit (Bio-Lite Luciferase Assay System, Vazyme, catalog number DD1201). Chemiluminescence signals were detected using a Tecan microplate reader, and the data were saved. A four-parameter logistic model was used to fit the dose-response data to the chemiluminescence signals using GraphPad Prism.

[0307] Some experimental results are shown in Figure 9. The results show that the anti-human FGFR2b antibody of the present invention can enhance the chemiluminescence signal in a dose-dependent manner, indicating that the NFAT signaling pathway is activated in a dose-dependent manner, which shows that the antibody has good ADCC activity.

[0308] Example 8: Indirect method for detecting the ADC killing activity of anti-human FGFR2b antibody

[0309] Using 293T-hFGFR2b cells as target cells, the target cells were cultured and collected according to the method described in Example 5, and then suspended in culture medium to a concentration of 2×10⁻⁶. 4 After the cells / mL were collected, 50 μL / well was added to a 96-well black clear-bottom cell culture plate (Beyotime, FCP965) for later use. The test antibody (the serially diluted anti-human FGFR2b chimeric antibody obtained in Example 2, starting at 8 nM, with 10-fold serial dilutions at 6 spots) was diluted with culture medium. With zero concentration without antibody as the background control, 25 μL / well was added to the culture plate and incubated at 4°C for 5 minutes. Anti-human IgG Fc secondary antibody (anti-HIgG(Fc)-C-MMAE ADC, Creative Biolabs, catalog number ADC-AA-003), C-terminally conjugated with MMAE toxin, was diluted to 4 μg / mL with culture medium. 25 μL / well was added to each well of the culture plate, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator. After 3 days, the cell culture plates were removed and allowed to return to room temperature. Cell viability was then assessed using a luciferase-based cell viability assay kit (Cell Counting-Lite 2.0 Luminescent Cell Viability Assay, Vazyme, catalog number DD1101). Chemiluminescence (RLU) signals from each well were read using a Tecan microplate reader, and a four-parameter logistic model was used to fit the dose-response data to the chemiluminescence signals using GraphPad Prism.

[0310] Some experimental results are shown in Figure 10. The results show that the anti-human FGFR2b antibody of the present invention can kill target cells in a dose-dependent manner, indicating that it can kill cells expressing FGFR2b antigen through ADC.

[0311] Example 9: Design of pH-dependent antibody mutation sites

[0312] An hFGFR2 antibody with pH-dependent binding properties was designed, featuring one or more histidine substitutions compared to the parent antibody (M15xi). The positions of the histidine-substituted amino acid residues were determined using computational methods. Based on the M15xi / hFGFR2b complex structure predicted by XtalFold (Shanghai) Co., Ltd., structural and binding analyses were performed using computational methods such as molecular dynamics simulations (e.g., Amber) supplemented by 3D visualization software (e.g., PyMol). Histidine substitutions were identified from multiple perspectives, including residue energy contribution and spatial relationship, with preference given to residues located at the antigen-antibody interface. The key amino acids for identification were determined using free energy perturbation (FEP) to calculate histidine residue mutations. This method assesses the binding strength between proteins based on physical models, thereby calculating the energy difference in binding between the antibody with histidine substitutions and the antigen under acidic and physiological conditions. Based on the design goal of pH-dependent binding described in this invention, the selected mutation sites are shown in Table 6-1, the amino acid sequences of the variable region and CDR region of the M15xi mutation are shown in Table 6-2, and the antigen-antibody predicted structure and site selection diagram are shown in Figure 11.

[0313] Table 6-1: Amino acid sites in M15xi predicted to be replaceable by histidine

[0314] Note: The positions in the table are the natural ordinal numbers of the M15xi light and heavy chains; according to the Kabat numbering system definition, the natural ordinal numbers Y32, Y33, F50, I51, R52, N56, E61, R101, L102, L103, Y104, A105 and F106 of the M15xi heavy chain variable region SEQ ID NO:1 in the above table correspond to the positions Y32, Y33, F50, I51, R52, N53, E58, R95, L96, L97, Y98, A99 and F100 in the Kabat numbering system; the natural ordinal numbers A32, S50, N53, Y91, S92 and Y94 of the M15xi light chain variable region SEQ ID NO:2 correspond to the positions A32, S50, N53, Y91, S92 and Y94 in the Kabat numbering system.

[0315] Table 6-2: Amino acid sequences of the variable region and CDR region of M15xi with histidine substitution

[0316] Note that the underlined part is the CDR determined according to the Kabat numbering system.

[0317] The pH-dependent antibody light and heavy chain variable regions designed based on the calculation results are shown in Table 7. The antibody was expressed and purified using the same method as in Example 3, and the expression results are shown in Table 7.

[0318] Table 7: pH-dependent mutations of M15xi and their expression results

[0319] Note: In the table, the mutation sites are the natural order sites of the variable regions of the light and heavy chains relative to M15xi. WT indicates that it is the same as the corresponding variable region of M15xi. For example, the light chain of antibody "B002" is formed by three point mutations of A32H, Y91H and S92H in the light chain of M15xi (LCDR1 contains the A32H mutation, A32H-LCDR1: KASQNVGTHVA (SEQ ID NO:38); LCDR3 contains the Y91H and S92H mutations, Y91H-S92H-LCDR3: QQHHTYPMYT (SEQ ID NO:39)). Its heavy chain is the same as the heavy chain of M15xi; others are similar.

[0320] Example 10. ELISA detection of the binding of anti-human FGFR2b antibody to human FGFR2b protein.

[0321] Human FGFR2b antigen protein was coated onto 96-well high-binding ELISA plates using the same method as in Example 4 of this invention. The next day, the antibody samples to be tested in Table 7 were diluted with phosphate buffer at pH 6.0 and pH 7.4, respectively, and 100 μL / well was added to the blocked ELISA plate. The plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, HRP (horseradish peroxidase)-labeled anti-human Kappa light chain secondary antibody (Sigma, A7164) diluted 1:5000 with phosphate buffer at the corresponding pH conditions was added. After incubating at 37°C for 1 hour, the plate was washed three times with PBST. 100 μL of TMB substrate (SURMODICS, TMBC-1000-01) was added to each well. After incubating at room temperature for 15 minutes, 50 μL of stop solution (Solarbio, C1058) was added to each well to stop the reaction. The OD 450 value was read using an ELISA reader (SpectraMax 384plus, Molecular Device), and the dose response data was fitted to the OD 450 value using a four-parameter logical model via GraphPad Prism.

[0322] Some experimental results are shown in Tables 8.1-8.2 and Figure 12. The results show that the M15xi mutant antibodies of the present invention can all bind to human FGFR2b protein. Compared with the parent antibody M15xi, the binding activity of B004, B005, B008, B010, and B014 to human FGFR2b protein at pH 6.0 is higher than that at pH 7.4, indicating that they have pH-dependent binding characteristics.

[0323] Table 8.1: Binding results of M15xi mutant antibody to human FGFR2b protein at pH 6.0

[0324] Table 8.2: Binding results of M15xi mutant antibody to human FGFR2b protein at pH 7.4

[0325] Note: The pH-dependent factor in the table is the ratio of the EC50 value of the antibody at pH 7.4 to the EC50 value of the antibody at pH 6.0.

[0326] Example 11: Humanization Design and Affinity Detection of Chimeric Antibody M15xi

[0327] The heavy and light chain CDRs of the chimeric antibody M15xi were grafted onto human germline-derived framework regions, respectively. The framework regions of the heavy chain were derived from IGHV 3-30, IGHV 3-72, and IGHJ2; the framework regions of the light chain were derived from IGKV 1-13 and IGKJ2. Xperhuman (Shanghai) Co., Ltd. was used to evaluate the humanized sequences. Through sequence alignment and structural analysis, information on potential immunogenic regions was obtained. Regions with potential risks were mutated and scored to increase the degree of humanization while maintaining activity. The final humanized heavy and light chain variable region sequences are shown in Table 9.

[0328] Table 9: Amino acid sequences of the light / heavy chain variable regions of the M15xi humanized antibody

[0329] Note: In the variable region of the M15xi humanized antibody, according to the Kabat numbering system, the HCDR2 sequence of 015_3_30-2 and 015_3_72-2 is FIRNKANIYTTEYAASVKG (SEQ ID NO:20), the HCDR2 sequence of 015_3_30-3 is FIRNKANIYTTEYADSVKG (SEQ ID NO:21), and the LCDR2 sequence of 015_1_13-3 and 015_1_13-4 is SASSRYT (SEQ ID NO:22). Other CDRs in the variable region of the M15xi humanized antibody are the same as the corresponding CDRs of M15.

[0330] The designed humanized light and heavy chain variable region sequences were combined to construct humanized antibodies M15-cu5~M15-cu24 (the amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO:30, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:31). Protein expression and purification were performed according to the same method as in Example 2 of this invention, and the affinity of each purified antibody for human FGFR2b protein was detected according to the same method as in Example 3 of this invention.

[0331] Some experimental results are shown in Table 10. The results show that the affinity constants of each humanized antibody molecule are all within 5 times that of the maternal chimeric antibody M15xi, and the immunogenicity scores are all lower than those of the maternal chimeric antibody.

[0332] Table 10: Immunogenicity scores and affinity constants of humanized molecules with human FGFR2b protein

[0333] Note: The immunogenicity score in the table is calculated using the XperHuman algorithm (sourced from XtalPi Technologies (Shanghai) Co., Ltd.). The lower the score, the lower the immunogenicity.

[0334] Example 12 Humanized and pH-dependent combined mutant molecules and their cell-binding activity assay

[0335] Humanized antibodies M15-cu08 and M15-cu23 were combined with pH-dependent antibody B008 to construct humanized antibodies M15-cu08-B008 and M15-cu23-B008, respectively. The amino acid sequences of their variable regions are shown in Table 11.

[0336] Table 11: Amino acid sequences of humanized and pH-dependent co-mutants of M15

[0337] Note: The underlined part is the CDR defined according to the Kabat numbering system. Among them, HCDR1-3 and LCDR3 are the same as the corresponding CDR of M15. The LCDR1 of M15-cu08-B008 and M15-cu23-B008 is KASQNVGTHVA (SEQ ID NO:27), the LCDR2 of M15-cu08-B008 is HASNRYT (SEQ ID NO:29), and the LCDR2 of M15-cu23-B008 is HASSRYT (SEQ ID NO:28).

[0338] M15-cu23-B008 heavy chain amino acid sequence (SEQ ID NO:49):

[0339] M15-cu23-B008 light chain amino acid sequence (SEQ ID NO:50):

[0340] M15-cu08-B008 heavy chain amino acid sequence (SEQ ID NO:51):

[0341] M15-cu08-B008 light chain amino acid sequence (SEQ ID NO:52):

[0342] The antibody was expressed and purified using the same method as in Example 2, and then the pH dependence of antibody binding to 293T-hFGFR2b cells was detected. Single-cell suspensions of 293T-hFGFR2b cells were cultured and collected using the same method as in Example 5. After centrifugation and discarding the supernatant, the cells were resuspended at 2 × 10⁻⁶ cells / mL in FACS buffer (PBS buffer containing 2% FBS at the corresponding pH values) at pH 6.0 and pH 7.4, respectively. 6 Cells / mL, 50 μL per well, were added to a 96-well V plate. An equal volume of FGFR2b antibody, diluted with FACS buffer at the corresponding pH, was added. Incubate on ice for 1 hour, wash twice with FACS buffer, and add 100 μL of goat anti-human-IgG (H+L)-AF488 secondary antibody (Invitrogen, A11013) diluted 1:1000 with FACS buffer at the corresponding pH to each well. Incubate on ice for 30 min. Wash twice with FACS buffer, and finally resuspend the cells in 100 μL of FACS buffer. Analyze using flow cytometry. iQue3 (SARTORIUS) detection and analysis results. Dose response data were fitted to fluorescence signals (MFI) using a four-parameter logistic model via GraphPad Prism.

[0343] Partial experimental results are shown in Figure 13. The results show that the maternal chimeric antibody M15xi and the humanized antibodies M15-cu08 and M15-cu23 can bind to 293T-FGFR2b cells in a dose-dependent manner, but there is no significant difference in binding activity under pH 6.0 and pH 7.4 conditions, indicating that these molecules do not have pH-dependent binding activity. However, the pH-dependent mutants M15-cu08-B008 and M15-cu23-B008 showed significantly stronger binding activity to cells under pH 6.0 conditions than under pH 7.4 conditions, indicating that the binding of these two molecules to FGFR2b is pH-dependent, with strong binding activity under acidic conditions and significantly reduced binding activity under neutral conditions.

[0344] Example 13 Affinity detection of humanized and pH-dependent combined mutant molecules with human FGFR2b protein

[0345] Using the same method as in Example 3, the affinity and binding kinetics between the molecule and human FGFR2b protein under different pH conditions were detected using Biocore 8K Plus. After capturing the candidate antibody at a concentration of 1 μg / mL using a ProteinA chip (Cytiva, 29127555), the hFGFR2b-His protein was diluted with PBS buffer at pH 6.0 and pH 7.4, respectively, and flowed through the sensor chip at a flow rate of 10 μL / min. After double subtraction (control channel and zero concentration), the association and dissociation curves were fitted using a "1:1 binding" model in the Biacore 8K Plus insight evaluation software.

[0346] Partial experimental results: Affinity and binding kinetic data are shown in Table 12, and the affinity detection fitting graph is shown in Figure 14. The results show that M15-cu08-B008 and M15-cu23-B008 showed almost no binding signal with FGFR2b protein under neutral conditions, while maintaining affinity with FGFR2b protein under pH 6.0 conditions, with affinity constants of 1.90 nM and 1.57 nM, respectively.

[0347] Table 12: Affinity constants of humanized and pH-dependent co-mutants of M15 with human FGFR2b protein

[0348] Example 14 Affinity maturation modification of anti-human FGFR2b antibody and its cell binding activity assay

[0349] Based on the M15-cu23-B008 molecule, yeast library construction and screening were conducted to identify molecules with enhanced affinity under pH 6.0 conditions. Finally, the molecule M15-cu23-B008-AM05, exhibiting good binding function, was obtained. The variable region amino acid sequence information of M15-cu23-B008-AM05 is shown in Table 13.

[0350] Table 13: Amino acid sequence of the variable region of M15-cu23-B008-AM05

[0351] Note: The underlined part is the CDR defined according to the Kabat numbering system. Among them, HCDR1-3 of M15-cu23-B008-AM05 is the same as HCDR1-3 of M15 (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 respectively), LCDR1 is KASQNVRTHVA (SEQ ID NO:55), LCDR2 is the same as LCDR2 of M15-cu23-B008 (SEQ ID NO:28), and LCDR3 is the same as LCDR3 of B010 (SEQ ID NO:42).

[0352] The amino acid sequence of the M15-cu23-B008-AM05 heavy chain (SEQ ID NO:56):

[0353] The amino acid sequence of the M15-cu23-B008-AM05 light chain (SEQ ID NO:57):

[0354] The M15-cu23-B008-AM05 antibody was expressed and purified using the same method as in Example 2 of this invention.

[0355] The pH dependence of antibody binding to 293T-hFGFR2b cells, SNU-16 cells, and KATO-III cells was detected. 293T-hFGFR2b and SNU-16 cells were cultured and collected according to the method described in Example 5 of this invention. KATO-III cells are a gastric cancer cell line highly expressing FGFR2b (purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection). The cells were semi-adherent and cultured statically at 37°C in a 5% (v / v) CO2 incubator using IMDM medium (Giboco, 31980030) containing 15% (v / v) fetal bovine serum (Biological Industries, 04-002-1A). After culturing to 90% confluence, cells suspended in the supernatant were collected. Adherent cells were washed twice with PBS buffer, then digested into single cells with Trypsin-EDTA (Gibco, 25200-072). The digestion reaction was terminated with culture medium, and the cell suspension was collected. The binding activity of antibody samples to cells was detected in 293T-hFGFR2b, SNU-16, and KATO-III cells at pH 6.0 and pH 7.4, respectively, according to the method described in Example 12 of this invention.

[0356] Some experimental results are shown in Figures 15-17. The results show that the affinity-matured antibody M15-cu23-B008-AM05 exhibits significantly stronger binding activity with 293T-FGFR2b cells, KATO-III cells, and SNU-16 cells at pH 6.0 than at pH 7.4, maintaining the pH-dependent binding of the antibody to FGFR2b. Furthermore, comparing the binding activity under the same pH conditions, the M15-cu23-B008-AM05 molecule shows stronger binding activity than the M15-cu23-B008 molecule, indicating a significant affinity maturation effect.

[0357] Example 15: Detection of endocytic activity of anti-human FGFR2b antibody

[0358] The activity of anti-human FGFR2b antibody endocytosis by 293T-FGFR2b cells, KATO-III cells and SNU-16 cells under different pH conditions was detected by flow cytometry.

[0359] Experimental Procedure: 293T-FGFR2b cells, KATO-III cells, and SNU-16 cells were collected using the same method as in Example 14 of this invention. After resuspending the cells in FACS buffer (pH 6.0 or pH 7.4), they were aliquoted into 96-well plates. The antibody to be tested was diluted to 400 nM with FACS buffer (pH 6.0 or pH 7.4) and mixed 1:1 with the cell suspension. The plates were then incubated at 37°C for cell endocytosis. After incubation for 0, 0.5, 1, 2, and 4 hours, the plates were placed on ice to terminate the endocytosis reaction. Cells were washed and stained with fluorescent secondary antibody and resuspended using the same method as in Example 12. The cells were then analyzed using flow cytometry. The fluorescence signal (MFI) was recorded under different pH and endocytosis time conditions using iQue3 (SARTORIUS) assays. The correlation between time and MFI was recorded using GraphPad Prism statistical analysis. In this experiment, Bemarituzumab antibody, known to have FGF1 blocking activity, was used as a positive control, and an isotype control (IgG1 isotype control unrelated to the FGFR2b antigen) was used as a negative control.

[0360] Some experimental results are shown in Figures 18 to 20. The results show that at the start of the experiment (i.e., 0 hours), the anti-human FGFR2b antibody of this invention exhibited a higher fluorescence signal at pH 6.0 than at pH 7.4 in 293T-FGFR2b cells, KATO-III cells, and SNU-16 cells. This result is consistent with the pH-dependent binding activity conclusion suggested in Example 15. With prolonged incubation time, the signal value at pH 6.0 decreased significantly, indicating that the molecule was internalized into the cells. At pH 7.4, the fluorescence signal value did not change significantly, indicating that the number of antibodies internalized into the cells was small. The positive control molecule did not show obvious pH-dependent characteristics; its binding signal and internalization effect were not significantly different in the three cell types and under different pH conditions. The experimental results demonstrate that pH-dependent molecules also exhibit pH-dependent characteristics in their internalization activity.

[0361] Example 16 Preparation of Anti-human FGFR2b Antibody-Drug Conjugate (ADC)

[0362] 1. Preparation of ADC with VC-MMAE as the drug-linker compound

[0363] Antibodies with a purity of ≥90% (e.g., M15-cu23-B008 and M15-cu23-B008-AM05 of this invention) were transferred to phosphate buffer (Gibco, C10010500BT) in 30 kDa ultrafiltration centrifuge tubes (Millipore, catalog number UFC503008) to a concentration of 5 mg / ml. 2.95 times the molar volume of the antibody was added to tris(2-carboxyethyl)phosphine (TCEP, MERCK, catalog number 51805-45-9), and the mixture was reacted at 37°C for 2 h to partially open the interchain disulfide bonds of the antibody. Then, add 8 times the molar number of the antibody toxin VC-MMAE (also known as MC-Val-Cit-PAB-MMAE or Maleimidocaproyl-Valine-Citrulline-PAB-MMAE, purchased from MCE, catalog number 646502-53-6), and react at 22°C for 1 hour. After the reaction, use an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa to change the medium to 20 mM histidine (purchased from Sigma-Aldrich, catalog number V900459-100G) buffer to remove unconjugated linker-toxin. Filter the product after medium change using a 0.22 μm sterile filter (purchased from Millipore, catalog number SLGPR33RB) for later use to obtain the ADC molecule Ab-VC-MMAE, with the structure shown in the formula: Ab-L-D1:

[0364] In the formula Ab-L-D1, Ab represents the antibody, and n represents the number of VC-MMAE conjugates for each antibody.

[0365] Linker-toxin VC-MMAE is a compound comprising a specific cleavable linker and a toxin load. The linker moiety includes: a maleimide hexanoyl group (MC) capable of forming a stable thioether bond with the coupling site of an antibody cysteine ​​residue; a dipeptide (VC) specifically recognized and cleaved by cathepsin B in tumor cells; and a self-eliminating spacer unit, p-aminobenzyloxycarbonyl (PAB), ensuring the release of the toxin in its active form. The toxin moiety is the cytotoxic load monomethylaurestatin E (MMAE). The specific structure of VC-MMAE is shown in Formula L-D1:

[0366] The structural formula for the MC part:

[0367] VC's structure:

[0368] The structural formula of PAB:

[0369] The structural formula of toxin MMAE:

[0370] 2. Preparation of ADC with GGFG-Dxd as the drug-linker compound

[0371] Antibodies with a purity of ≥90% (e.g., M15-cu23-B008, M15-cu23-B008-AM05, and M15-cu23 of this invention) were transferred to phosphate buffer using an ultrafiltration centrifuge tube at a concentration of 5 mg / ml. Tris(2-carboxyethyl)phosphine (TCEP) was added in 12 times the molar amount of antibody, and the reaction was carried out at 37°C for 2 hours to completely open the interchain disulfide bonds. The reduced reaction solution was then desalted using a desalting column (Thermo Scientific, catalog number A57759) to remove excess unreacted TCEP. Then, a linker-toxin (GGFG-Dxd (also known as MC-GGFG-DXD, purchased from MCE, catalog number 1599440-13-7) in 12 times the molar amount of antibody was added, and the reaction was carried out at 22°C for 1 hour. After the reaction, the medium was changed to 20 mM histidine buffer using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa. Then, activated charcoal (Sigma, catalog number C6241-5G) washed with histidine buffer was added to remove uncoupled linker-toxins. The final product was filtered through a 0.22-micron sterile filter and used as the ADC molecule Ab-GGFG-Dxd, with the following structure: Formula Ab-L-D2:

[0372] In the formula Ab-L-D2, Ab represents the antibody, and n represents the number of VC-MMAE conjugates for each antibody.

[0373] Linker-toxin GGFG-Dxd is a compound containing a specific cleavable linker and a toxin loading Dxd. The linker moiety includes a maleimide hexanoyl group (MC) capable of forming a stable thioether bond with the coupling site of antibody cysteine ​​residues, and a tetrapeptide linker (GGFG) specifically recognized and cleaved by cathepsin B in tumor cells. The toxin moiety is a novel topoisomerase I inhibitor (Dxd).

[0374] The specific structure of GGFG-Dxd is as follows: Formula L-D2:

[0375] The structural formula for the MC part:

[0376] The structural formula of the GGFG part:

[0377] The structural formula of toxin Dxd:

[0378] 3. Drug / antibody ratio (DAR) and purity testing of ADC molecules.

[0379] The average DAR value of ADC molecules was determined by hydrophobic interaction chromatography (HIC): An Agilent 1260 Infinity II chromatograph was used with a TSKgel Butyl-NRP column. Mobile phase A was phosphate buffer containing 1.5M ammonium sulfate (Adamas Life, catalog number 7783-20-2), and mobile phase B was phosphate buffer containing 25% isopropanol (Energy Chemical, catalog number 67-63-0). The liquid phase gradient was 100% phase A to 100% phase B, as shown in Table 14 below. Peaks with different retention times corresponded to antibody molecules linked to different numbers of toxins, from which the DAR value could be calculated.

[0380] Table 14: Hydrophobic Interaction Chromatographic Mobile Phase Gradient Settings

[0381] The average DAR value of ADC molecules was determined by liquid chromatography-mass spectrometry (LC / MS): An ultra-high performance liquid chromatograph (Vanquish instrument, Agilent-PL1312-1502 column) and a high-resolution mass spectrometer (Q Exactive instrument) were coupled. ADC samples were reduced with 20 mM dithiothreitol (DTT, Sigma-Aldrich, catalog number 43815-5G) for 1 hour before being directly introduced into the ultra-high performance liquid chromatography system for separation. Solution A was a 0.1% formic acid (MERCK, catalog number 900667) aqueous solution, and solution B was a 0.1% formic acid acetonitrile (Thermo Fisher, catalog number 28905) solution. The mobile phase gradient is shown in Table 15 below. After processing, the samples were separated by ultra-high performance liquid chromatography and then detected and scanned using a Q Exactive high-resolution mass spectrometer. All raw data were analyzed to characterize the protein molecular weight using the deconvolution software Biopharma Finder. Peaks with different relative molecular masses corresponded to antibody molecules with different numbers of toxins, from which the DAR value could be calculated.

[0382] Table 15: Mobile Phase Gradient and Parameter Settings for Liquid Chromatography-Mass Spectrometry

[0383] Size exclusion chromatography (SEC) was used to determine the monomer purity of the ADC molecules: an Agilent 1260 Infinity II chromatograph was used with a TSKgel G300SWXL column and a phosphate buffer containing 15% isopropanol as the mobile phase, with isocratic elution. Peaks at different retention times corresponded to antibody molecules of different relative molecular masses, thus determining the monomer purity of the ADC molecules.

[0384] The average DAR value and purity of the prepared ADC molecules are shown in Table 16.

[0385] Table 16: Preparation, DAR value and purity of ADC

[0386] Note: Isotype control refers to isotype IgG unrelated to the FGFR2b target.

[0387] Example 17: pH-dependent cell-binding activity of anti-human FGFR2b antibody-drug conjugate

[0388] To detect whether the ADC molecule can maintain the pH-dependent binding activity of its antibody portion, the ADC molecule prepared in Example 16 of this invention was tested for cell binding activity with 293T-hFGFR2b cells and SNU-16 cells at pH 6.0 and pH 7.4, respectively, using the same method as described in Example 12.

[0389] Some experimental results are shown in Figures 21-22. The results show that the control antibodies Bemarituzumab and the ADC molecules M15-cu23-Dxd and Bema-Dxd constructed from M15-Cu23, which exhibit no significant pH-dependent binding, maintained their binding activity with 293T-hFGFR2b or SNU-16 cells under both pH 7.4 and pH 6.0 conditions. However, the M15-cu23-B008-MMAE, M15-cu23-B008-Dxd, M15-cu23-B008-AM05-MMAE, and M15-cu23-B008-AM05-Dxd molecules of this invention maintained the pH-dependent binding activity of their antibody portions.

[0390] Example 18: In vitro ligand blocking activity of anti-human FGFR2b antibody-drug conjugate

[0391] To determine whether the ADC molecule retained its pH-dependent ligand blocking activity, the antibody of this invention and the ADC molecule prepared in Example 16 were tested for their blocking activity against ligand FGF7 and 293T-hFGFR2b cells at pH 6.0 and pH 7.4, respectively, following the method described in Example 6. 293T-FGFR2b cells were collected, washed twice with PBS, and resuspended in FACS buffer at the corresponding pH values. 5Cells were seeded at a density of 1 / 2 well in 96-well V plates. FGF7-His protein was diluted to a final concentration of 69 nM ng / mL with FACS buffer at the appropriate pH and added to each well. Simultaneously, serially diluted (starting at 100 nM, 5-fold serial dilutions) of the antibody or ADC molecule to be tested was added. Cells were incubated on ice for 1 hour, washed twice with FACS buffer, and 100 μL of diluted anti-His tag-AF488 (GenScript, A01800-100) was added to each well. Cells were incubated on ice for 1 hour, washed twice with FACS buffer, and resuspended in 100 μL of FACS buffer. Results were detected and analyzed by flow cytometry.

[0392] Partial experimental results are shown in Figure 23. The results indicate that the naked antibody M15-cu23-B008-AM05 and its ADC molecule M15-cu23-B008-AM05-Dxd could not block the binding of FGF7 to FGFR2b at pH 7.4, but could block the binding of FGF7 to FGFR2b with weak activity at pH 6.0. The pH-independent naked antibodies to Bemarituzumab and M15-cu23, and their ADC molecules M15-cu23-Dxd and Bema-Dxd, exhibited blocking activity under both conditions.

[0393] In addition, using a similar detection method, the activity of the antibody of this invention and the ADC molecule prepared in Example 16 in blocking FGF10 ligand and 293T-hFGFR2b cells was detected. Since FGF10 ligand hardly binds to 293T-hFGFR2b cells at pH 6.0, the blocking activity of each molecule was only evaluated at pH 7.4. 293T-FGFR2b cells were resuspended in FACS buffer at pH 7.4 and then... 5 Seeds were placed at a density of / wells into 96-well V plates. Biotin-labeled FGF-10 protein (purchased from Beijing Bioscience, catalog number FG0-H81Q7) was diluted to a final concentration of 60 ng / mL with FACS buffer at pH 7.4 and added to the 96-well plates. The target antibody or ADC molecule was then added in a serially diluted manner (starting at 100 nM, 5-fold dilution). The binding of FGF10 molecules to the cells was detected using antibiotin-PE (eBioscience, 12-9895-82) secondary antibody.

[0394] Some experimental results are shown in Figure 24. The results show that, under pH 7.4 conditions, both the naked Bemarituzumab antibody and its ADC molecule Bema-Dxd can block the binding of FGF10 to 293T-hFGFR2b. The FGF10 blocking activity of the M15-cu23 naked antibody and its ADC molecule M15-cu23-Dxd of the present invention is weaker than that of Bemarituzumab, while the M15-cu23-B008-AM05 naked antibody and its ADC molecule M15-cu23-B008-AM05-Dxd have extremely weak FGF10 ligand blocking activity.

[0395] Example 19: In vivo efficacy of anti-human FGFR2b ADC molecule in gastric cancer-bearing mice

[0396] The FGFR2b-positive human gastric cancer cell line SNU-16 (ATCC, CRL-5974) was selected to establish an in vivo mouse model to evaluate the antitumor efficacy of candidate molecules in vivo. Six- to eight-week-old female CB17-SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were housed in a specific pathogen-free animal facility and allowed to acclimatize for 3 days before experiments. Human gastric cancer SNU16 cells were cultured in RPMI1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C under 5% CO2 atmosphere, and passaged routinely 2-3 times per week. Cells in the exponential growth phase were collected and counted, and 6 x 10⁶ cells were collected. 6 One SNU-16 tumor cell was resuspended in 0.1 mL of PBS, thoroughly mixed with 0.1 mL of matrigel, and then subcutaneously injected into the right side of each mouse to form a tumor. The average tumor volume of the tumor-bearing mice was approximately 100-150 mm. 3 Tumor volume was measured in all mice. Suitable tumor-bearing mice were randomly divided into three groups: the test group, the positive control group (Bemarituzumab), and the blank control group (DPBS), with six mice in each group. The dosage for each group was 5 mg / kg. The blank control group received a tail vein injection of DPBS, while the other groups received a tail vein injection of the corresponding test drug, all administered as a single dose. The observation period was 4 weeks, during which tumor volume and animal weight were monitored twice weekly. The tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = [1 - (Ti - T0) / (Ci - C0)] × 100%, where Ti is the mean tumor volume in the treatment group on day i of administration; T0 is the mean tumor volume in the treatment group on day 0 of administration; Ci is the mean tumor volume in the blank control group on day i of administration; and C0 is the mean tumor volume in the blank control group on day 0 of administration.

[0397] Some experimental results are shown in Table 17 and Figures 25 and 26. The results show that there was no significant difference in animal body weight before and after the experiment among the groups. Compared with the blank control group, the anti-human FGFR2b ADC molecule of the present invention and the positive control Bemarituzumab can effectively inhibit tumor growth. Among them, the TGI of the M15-cu23-B008-MMAE, M15-cu23-B008-AM05-MMAE and M15-cu23-B008-AM05-Dxd groups were 142%, 144% and 132%, respectively, which were stronger than the M15-cu23-B008-Dxd group (82%) and the positive control group (72%).

[0398] Table 17: Efficacy of anti-human FGFR2b ADC molecules in SNU-16 gastric cancer-bearing mice

[0399] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-human FGFR2b antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to the HCDR1-3 in a VH set forth in SEQ ID NO: 53, 23, 25, 11-15, 1, or 9, or are identical to the HCDR1-3 in a VH having at least 80% sequence identity to SEQ ID NO: 53, 23, 25, 11-15, 1, or 9; and, the LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to the LCDR1-3 in a VL set forth in SEQ ID NO: 54, 24, 26, 16-18, 2, or 10, or are identical to the LCDR1-3 in a VL having at least 80% sequence identity to SEQ ID NO: 54, 24, 26, 16-18, 2, or 10; optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat, IMGT, Abm, Contact, or Chothia numbering system.

2. The antibody of claim 1, wherein: A) the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to the HCDR1-3 in SEQ ID NO: 53, respectively; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to the LCDR1-3 in SEQ ID NO: 54, respectively; B) the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to the HCDR1-3 in SEQ ID NO: 23, respectively; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to the LCDR1-3 in SEQ ID NO: 24, respectively; C) the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to the HCDR1-3 in SEQ ID NO: 25, respectively; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to the LCDR1-3 in SEQ ID NO: 26, respectively; D) the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to the HCDR1-3 in SEQ ID NO: 11, 12, 13, 14, or 15, respectively; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to the LCDR1-3 in SEQ ID NO: 16, 17, or 18, respectively; E) the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are identical to the HCDR1-3 in SEQ ID NO: 1, respectively; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are identical to the LCDR1-3 in SEQ ID NO: 2, respectively; or F) the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are identical to HCDR1-3 in SEQ ID NO: 9, respectively; the LCDR1, LCDR2 and LCDR3 of the light chain variable region are identical to LCDR1-3 in SEQ ID NO: 10, respectively; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system.

3. The antibody of claim 1 or 2, wherein: a) the HCDR1 of the heavy chain variable region comprises DX 20 X1MN (SEQ ID NO: 32), wherein X1is Y or H, X 20 is Y or H; the HCDR2 comprises X2X3X4NKAX5IYTTX6YX 18 X 19 SVKG (SEQ ID NO: 33), wherein X2is F or H, X3is I or H, X4is R or H, X5is N or H, X6is E or H, X 18 is S or A, X 19 is A or D; and the HCDR3 comprises X7X8X 21 X9X 10 X 11 AMDY (SEQ ID NO: 34), wherein X7is R or H, X8is L or H, X9is Y or H, X 10 is A or H, X 11 is F or H, X 21 is L or H; and b) the LCDR1 of the light chain variable region comprises KASQNVX 22 TX 12 VA (SEQ ID NO: 35), wherein X 12 is A or H, X 22 is G or R; the LCDR2 comprises X 13 ASX 14 RYT (SEQ ID NO: 36), wherein X 13 is S or H, X 14 is N, S or H; and the LCDR3 comprises QQX 15 X 16 TX 17 PMYT (SEQ ID NO: 37), wherein X 15 is Y or H, X 16 is S or H, X 17 is Y or H; Optionally, wherein at least one of X1to X 17 , X 20 , and X 21 is H. Optionally, A) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 55, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 42; B) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 8; C) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 27, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 8; D) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 4, 20 or 21, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 7 or 22, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 8; E) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:20, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8; F) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:21, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8; G) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:22, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8; H) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:20, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:22, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8; I) the HCDR1 of the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:3, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; and, the LCDR1 of the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:38, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:39; J) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:39; K) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:44, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:45; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; L) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:19, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; M) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:46; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; N) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; O) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:40; P) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:42; Q) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:47; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; R) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:48, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; S) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:43, HCDR2 includes the amino acid sequence shown in SEQ ID NO:44, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; or T) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; Optionally, i) The heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, 4 and 5 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:55, 28 and 42 respectively. ii) The heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:27, 28, and 8, respectively; or iii) The heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, 4 and 5 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:27, 29 and 8 respectively.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

5. The antibody according to any one of claims 1 to 4, wherein: The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:53, 23, 25, 11-15, 1 or 9, or having at least 80% sequence identity with SEQ ID NO:53, 23, 25, 11-15, 1 or 9; and / or The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:54, 24, 26, 16-18, 2 or 10, or having at least 80% sequence identity with SEQ ID NO:54, 24, 26, 16-18, 2 or 10; Optionally, the anti-human FGFR2b antibody comprises: A) The heavy chain variable region as shown in SEQ ID NO:53, and the light chain variable region as shown in SEQ ID NO:54; B) The heavy chain variable region as shown in SEQ ID NO:23, and the light chain variable region as shown in SEQ ID NO:24; C) The heavy chain variable region as shown in SEQ ID NO:25, and the light chain variable region as shown in SEQ ID NO:26; D) Heavy chain variable regions as shown in SEQ ID NO:11, 12, 13, 14 or 15, and light chain variable regions as shown in SEQ ID NO:16, 17 or 18; E) The heavy chain variable region as shown in SEQ ID NO:1, and the light chain variable region as shown in SEQ ID NO:2; or F) The heavy chain variable region as shown in SEQ ID NO:9, and the light chain variable region as shown in SEQ ID NO:

10.

6. An anti-human FGFR2b antibody, said antibody having pH-dependent binding function to human FGFR2b antigen; Optionally, the antibody exhibits weaker binding activity to the FGFR2b antigen under neutral pH conditions than under acidic pH conditions. Optionally, at least one of the amino acids at positions 32, 33, 50, 51, 52, 53, 58, 95, 96, 97, 98, 99, and 100 of the heavy chain variable region of the antibody is histidine (H); and / or at least one of the amino acids at positions 32, 50, 53, 91, 92, and 94 of the amino acid sequence of SEQ ID NO:2 is histidine; the amino acid positions are the corresponding positions numbered according to the Kabat numbering system. Optionally, the variable region of the light chain of the antibody is at least one histidine in amino acids at positions 32, 50, 91 and 92, wherein the amino acid positions are the corresponding positions numbered according to the Kabat numbering system. Optionally, the variable region of the light chain of the antibody has histidine at amino acid positions 32, 50, 91 and 92, the variable region of the light chain of the antibody has histidine at amino acid positions 32 and 50, or the variable region of the light chain of the antibody has histidine at amino acid positions 50 and 92, wherein the amino acid positions are the corresponding positions numbered according to the Kabat numbering system. Optionally, the heavy chain variable region of the antibody has at least one histidine in amino acids 33, 51, 96 and 98, wherein the amino acid position is the corresponding position numbered according to the Kabat numbering system. Optionally, the heavy chain variable region of the antibody has histidine at positions 33 and 51, or the heavy chain variable region of the antibody has histidine at positions 33, 51, 96 and 98, wherein the amino acid positions are the corresponding positions numbered according to the Kabat numbering system. Optionally, a) the HCDR1 of the heavy chain variable region comprises DX 20 X1MN (SEQ ID NO: 32), wherein X1is Y or H, X 20 is Y or H; the HCDR2 comprises X2X3X4NKAX5IYTTX6YX 18 X 19 SVKG (SEQ ID NO: 33), wherein X2is F or H, X3is I or H, X4is R or H, X5is N or H, X6is E or H, X 18 is S or A, X 19 is A or D; and the HCDR3 comprises X7X8X 21 X9X 10 X 11 AMDY (SEQ ID NO: 34), wherein X7is R or H, X8is L or H, X9is Y or H, X 10 is A or H, X 11 is F or H, X 21 is L or H; and b) the LCDR1 of the light chain variable region comprises KASQNVX 22 TX 12 VA (SEQ ID NO: 35), wherein X 12 is A or H, X 22 is G or R; the LCDR2 comprises X 13 ASX 14 RYT (SEQ ID NO: 36), wherein X 13 is S or H, X 14 is N, S or H; and the LCDR3 comprises QQX 15 X 16 TX 17 PMYT (SEQ ID NO: 37), wherein X 15 is Y or H, X 16 is S or H, X 17 is Y or H; wherein at least one of X1to X 17 , X 20 and X 21 is H; Optionally, a) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:55, LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:42; b) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:27, LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; c) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:27, LCDR2 includes the amino acid sequence shown in SEQ ID NO:29, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; d) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:39; e) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:38, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:8; or f) The heavy chain variable region HCDR1 includes the amino acid sequence shown in SEQ ID NO:3, HCDR2 includes the amino acid sequence shown in SEQ ID NO:4, and HCDR3 includes the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region LCDR1 includes the amino acid sequence shown in SEQ ID NO:6, LCDR2 includes the amino acid sequence shown in SEQ ID NO:41, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:42; Optionally, the antibody comprises a heavy chain variable region as shown in SEQ ID NO:53 and a light chain variable region as shown in SEQ ID NO:54; or the antibody comprises a heavy chain variable region as shown in SEQ ID NO:23 and a light chain variable region as shown in SEQ ID NO:24; or the antibody comprises a heavy chain variable region as shown in SEQ ID NO:25 and a light chain variable region as shown in SEQ ID NO:

26.

7. The antibody according to any one of claims 1 to 6, comprising an antibody constant region; Optionally, the heavy chain constant region of the antibody is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4, and the light chain constant region of the antibody is selected from the κ or λ chain constant region. Optionally, the heavy chain constant region comprises a sequence as shown in SEQ ID NO:30 or having at least 80% sequence identity with SEQ ID NO:30, and / or the light chain constant region comprises a sequence as shown in SEQ ID NO:31 or having at least 80% sequence identity with SEQ ID NO:31; Optionally, the antibody comprises a heavy chain and a light chain, wherein: i) The heavy chain comprises a sequence as shown in SEQ ID NO:56 or having at least 80% sequence identity with SEQ ID NO:56, and / or the light chain comprises a sequence as shown in SEQ ID NO:57 or having at least 80% sequence identity with SEQ ID NO:57; or ii) The heavy chain comprises a sequence as shown in SEQ ID NO:49 or having at least 80% sequence identity with SEQ ID NO:49, and / or the light chain comprises a sequence as shown in SEQ ID NO:50 or having at least 80% sequence identity with SEQ ID NO:50; or iii) The heavy chain comprises a sequence as shown in SEQ ID NO:51 or having at least 80% sequence identity with SEQ ID NO:51, and / or the light chain comprises a sequence as shown in SEQ ID NO:52 or having at least 80% sequence identity with SEQ ID NO:52; Optionally, the antibody comprises a heavy chain as in SEQ ID NO:49 and a light chain as in SEQ ID NO:50; or the antibody comprises a heavy chain as in SEQ ID NO:51 and a light chain as in SEQ ID NO:52; or the antibody comprises a heavy chain as in SEQ ID NO:56 and a light chain as in SEQ ID NO:

57.

8. The antibody according to any one of claims 1 to 7, wherein the anti-human FGFR2b antibody is a full-length antibody or an antigen-binding fragment selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, scFab and (dsFv)2.

9. An anti-human FGFR2b antibody that competes with the antibody of any one of claims 1 to 8 for binding to human FGFR2b or that binds to the same antigenic epitope as the antibody of any one of claims 1 to 8.

10. The antibody according to any one of claims 1 to 9, wherein the antibody has at least one of the following functions (1) to (6): (1) The antibody can specifically bind to human FGFR2b antigen; optionally, the antibody can bind to human FGFR2b protein with an EC50 value of less than 10 nM, and the EC50 value is detected by ELISA; optionally, the antibody can bind to human FGFR2b protein with a KD value of less than 10 E-09 M, and the KD value is detected by surface plasmon resonance assay. (2) The antibody can specifically bind to the FGFR2b antigen of cynomolgus monkeys and / or mice; optionally, the antibody can bind to the FGFR2b protein of monkeys and / or mice with an EC50 value of less than 100 nM, and the EC50 value is detected by ELISA. (3) the antibody is capable of pH-dependent binding to human FGFR2b antigen; optionally, the antibody has a weaker binding activity to FGFR2b antigen at neutral pH (e.g., 7.4) than at acidic pH (e.g., 6.0); Optionally, the ratio of the EC50 value of the antibody binding to the human FGFR2b antigen at pH 7.4 to the EC50 value of the antibody binding to the human FGFR2b antigen at pH 6.0 is greater than 1, and the EC50 value is detected by ELISA. (4) The antibody can bind to cells expressing human FGFR2b protein; optionally, the cells are SNU-16 cells, 293T cells, or KATO-III cells expressing FGFR2b; optionally, the antibody can bind to cells expressing human FGFR2b protein with an EC50 value of less than 10 nM, wherein the EC50 value is detected by ELISA; optionally, the antibody can bind to cells expressing FGFR2b in a pH-dependent manner. Optionally, the antibody exhibits weaker binding activity against FGFR2b-expressing cells under neutral pH (e.g., 7.4) conditions compared to its binding activity against FGFR2b-expressing cells under acidic pH (e.g., 6.0) conditions. (5) The antibody can block the binding of FGF1 / 7 to FGFR2b; or (6) The antibody has ADCC activity.

11. A nucleic acid molecule encoding an anti-human FGFR2b antibody as described in any one of claims 1 to 10.

12. A vector comprising the nucleic acid of claim 11.

13. A host cell comprising the nucleic acid of claim 11 or the vector of claim 12.

14. A method for preparing an antibody binding to human FGFR2b, comprising culturing the host cell of claim 13 under conditions suitable for expressing the antibody, and recovering the antibody from the cultured host cell culture.

15. A multispecific molecule comprising the anti-human FGFR2b antibody according to any one of claims 1 to 10; Optionally, the multispecific molecule specifically binds to human FGFR2b and specifically binds to one or more other antigens.

16. A conjugate comprising the anti-human FGFR2b antibody according to any one of claims 1 to 10, and a conjugation moiety; Optionally, the coupling portion is selected from protein tags, detectable markers, or therapeutic agents; Optionally, the conjugation portion is a cytotoxic drug, and the conjugation portion is linked to the antibody of any one of claims 1 to 10 via a linker.

17. The conjugate according to claim 16, having the structure shown in formula a: Formula a: Ab-(LD)m In formula a, Ab is the anti-human FGFR2b antibody according to any one of claims 1 to 10; L is a linker unit connecting Ab and D; D is a cytotoxic drug; m is an integer selected from 1 to 10; Optionally, the anti-human FGFR2b antibody comprises a heavy chain variable region and a light chain variable region, wherein a) the heavy chain variable region comprises HCDR1 comprising DX 20 X1MN (SEQ ID NO: 32), wherein X1is Y or H, X 20 is Y or H; HCDR2 comprises X2X3X4NKAX5IYTTX6YX 18 X 19 SVKG (SEQ ID NO: 33), wherein X2is F or H, X3is I or H, X4is R or H, X5is N or H, X6is E or H, X 18 is S or A, X 19 is A or D; and HCDR3 comprises X7X8X 21 X9X 10 X 11 AMDY (SEQ ID NO: 34), wherein X7is R or H, X8is L or H, X9is Y or H, X 10 is A or H, X 11 is F or H, X 21 is L or H; and b) the light chain variable region comprises LCDR1 comprising KASQNVX 22 TX 12 VA (SEQ ID NO: 35), wherein X 12 is A or H, X 22 is G or R; LCDR2 comprises X 13 ASX 14 RYT (SEQ ID NO: 36), wherein X 13 is S or H, X 14 is N, S or H; and LCDR3 comprises QQX 15 X 16 TX 17 PMYT (SEQ ID NO: 37), wherein X 15 is Y or H, X 16 is S or H, X 17 is Y or H; wherein, at least one of X1to X 17 , X 20 and X 21 is H; Optionally, the heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:55, 28, and 42, respectively; or the heavy chain variable region of the anti-human FGFR2b antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:27, 28, and 8, respectively; Optionally, the heavy chain variable region of the anti-human FGFR2b antibody contains an amino acid sequence as shown in SEQ ID NO:53, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:54; or the heavy chain variable region of the anti-human FGFR2b antibody contains an amino acid sequence as shown in SEQ ID NO:23, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

24. Optionally, the heavy chain of the anti-human FGFR2b antibody comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain comprises the amino acid sequence shown in SEQ ID NO:57; or the heavy chain of the anti-human FGFR2b antibody comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

50.

18. The conjugate of claim 17, the linker unit L comprising one or more of the following structures: maleimidocaproyl (MC), valine-citrulline (Val-Cit), p-aminobenzyloxycarbonyl (PAB), glycine-glycine-phenylalanine-glycine (GGFG), maleimidyl, carbonyl, amino, amido, aminoacyl, -(PEG)n- (where n is independently selected from an integer from 1 to 20), C 1-20 alkylene, C 1-20 heteroalkylene, -(CºC)-, -(CH=CH)-, -0-, -S-, maleimidopropionyl (MP), methylsulfonylpyrimidinyl, valine-alanine (Val-Ala), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), dimethyl ethylenediamine (DMED), N-succinimidyl 4-(2-pyridylthio)valerate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyl disulfide) butyrate (SPDB), N-succinimidyl 3-(pyridine-2-yl dithio)-propionate (SPDP), or acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC); Optionally, the adapter unit L comprises: an extension unit for attaching an antibody to the adapter portion, a protease cleavage unit, and a spacer unit for attaching a drug to the adapter portion; the extension unit is selected from: maleimide hexanoyl, maleimide propionyl, or maleimide; the protease cleavage unit is selected from: valine-citrulline, valine-alanine, alanine-phenylalanine, phenylalanine-lysine, or glycine-glycine-phenylalanine-glycine; the spacer unit is selected from aminobenzyloxycarbonyl or a peptide bond; Optionally, the connector unit L comprises: maleimide hexanoyl, valine-citrulline, and p-aminobenzyloxycarbonyl; or comprises: maleimide and glycine-glycine-phenylalanine-glycine; Optionally, the connector unit L is MC-Val-Cit-PAB or MC-GGFG.

19. The conjugate according to claim 17 or 18, wherein the cytotoxic drug is selected from one or more of auroretamine compounds, camptothecin compounds, or maytansine alkaloids; Optionally, the cytotoxic drug D is MMAE or Dxd; Optionally, the cytotoxic drug D has the following structural formula D1: Optionally, the cytotoxic drug D has the following formula D2:

20. The conjugate according to any one of claims 17 to 19, wherein the moiety (L-D) is derived from a structure according to formula L-D1 or L-D2:

21. The conjugate of any one of claims 17-20, having the structure of Formula Ab-L-D1 or Formula Ab-L-D2: In formulas Ab-L-D1 and Ab-L-D2, Ab is the anti-human FGFR2b antibody according to any one of claims 1 to 10; n is an integer selected from 1 to 10; Optionally, the heavy chain of the anti-human FGFR2b antibody is as shown in SEQ ID NO:56, and the light chain is as shown in SEQ ID NO:57; or the heavy chain of the anti-human FGFR2b antibody is as shown in SEQ ID NO:49, and the light chain is as shown in SEQ ID NO:

50. Optionally, the heavy chain of the anti-human FGFR2b antibody is as shown in SEQ ID NO:56, and the light chain is as shown in SEQ ID NO:

57.

22. A pharmaceutical composition comprising the anti-human FGFR2b antibody of any one of claims 1 to 10, the multispecific molecule of claim 15, the conjugate of any one of claims 16 to 21, the nucleic acid molecule of claim 11, the carrier of claim 12 or the cell of claim 13, and a pharmaceutically acceptable carrier; Optionally, the pharmaceutical composition may further comprise additional agents.

23. A method of treating a tumor, the method comprising administering to a subject in need a therapeutically effective amount of the anti-human FGFR2b antibody of any one of claims 1 to 10, the multispecific molecule of claim 15, the conjugate of any one of claims 16 to 21, the nucleic acid molecule of claim 11, the carrier of claim 12, the cell of claim 13, or the pharmaceutical composition of claim 17; Optionally, the tumor is a disease associated with high expression of FGFR2b; Optionally, the subject is a human being.

24. Use of the anti-human FGFR2b antibody of any one of claims 1 to 10, the multispecific molecule of claim 15, the conjugate of any one of claims 16 to 21, the nucleic acid molecule of claim 11, the carrier of claim 12, the cell of claim 13, or the pharmaceutical composition of claim 17 in the preparation of a medicament; Optionally, the drug is used to treat tumors; Optionally, the antibody of any one of claims 1 to 10, the multispecific molecule of claim 15, the conjugate of any one of claims 16 to 21, the nucleic acid molecule of claim 11, the carrier of claim 12, the cell of claim 13, or the pharmaceutical composition of claim 17 may be used alone or in combination with other pharmaceutical agents.

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