Anti-FGFR2 antibody and multispecific binding molecule comprising same

Abstract

An isolated antibody or antigen-binding fragment that specifically binds to an FGFR2 protein, and a multispecific binding molecule comprising an isolated antibody or antigen-binding fragment that specifically binds to an FGFR2 protein. The present invention also relates to a nucleic acid encoding the isolated antibody or antigen-binding fragment that specifically binds to an FGFR2 protein or the multispecific binding molecule, and a host cell comprising the nucleic acid encoding the isolated antibody or antigen-binding fragment that specifically binds to an FGFR2 protein or the multispecific binding molecule, and a method for preparing the isolated antibody or antigen-binding fragment that specifically binds to an FGFR2 protein or the multispecific binding molecule. In addition, the present invention further relates to the diagnostic, prophylactic and / or therapeutic use of the isolated antibody or antigen-binding fragment that specifically binds to an FGFR2 protein or the multispecific binding molecule.

Description

Anti-FGFR2 antibodies and multispecific binding molecules comprising the same Technical Field

[0001] The present invention generally relates to binding molecules and their uses. More specifically, the present invention relates to isolated antibodies or antigen-binding fragments that specifically bind to FGFR2, as well as multispecific binding molecules (e.g., comprising such antibodies or antigen-binding fragments). The present invention also relates to diagnostic, prophylactic, and / or therapeutic uses of isolated antibodies or antigen-binding fragments and multispecific binding molecules that specifically bind to FGFR2. Background Art

[0002] Fibroblast growth factor receptor 2 (FGFR2) is a tyrosine kinase receptor, which has various forms of variants. There is alternative splicing in the FGFR2 extracellular domain, thereby producing a variant comprising all three Ig-like domains (alpha isotype) or only two Ig-like domains (beta isotypes) [Toshiyuki Ishiwata, Role of fibroblast growth factor receptor-2splicing in normal and cancer cells. Frontiers In Bioscience, Landmark, 23, 626-639, January 1, 2018]. According to the difference in exon sources, FGFR2 can be divided into two types, FGFR2IIIb and FGFR2IIIc. When the second half of the C-terminal amino acids of the extracellular domain III are encoded by exon 8, the FGFR2IIIb variant is generated, while when the second half of the C-terminal amino acids are encoded by exon 9, the FGFR2IIIc variant is generated [Toshiyuki Ishiwata, Role of fibroblast growth factor receptor-2 splicing in normal and cancer cells. Frontiers In Bioscience, Landmark, 23, 626-639, January 1, 2018]. FGFR2IIIb is mainly expressed in epithelial cells, and its ligand is FGF7. It also binds to FGF10, FGF22, etc. Ligand binding to FGFR2 triggers receptor dimerization, followed by phosphorylation of the kinase domain, leading to docking of adaptor proteins and activation of four key downstream pathways: RAS-RAF-MAPK, PI3K-AKT, signal transducer and activator of transcription (STAT), and phospholipase Cγ (PLCγ) [Nicholas Turner and Richard Grose, Fibroblast growth factor signaling: from development to cancer. DOI: 10.1038 / nrc2780], thereby affecting cell proliferation, survival, migration, and invasion. FGFR2IIIb is overexpressed in various cancers, including gastric, breast, lung, esophageal, pancreatic, and colorectal cancers.

[0003] Gastric cancer is the fifth most commonly diagnosed malignancy worldwide, with over one million new cases annually and 784,000 deaths worldwide in 2018 [Elizabeth C. Smyth, et al., Gastric cancer. Lancet 2020;396:635–48]. Gastric cancer can be divided into early-stage and advanced-stage gastric cancer. Because gastric cancer is often diagnosed at an advanced stage, it has a high mortality rate and is the third most common cause of cancer-related death. Despite the development of a variety of targeted therapies for gastric cancer, surgery remains the primary treatment option in clinical practice, leaving a significant unmet clinical need.

[0004] Bemarituzumab, a humanized, defucosylating monoclonal antibody targeting FGFR2IIIb, has two proven mechanisms of action: blocking FGFR2b signaling through competitive binding inhibition of FGF, and antibody-dependent cell-mediated cytotoxicity (ADCC) induced by FGFR2b-overexpressing tumor cells. In animal models, it has been shown to inhibit the growth of FGFR2b-amplified gastric cancer cells [Hong Xiang et al., Preclinical characterization of bemarituzumab, an anti-FGFR2b antibody for the treatment of cancer. MABS, 2021, Vol. 13, No. 1, e1981202 (16 pages). DOI: 10.1080 / 19420862.2021.1981202]. It is currently in Phase III clinical trials, being studied in combination with mFOLFOX6 for the treatment of advanced gastric cancer and gastroesophageal junction cancer. RLY-4008 is a highly selective FGFR2 inhibitor that can inhibit the growth of tumors harboring a variety of FGFR2 mutations in vivo. It is currently in Phase I clinical trials for unresectable or metastatic cholangiocarcinoma and other solid tumors [Jessica Casaletto et al., RLY-4008, a Novel Precision Therapy for FGFR2-Driven Cancers Designed to Potently and Selectively Inhibit FGFR2 and FGFR2 Resistance Mutations. AACR, 2021, Poster 1455]. In addition, several pan-FGFR inhibitors are already on the market. Erdafitinid is the first FDA-approved oral FGFR inhibitor. It targets FGFR isoforms 1, 2, and 3 and is indicated for the treatment of adult patients with locally advanced or metastatic bladder cancer harboring FGFR3 or FGFR2 mutations whose disease has progressed after platinum-based chemotherapy. Other clinical indications currently under investigation include non-small cell lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, and multiple myeloma.

[0005] Vascular endothelial growth factor A (VEGFA) is a heparin-binding homodimeric glycoprotein with a molecular weight of 40-45 kDa. It belongs to the growth factor family that also includes VEGFB, VEGFC, VEGFD, and placental growth factor (PlGF). The human VEGFA gene is located on chromosome 6p21.1 and consists of eight exons and seven introns. VEGF exerts its effects by interacting with two highly related tyrosine kinase receptors, VEGFR1 (also known as Flt1) and VEGFR2 (also known as Flk1 or KDR). These receptors are primarily expressed in endothelial cells, a wide range of non-endothelial cells, and cells from various tumor types [Daniela Frezzetti et al., VEGF as a potential target in lung cancer. EXPERT OPINION ON THERAPEUTIC TARGETS, 2017, Vol. 21, No. 10, 959–966]. VEGFR1 has a higher affinity for VEGFA, but VEGFA binds to VEGFR2 to induce homo- or heterodimerization, which can lead to phosphorylation of the tyrosine kinase domain and activation of several signaling proteins, including mitogen-activated protein kinase and PI-3-kinase (PI-3-K), to regulate different cellular functions, including cell proliferation, survival, and migration. It is the main pathway of angiogenesis [Chloe J, et al., Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2. Int. J. Mol. Sci. 2018, 19, 1264; Doi: 10.3390 / ijms19041264].

[0006] Bevacizumab is an FDA-approved anti-angiogenic drug. It is a recombinant humanized monoclonal antibody targeting VEGFA. It binds to VEGFA and blocks its biological activity, thereby inhibiting angiogenesis. Studies have shown that bevacizumab's inhibition of vascular endothelial growth factor ligands can prevent tumor growth. Bevacizumab has been approved for the following clinical conditions: gastric cancer, metastatic colorectal cancer, non-squamous cell lung cancer, metastatic breast cancer, and metastatic renal cell carcinoma.

[0007] Therefore, the present invention provides a need to develop antibodies against FGFR2b, as well as multispecific binding molecules constructed based thereon.

[0008] Summary of the Invention

[0009] In one aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to FGFR2 protein.

[0010] Another aspect of the present invention relates to a multispecific binding molecule, such as a bispecific binding molecule or a fusion protein, comprising an antibody or antigen-binding fragment thereof that specifically binds to an FGFR2 protein. In one embodiment, the multispecific binding molecule (e.g., a bispecific binding molecule or a fusion protein) specifically binds to an FGFR2 protein and a VEGF, such as a VEGFA, such as a VEGF165 molecule.

[0011] Other aspects of the present invention also relate to nucleic acids encoding the isolated antibodies or antigen-binding fragments that specifically bind to FGFR2 proteins, the multispecific binding molecules, and host cells containing the isolated antibodies or antigen-binding fragments that specifically bind to FGFR2 proteins, the multispecific binding molecules, or the nucleic acids, as well as methods for preparing the isolated antibodies or antigen-binding fragments that specifically bind to FGFR2 proteins, and the multispecific binding molecules. Furthermore, the present invention relates to diagnostic, prophylactic, and / or therapeutic uses of the isolated antibodies or antigen-binding fragments that specifically bind to FGFR2 proteins, and the multispecific binding molecules.

[0012] The multispecific binding molecules obtained by the present invention for FGFR2b and VEGFA, such as humanized antibody fusion proteins comprising anti-FGFR2 antibodies and VEGFR1 domains, have an antibody portion capable of blocking FGF-mediated FGFR2b signal transduction and inducing antibody-dependent cell-mediated cytotoxicity, while the VEGFR1 portion can capture VEGFA factors through a "trap" function, thereby blocking the generation of VEGFR2 downstream signals and thereby inhibiting angiogenesis in tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0014] Figures 1 and 2 show the binding of murine chimeric anti-FGFR2 antibodies (i.e., antibody E3A1, antibody D12D8, antibody F6B6, antibody A1B2, antibody A5H12, antibody F3F8, antibody G11B8, antibody H3F7-a) to gastric cancer tumor cells KATO-III expressing human FGFR2 at the cellular level.

[0015] Figures 3, 4, 5, 6, 7, and 8 show the binding detection of murine chimeric anti-FGFR2 antibodies (i.e., antibody F3F8, antibody D12D8, antibody G11B8, antibody A1B2, antibody H3F7-a, antibody E3A1, antibody A5H12, antibody F6B6) to human FGFR2 homologous family proteins FGFR1, FGFR3, and FGFR4, respectively.

[0016] FIG9 shows the binding detection of murine chimeric anti-FGFR2 antibodies (ie, antibody G11B8, antibody A1B2, antibody H3F7-a, antibody A5H12) to human FGFR2-IIIc (FGFR2c).

[0017] Figures 10 and 11 show the binding assay of humanized anti-FGFR2 antibodies (ie, humanized antibodies huG1B8-1 to huG11B8-12) to gastric cancer tumor cells KATO-III expressing human FGFR2 at the cellular level.

[0018] Figures 12, 13 and 14 respectively show that the humanized anti-FGFR2 antibody (ie, humanized antibody huG11B8-5) can block the binding of human FGFR2 to its ligands FGF7, FGF10 and FGF22.

[0019] FIG15 shows the results of a blocking test on the binding effect of VEGF165 and VEGFR2 by an anti-human FGFR2-VEGFR1 antibody fusion protein (ie, antibody huG11B8-VEGFR1) in the presence of engineered 293T cells that highly express human VEGFR2.

[0020] Figure 16 shows antibody-mediated receptor internalization and endocytosis.

[0021] FIG17 shows the results of killing of FGFR2-expressing Ba / F3 cells by NK92MI-CD16aV induced by FGFR2 antibody and its fusion protein.

[0022] Figures 18 and 20 show the changes in tumor volume after huG11B8-VEGFR1 was administered to tumor-bearing mice.

[0023] Figures 19 and 21 show the changes in body weight of tumor-bearing mice after administration of huG11B8-VEGFR1.

[0024] FIG22 shows exemplary fusion protein structures. DETAILED DESCRIPTION

[0025] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be varied. In addition, the terminology used herein is only for describing specific embodiments and is not intended to be limiting.

[0026] I. Definition

[0027] For the purpose of interpreting this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0029] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0030] As used herein, the term "comprising" means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps. In this article, when the term "comprising" is used, unless otherwise indicated, it also encompasses the situation of consisting of the recited elements, integers, or steps. For example, when it is mentioned that an antibody variable region "comprising" a specific sequence, it is also intended to encompass an antibody variable region consisting of the specific sequence.

[0031] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0032] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells can also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).

[0033] The term "FGFR2" refers to fibroblast growth factor receptor 2 (e.g., human FGFR2 or cynomolgus monkey FGFR2, such as the human FGFR2 protein under accession number NP_001138385.1 or the cynomolgus monkey FGFR2 protein under XP_028682763.1). In one embodiment, the FGFR2 of the present invention is FGFR2IIIb, such as human FGFR2IIIb. In one embodiment, the human FGFR2 protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 94 (or its amino acid sequence without a His tag), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of said sequence. In one embodiment, the cynomolgus monkey FGFR2 protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 95 (or its amino acid sequence without the His tag), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of said sequence.

[0034] As used herein, the terms "anti-FGFR2 antibody," "anti-FGFR2," "FGFR2 antibody," or "antibody that binds to FGFR2" refer to an antibody that is capable of binding to a FGFR2 protein or a fragment thereof with sufficient affinity. In one embodiment, the extent of binding of an anti-FGFR2 antibody to a non-FGFR2 protein is less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the binding of the antibody to FGFR2. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), thin-layer interferometry (BLI), biointerferometry, MSD assay, or surface plasmon resonance (SPR), or flow cytometry.

[0035] In one embodiment, the antigen-binding region that binds to FGFR2 in the anti-FGFR2 antibody, or antigen-binding fragment, or binding molecule of the present invention has high affinity binding activity to cells expressing human FGFR2 or cynomolgus monkey FGFR2 or their extracellular domains. In one embodiment, the antigen-binding region that binds to FGFR2 in the anti-FGFR2 antibody, or antigen-binding fragment, or binding molecule of the present invention has cross-reactivity to human and monkey (e.g., cynomolgus monkey) FGFR2 or their extracellular domains, i.e., it can bind to human and monkey (e.g., cynomolgus monkey) FGFR2.

[0036] The term "VEGF" as used herein refers to vascular growth factor. VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) and endocrine gland-derived VEGF (EG-VEGF). The active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members. VEGF-A exists in six isoforms generated by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. These isoforms differ primarily in their bioavailability, with VEGF165 being the predominant isoform. In some embodiments, the VEGFA of the present invention refers to VEGFA from humans, such as VEGF165 from humans. In one embodiment, the amino acid sequence of the VEGFA of the present invention is the amino acid sequence with accession number P15692 (uniprot database).

[0037] The term "VEGF receptor" or "VEGFR" refers to a cellular receptor for VEGF, typically a cell surface receptor found on vascular endothelial cells, and variants thereof that retain the ability to bind hVEGF. Three types of VEGFR subtypes, VEGFR-1 (flt-1), VEGFR-2 (KDR), and VEGFR-3, make up the VEGFR family. An example of a VEGF receptor is VEGFR1, i.e., fms-like tyrosine kinase (flt-1), a transmembrane receptor in the tyrosine kinase family. The Flt-1 receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain is involved in the binding of VEGF, while the intracellular domain is involved in signal transduction. Another example of a VEGF receptor is VEGFR-2, i.e., flk-1 receptor (also known as KDR). The binding of VEGF to the flt receptor results in the formation of at least two high molecular weight complexes with apparent molecular weights of 205,000 daltons and 300,000 daltons. It is believed that the 300,000 dalton complex is a dimer comprising two receptor molecules bound to a single molecule of VEGF. In one embodiment, the VEGFR is the VEGF-A receptor flt-1 or flk-1. In one embodiment, the VEGFR is flt-1. Wild-type VEGFR1 can be the polypeptide under reference number P17948 in UniProt. In one embodiment of the present invention, "VEGFR" or "VEGF receptor" means a polypeptide having the amino acid sequence of a wild-type human VEGF receptor, or a polypeptide having a sequence substantially identical to the wild-type amino acid sequence. The VEGFR can retain at least 75%, 80%, 85%, 90%, 95%, or 99% of the VEGF binding activity of the wild-type sequence, or be equal to or greater than the binding activity of the wild-type sequence. In one embodiment, the VEGFR1 of the present invention comprises the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 101, or comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 101 and comprises the amino acid sequence of SEQ ID NO: 100 or 102.

[0038] "Extracellular domain" or "ECD" refers to the extracellular domain of VEGFR, which can bind to VEGF (e.g., VEGFA) and block its activity. In some embodiments, ECD is determined based on the Topo logical domain of Uniprot. In some embodiments, the ECD described herein also encompasses variant ECDs that retain the function of the original extracellular domain but undergo certain modifications. In some embodiments, the ECD of a VEGFR generally comprises the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 102, or comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 102 and comprises ECD2 (e.g., having the amino acid sequence of SEQ ID NO: 100). In some embodiments, the ECD of a VEGFR comprises ECD2. In some embodiments, the ECD2 of VEGFR generally comprises, or consists of, the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 100.

[0039] The term "functional fragment" of a protein includes a portion of a protein that is a portion or segment of a complete or complete protein that has fewer amino acid residues than the complete or complete protein, but still retains the desired protein function. In some embodiments, the functional fragment comprises or consists of an extracellular domain. In some embodiments, the functional fragment comprises or consists of extracellular domain 2 (ECD2).

[0040] As used herein, a "VEGF antagonist" refers to a compound that can reduce or inhibit the activity of VEGF in vivo. A VEGF antagonist can bind to one or more VEGFs or VEGF receptors or block the binding of one or more VEGF proteins to one or more VEGF receptors. A VEGF antagonist can be, for example, a small molecule, an anti-VEGF antibody or antigen-binding fragment thereof, one or more VEGF receptors or their extracellular domains, or an anti-VEGF receptor antibody or antigen-binding fragment thereof. In one embodiment, the VEGF antagonist is any licensed anti-VEGF drug, such as brolucizumab, ranibizumab, or aflibercept. In one embodiment, the VEGF antagonist is an anti-VEGF antibody (such as brolucizumab or ranibizumab or bevacizumab or a bispecific antibody such as farximab) or an anti-VEGF DARPin (such as abicipar) or a soluble VEGF receptor or a fragment thereof, such as a functional fragment, such as ECD or ECD2.

[0041] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, known as complementarity determining regions (CDRs), interspersed with more conserved regions, known as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in binding the antibody to the antigen, but exhibits various effector functions.

[0042] In some embodiments, the antibody heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. In some preferred embodiments, the antibody heavy chain constant region HC of the present invention is

[0043] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 91;

[0044] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 91; or

[0045] (iii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 91. In some embodiments, the heavy chain constant region of the present invention further comprises one or more modifications that can alter the effector function of the antibody, such as (ADCC and / or CDC) or alter its half-life.

[0046] In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region.

[0047] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 92;

[0048] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 92; or

[0049] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 92.

[0050] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0051] The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody that has fewer amino acid residues than the intact or complete antibody and that is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, and single-domain antibodies (sdAbs). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. For example, Fab fragments can be obtained by digesting an intact antibody with papain. In addition, digesting an intact antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab', a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, edited by WE Paul, Raven Press, NY (1993)). The Fv fragment consists of the VL and VH domains of a single antibody arm. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, recombinant methods can be used to connect them via a synthetic linker peptide that enables the two domains to be produced as a single protein chain, in which the VL and VH regions are paired to form a single-chain Fv (scFv). The antibody fragment can be obtained by chemical methods, recombinant DNA methods, or protease digestion.

[0052] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0053] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor.

[0054] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA.

[0055] As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule.

[0056] As used herein, the term "target binding region" refers to the portion of a multispecific binding molecule (e.g., a fusion protein), such as a bispecific binding molecule, that binds to a specific target or antigen. The target binding region may be an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the rest of the binding molecule and may or may not bind to its target as a separate entity. The target binding region may also be a receptor or ligand, or a domain of a receptor that is capable of binding to a ligand. When the target binding region specifically binds to an antigen, the "target binding region" is also referred to as an "antigen binding region." In one embodiment, the antigen binding region for a multispecific binding molecule of the present invention comprises a VH / VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH / VL pair may be contained in a single polypeptide chain (e.g., in an scFv) or contained in two separate polypeptide chains (e.g., in a Fab heavy chain and a Fab light chain, respectively).

[0057] When referring to "the target binding region is derived from an antibody", it means that the binding domain constituting the target binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, the heavy chain variable region and / or light chain variable region of the target binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the target binding region are CDRs of the antibody.

[0058] The term "derived from" means that the fragment in the target binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion, or addition, at one or more sites. In a specific embodiment, the mutation is not in the CDR of the antibody. In a specific embodiment, the mutation is not in the variable region of the antibody.

[0059] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding regions, each of which binds to the same epitope of the same antigen. For example, the present invention provides a monospecific antibody against FGFR2.

[0060] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target binding region and a second target binding region, wherein the first target binding region binds one target and the second target binding region binds another target. Thus, the multispecific binding molecules according to the present invention comprise specificities for at least two different targets. The molecules according to the present invention also encompass multispecific molecules comprising multiple target binding regions, such as trispecific binding molecules.

[0061] In some embodiments, when the target binding regions of the binding molecules are all derived from antibodies, multispecific binding molecules are also referred to as multispecific antibodies. In the case of multispecific antibodies, the target is an antigen. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies. As used herein, the term "multispecific antibody" refers to an antibody having at least two antigen-binding regions, each of which is bound to a different epitope of the same antigen or to a different epitope of different antigens. Multispecific antibodies are antibodies that have binding specificity to at least two different antigenic epitopes. In one embodiment, bispecific antibodies are provided herein that have binding specificity for a first antigen and a second antigen. For example, the present invention provides bispecific antibodies for FGFR2 and other antigens (e.g., VEGFR2).

[0062] In some embodiments, when a multispecific binding molecule of the invention further comprises a target binding region derived from a protein other than an antibody, the multispecific binding molecule is also referred to as a "fusion protein." For example, in some embodiments, the multispecific binding molecule of the invention is a fusion protein comprising a target binding region derived from an antibody or antigen-binding fragment thereof of the invention and a target binding region derived from another protein, such as a ligand or receptor, such as a VEGF receptor or a functional fragment thereof (ECD or ECD2).

[0063] As used herein, the terms "antibody," "multispecific binding molecule," "multispecific antibody," and "fusion protein" are not mutually exclusive when referred to herein.

[0064] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions.

[0065] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGene Tics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the definition of NorthCDRs based on affinity propagation clustering using a large number of crystal structures.

[0066] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0067] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position of the Kabat numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0068] In one embodiment, the HCDRs and LCDRs in the antibodies of the invention are defined according to the Kabat scheme.

[0069] The term "Fc domain" or "Fc region" or "Fc fragment" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD classes of antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE classes of antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. As used herein, the term "Fc domain" or "Fc region" or "Fc fragment" excludes the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 (without the hinge region) and the light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In some embodiments, the heavy chain constant region Fc suitable for use in the present invention is derived from an antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3, or IgG4, preferably the constant region of IgG1. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO: 93, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence. In some embodiments of the present invention, the Fc fragments dimerize to form Fc dimers. In some embodiments, the Fc fragments heterodimerize to form Fc heterodimers. In the case where the Fc fragments heterodimerize to form heterodimers, the Fc fragments may comprise mutations for heterodimerization, such as knob into hole mutations. In some embodiments, the lysine at the C-terminus of the Fc region may be substituted with alanine.In some embodiments, Fc comprises the amino acid sequence shown in SEQ ID NO:97, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0070] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 216 (EU numbering system). In some cases, such as when constructing a Fab, the CH1 region may also include part of the hinge region, such as EPKSC. Thus, in some embodiments, the term "CH1" region refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 96).

[0071] Examples of "effector functions" of immunoglobulins include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B-cell receptor), and B-cell activation.

[0072] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0073] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a murine antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing portions of the variable region that are not involved in binding with the corresponding portions of a human antibody).

[0074] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0075] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay.

[0076] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes or be conservatively modified variants.

[0077] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.

[0078] As used herein, the term "joint" refers to any molecule that enables the direct connection of the different parts of a bispecific binding molecule. The example of a covalently linked joint between different molecular parts includes a peptide joint and a non-protein polymer, including but not limited to a copolymer of polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol. In some embodiments, the joint is a peptide joint (also referred to as a "connecting peptide"), which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or a hinge region from an immunoglobulin, for connecting the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule. For example, a peptide joint can connect the first target binding region of the binding molecule to the second target binding region. For example, a peptide joint can also connect a part of an antibody to another part of the antibody, such as connecting a light chain variable region to a heavy chain variable region. Preferably, the peptide joint has a length that is sufficient to connect two entities in a manner such that they maintain their conformations relative to each other so as not to hinder the desired activity. In one embodiment, the connecting peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (GS)n(SEQ ID NO:112), (GGS)n(SEQ ID NO:113), (GSGGS)n(SEQ ID NO:114), (GGGGS)n(SEQ ID NO:115), (GGGS)n(SEQ ID NO:116) and (GGGGS)nG(SEQ ID NO:117), wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers and other flexible linkers. In some embodiments, the peptide linker is (GGGGS)n, wherein n=an integer of 1-10, for example, 1, 2, 3, 4, 5 or 6, for example, the sequence shown in SEQ ID NO:90.

[0079] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0080] The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences that are operatively linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which recombinant polynucleotides are incorporated.

[0081] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0082] The term "treat," ...

[0083] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0084] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immunosuppressants).

[0085] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0086] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0087] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0088] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0089] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0090] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0091] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0092] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein. The term "tumor" encompasses solid tumors and hematologic tumors. The term "anti-tumor effect" or "tumor inhibitory effect" or "tumor suppression effect" refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0093] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0094] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0095] The term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody or multispecific binding molecule of the invention, and (ii) other therapeutic agent) are administered to a patient as separate entities simultaneously, without specific time restrictions, or sequentially at equal or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect in the treatment of a disease or condition that is greater than that achieved by using any one of the ingredients alone. The ingredients can each be in the form of a separate formulation, which can be the same or different.

[0096] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0097] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. In some embodiments, the tissue sample is tumor tissue. The tissue sample may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0098] II. Anti-FGFR2 Antibodies

[0099] In one aspect, the present invention provides an FGFR2 antibody having a higher binding affinity to FGFR2. In some embodiments, the FGFR2 antibody of the present invention is suitable for constructing a target binding region in a multispecific binding molecule (eg, a fusion protein).

[0100] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention bind to FGFR2 (e.g., human FGFR2 or monkey FGFR2, such as cynomolgus monkey FGFR2) with higher affinity. In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention are capable of binding to both human FGFR2 and monkey FGFR2, such as cynomolgus monkey FGFR2. In some embodiments, the affinity of the antibody is determined by thin-layer interferometry or surface plasmon resonance. In some embodiments, the anti-FGFR2 antibodies of the present invention have an equilibrium dissociation constant (K) of D ) binds to human FGFR2 or monkey FGFR2, such as cynomolgus monkey FGFR2, the K D Less than about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, 0.01 nM, or 1 pM.

[0101] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention bind to human or cynomolgus monkey FGFR2 expressed in cells. In some embodiments, the affinity of the anti-FGFR2 antibodies to human or cynomolgus monkey FGFR2 expressed in cells is determined by flow cytometry.

[0102] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention are capable of blocking the binding of FGFR2 to its ligand (eg, FGF7, FGF10, and / or FGF22).

[0103] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention are capable of mediating the internalization of FGFR2 on the surface of cells (eg, tumor cells).

[0104] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention are capable of mediating effector cell cytotoxicity (ADCT) against tumor cells.

[0105] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention are capable of killing tumor cells.

[0106] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0107] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0108] In some embodiments, an anti-FGFR2 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0109] In some aspects, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VL). In some aspects, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0110] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region HC. In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region LC. In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region HC and a light chain constant region LC.

[0111] In some embodiments, the heavy chain variable region VH of the anti-FGFR2 antibody of the present invention is

[0112] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104; or

[0113] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104; or

[0114] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions.

[0115] In some embodiments, the light chain variable region VL of the anti-FGFR2 antibody of the present invention is

[0116] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105; or

[0117] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105; or

[0118] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions.

[0119] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the anti-FGFR2 antibody of the present invention, HCDR1, HCDR2 and HCDR3 are

[0120] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH represented by SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104, or

[0121] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of (i),

[0122] Preferably, the HCDRs are defined according to Kabat.

[0123] In some embodiments, the three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, of the anti-FGFR2 antibodies of the invention are from the light chain variable region.

[0124] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105, or

[0125] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of (i);

[0126] Preferably, the LCDR is determined according to Kabat.

[0127] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, 13, 23, 28, 45 or 64, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0128] In some embodiments, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, 14, 24, 29, 46, 55, 65 or 66, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0129] In some embodiments, HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5, 15, 25, 30, 39, 47, 56 or 67, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0130] In some embodiments, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, 18, 33, 50, 59 or 82, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0131] In some embodiments, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, 19, 34, 42, 51, 60 or 83, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therefrom.

[0132] In some embodiments, LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, 20, 35, 52, 61 or 84, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0133] In some embodiments, in the anti-FGFR2 antibodies of the present invention,

[0134] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 65 or 66; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 67; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 82; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 83; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 84;

[0135] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 5; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10;

[0136] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 23; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 24; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 25; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 82; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 83; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 84;

[0137] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 38; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 39; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10;

[0138] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 28; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 29; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 30; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 33; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 34; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 35;

[0139] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20;

[0140] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:45; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:46; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:50; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:51; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:52; or

[0141] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 55; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 56; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 59; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 60; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 61.

[0142] In some specific embodiments of the present invention, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0143] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0144] the VH comprising, or consisting of, the amino acid sequence of SEQ ID NO:22, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprising, or consisting of, the amino acid sequence of SEQ ID NO:81, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0145] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:37, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:41, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0146] the VH comprising, or consisting of, the amino acid sequence of SEQ ID NO:27, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprising, or consisting of, the amino acid sequence of SEQ ID NO:32, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0147] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0148] the VH comprising, or consisting of, the amino acid sequence of SEQ ID NO: 44 or 104, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprising, or consisting of, the amino acid sequence of SEQ ID NO: 49 or 105, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0149] the VH comprising, or consisting of, the amino acid sequence of SEQ ID NO:44, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprising, or consisting of, the amino acid sequence of SEQ ID NO:49, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0150] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0151] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:54, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:58, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0152] the VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 63, 69, 71, 73, 75, 77 or 79, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 81, 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0153] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:63, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:81, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0154] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0155] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0156] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0157] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0158] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0159] The VH comprises the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0160] In some embodiments of the present invention, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises

[0161] The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 2, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 7;

[0162] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 22, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 81;

[0163] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 37, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 41;

[0164] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 27, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 32;

[0165] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 12, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 17;

[0166] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 44 or 104, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 49 or 105;

[0167] The three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 54, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 58;

[0168] The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 22, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 81; or

[0169] The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:63, 69, 71, 73, 75, 77 or 79, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:81, 86 or 88.

[0170] In some embodiments of the present invention, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises:

[0171] HCDR1 as set forth in SEQ ID NO:64, HCDR2 as set forth in SEQ ID NO:65 or 66, HCDR3 as set forth in SEQ ID NO:67; LCDR1 as set forth in SEQ ID NO:82, LCDR2 as set forth in SEQ ID NO:83, and LCDR3 as set forth in SEQ ID NO:84;

[0172] HCDR1 as set forth in SEQ ID NO:3, HCDR2 as set forth in SEQ ID NO:4, HCDR3 as set forth in SEQ ID NO:5; LCDR1 as set forth in SEQ ID NO:8, LCDR2 as set forth in SEQ ID NO:9, and LCDR3 as set forth in SEQ ID NO:31;

[0173] HCDR1 as set forth in SEQ ID NO:23, HCDR2 as set forth in SEQ ID NO:24, HCDR3 as set forth in SEQ ID NO:25; LCDR1 as set forth in SEQ ID NO:82, LCDR2 as set forth in SEQ ID NO:83, and LCDR3 as set forth in SEQ ID NO:84;

[0174] HCDR1 as set forth in SEQ ID NO: 3, HCDR2 as set forth in SEQ ID NO: 38, HCDR3 as set forth in SEQ ID NO: 39; LCDR1 as set forth in SEQ ID NO: 8, LCDR2 as set forth in SEQ ID NO: 42, and LCDR3 as set forth in SEQ ID NO: 10;

[0175] HCDR1 as set forth in SEQ ID NO:28, HCDR2 as set forth in SEQ ID NO:29, HCDR3 as set forth in SEQ ID NO:30; LCDR1 as set forth in SEQ ID NO:33, LCDR2 as set forth in SEQ ID NO:34, and LCDR3 as set forth in SEQ ID NO:35;

[0176] HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 14, HCDR3 as set forth in SEQ ID NO: 15; LCDR1 as set forth in SEQ ID NO: 18, LCDR2 as set forth in SEQ ID NO: 19, and LCDR3 as set forth in SEQ ID NO: 20;

[0177] HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, HCDR3 as shown in SEQ ID NO:47; LCDR1 as shown in SEQ ID NO:50, LCDR2 as shown in SEQ ID NO:51, and LCDR3 as shown in SEQ ID NO:52; or

[0178] HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 55, HCDR3 as shown in SEQ ID NO: 56; LCDR1 as shown in SEQ ID NO: 59, LCDR2 as shown in SEQ ID NO: 60 and LCDR3 as shown in SEQ ID NO: 61.

[0179] In some specific embodiments of the present invention, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0180] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 7;

[0181] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81;

[0182] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 41;

[0183] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 32;

[0184] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0185] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 44 or 104, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or 105;

[0186] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 44, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 49;

[0187] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 104, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 105;

[0188] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 54, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 58;

[0189] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 63, 69, 71, 73, 75, 77 or 79, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81, 86 or 88;

[0190] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81;

[0191] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 69, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 86 or 88;

[0192] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 71, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 86 or 88;

[0193] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 73, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 86 or 88;

[0194] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 86 or 88;

[0195] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 77, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 86 or 88; or

[0196] The VH contains or consists of the amino acid sequence shown in SEQ ID NO: 79, and the VL contains or consists of the amino acid sequence shown in SEQ ID NO: 86 or 88.

[0197] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0198] In some embodiments, mutations can be made in the CDR portion to eliminate deamidation, such as mutating amino acid N to other amino acids, such as including an N54E mutation in HCDR2.

[0199] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:

[0200] (i) exhibiting the same or similar binding affinity and / or specificity for FGFR2 as the antibodies of the present invention;

[0201] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to FGFR2;

[0202] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0203] (iv) competing with the antibodies of the present invention for binding to FGFR2;

[0204] (v) possess one or more biological properties of an antibody of the invention.

[0205] In some embodiments, the anti-FGFR2 antibody or antigen-binding fragment thereof of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4, or an antigen-binding fragment thereof, for example, an antibody in the form of IgG1, or an antigen-binding fragment thereof. In some embodiments, the heavy chain constant region of the anti-FGFR2 antibody of the present invention is the constant region of IgG1, IgG2, IgG3, or IgG4, for example, the constant region of IgG1.

[0206] In some embodiments, the light chain constant region of an anti-FGFR2 antibody of the invention is a kappa or lambda light chain constant region, such as a kappa light chain constant region.

[0207] In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention comprise an antibody heavy chain. In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention comprise an antibody light chain. In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain. In some embodiments, the anti-FGFR2 antibodies or antigen-binding fragments thereof of the present invention comprise two heavy chains and two light chains.

[0208] In one embodiment, the heavy chain of the anti-FGFR2 antibody of the invention

[0209] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, 11, 21, 26, 36, 43, 53, 62, 68, 70, 72, 74, 76, 78 or 106; or

[0210] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 1, 11, 21, 26, 36, 43, 53, 62, 68, 70, 72, 74, 76, 78 or 106; or

[0211] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 1, 11, 21, 26, 36, 43, 53, 62, 68, 70, 72, 74, 76, 78 or 106, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions, more preferably said amino acid changes do not occur in the variable regions.

[0212] In some embodiments, the lysine at the C-terminus of the heavy chain can be substituted with alanine, particularly when used to construct multispecific binding molecules, e.g., when linked to other target binding regions at the C-terminus with or without a linker.

[0213] In some embodiments, the light chain of the anti-FGFR2 antibody of the invention

[0214] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, 16, 31, 40, 48, 57, 80, 85, 87 or 107; or

[0215] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 6, 16, 31, 40, 48, 57, 80, 85, 87 or 107; or

[0216] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 6, 16, 31, 40, 48, 57, 80, 85, 87 or 107, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions, more preferably said amino acid changes do not occur in the variable regions.

[0217] In some specific embodiments of the present invention, the anti-FGFR2 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0218] i. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0219] ii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0220] iii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0221] iv. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0222] v. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0223] vi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 43 or 106, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 48 or 107, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0224] vii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0225] viii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0226] ix. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0227] x. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 62, 68, 70, 72, 74, 76 or 78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 80, 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0228] xi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:62, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:80, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0229] xii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:68, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0230] xiii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:70, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0231] xiv. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0232] xv. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:74, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0233] xvi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0234] xvii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0235] xviii. The heavy chain and light chain of any one of (i) to (xvii) above, wherein the C-terminal lysine of the heavy chain is substituted with alanine.

[0236] In some specific embodiments of the present invention, the anti-FGFR2 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0237] i. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 6;

[0238] ii. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 21, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 80;

[0239] iii. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 40;

[0240] iv. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 26, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 31;

[0241] v. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 16;

[0242] vi. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 43 or 106, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 48 or 107;

[0243] vii. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 43, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 48;

[0244] viii. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 106, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 107;

[0245] ix. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 53, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 57;

[0246] x. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 62, 68, 70, 72, 74, 76 or 78, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, 85 or 87;

[0247] xi. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 62, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 80;

[0248] xii. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 68, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 85 or 87;

[0249] xiii. The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 70, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 85 or 87;

[0250] xiv. The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 72, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 85 or 87;

[0251] xv. The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 74, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 85 or 87;

[0252] xvi. The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 76, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 85 or 87;

[0253] xvii. the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 78, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 85 or 87; or

[0254] xviii. The heavy chain and light chain of any one of (i) to (xvii) above, wherein the C-terminal lysine of the heavy chain is substituted with alanine.

[0255] In some embodiments, the anti-FGFR2 antibody is a monoclonal antibody.

[0256] In some embodiments, the anti-FGFR2 antibody is humanized.

[0257] In one embodiment, the anti-FGFR2 antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), heavy chain antibodies, or linear antibodies. In one embodiment, the anti-FGFR2 antibody fragment of the present invention is Fab, comprising VH and VL as described herein, as well as CH1 and CL.

[0258] In one embodiment, the anti-FGFR2 antibody of the present invention may also be a bispecific antibody or a multispecific antibody that specifically binds to FGFR2 and one or more other antigens, such as VEGFA or VEGFR2.

[0259] III. Multispecific Binding Molecules

[0260] In some embodiments, the invention relates to a multispecific binding molecule, eg, comprising one binding specificity for FGFR2 and other binding specificities for one or more molecules, eg, VEGFA, eg, VEGF165, or eg, VEGFR2.

[0261] In some embodiments, the multispecific binding molecule is a bispecific binding molecule. In some embodiments, the multispecific binding molecule, eg, a bispecific binding molecule, is a fusion protein.

[0262] Thus, one aspect of the present invention relates to a bispecific binding molecule comprising

[0263] A first target binding region and a second target binding region, wherein the first target binding region specifically binds FGFR2, and / or the second target binding region specifically binds VEGF (eg, VEGFA, eg, VEGF165; or eg, VEGFR2).

[0264] In some embodiments, the first target binding region is derived from an anti-FGFR2 antibody or an antigen-binding fragment thereof, for example, an Fab of an anti-FGFR2 antibody. In some embodiments, the anti-FGFR2 antibody or an antigen-binding fragment thereof is an anti-FGFR2 antibody or an antigen-binding fragment thereof of the present invention.

[0265] The first target binding region of the bispecific binding molecules suitable for use in the present invention may comprise or consist of an anti-FGFR2 full-length antibody or an antigen-binding fragment thereof, as long as it can specifically bind to FGFR2, including but not limited to, for example, full-length antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), heavy chain antibodies or linear antibodies that specifically bind to FGFR2.

[0266] In some embodiments, the second target binding region that specifically binds to VEGF (e.g., VEGFA, e.g., VEGF165) is derived from a VEGF antagonist. In some embodiments, the VEGF antagonist is an anti-VEGF or anti-VEGFR2 antibody or antigen-binding fragment thereof, a soluble VEGF receptor, or a fusion protein comprising the same or a functional fragment thereof.

[0267] In some embodiments, the second target binding region of the bispecific binding molecules suitable for use in the present invention may comprise or consist of a full-length anti-VEGF or anti-VEGFR2 antibody or an antigen-binding fragment thereof, as long as it can specifically bind to VEGF or VEGFR2, including but not limited to, for example, full-length antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), heavy chain antibodies or linear antibodies that specifically bind to VEGF or VEGFR2.

[0268] In some embodiments, the second target binding region of the bispecific binding molecules suitable for use in the present invention may comprise a VEGF receptor or a functional fragment thereof, e.g., the extracellular domain (ECD) of a VEGF receptor or a functional fragment thereof (e.g., extracellular domain 2). In some embodiments, the VEGF receptor is VEGFR1. In some embodiments, the second target binding region may comprise or consist of VEGFR1 or a functional fragment thereof, e.g., the extracellular domain of VEGFR1 or a fragment thereof, e.g., extracellular domain 2.

[0269] In some embodiments, the VEGF receptor is VEGFR1, which comprises or consists of the amino acid sequence set forth in SEQ ID NO:101, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:101 and comprises the amino acid sequence set forth in SEQ ID NO:100 or SEQ ID NO:102.

[0270] In some embodiments, the extracellular domain of the VEGF receptor is the extracellular domain of VEGFR1, for example, comprising or consisting of the following amino acid sequence:

[0271] The amino acid sequence set forth in SEQ ID NO: 102; an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 102; or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 102 and comprises the amino acid sequence set forth in SEQ ID NO: 100.

[0272] In some embodiments, the functional fragment of the extracellular domain of the VEGF receptor is extracellular domain 2 (ECD2). In some embodiments, extracellular domain 2 is extracellular domain 2 of VEGFR1, for example, comprising the amino acid sequence shown in SEQ ID NO: 100, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting of said amino acid sequence.

[0273] In some embodiments, when the second target binding region comprised by a multispecific binding molecule of the invention (e.g., a bispecific binding molecule) comprises a VEGF receptor or a functional fragment thereof (e.g., an extracellular domain or a fragment thereof, such as extracellular domain 2), the binding molecule is also referred to as a "fusion protein" or a "fusion protein molecule."

[0274] In some embodiments, a multispecific binding molecule (eg, a bispecific binding molecule) of the invention comprises one or two or more Fc regions, eg, comprises two Fc regions.

[0275] The Fc region fragments suitable for antibody molecules of the present invention can be any antibody Fc region. The Fc region can include native sequence Fc regions and variant Fc regions. The native sequence Fc domain covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc regions of their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of an antibody of the present invention can include two or three constant domains, i.e., CH2 domain, CH3 domain and optional CH4 domain. In some embodiments, the antibody Fc region can also have an IgG hinge region or a partial IgG hinge region at the N-terminus, such as an IgG1 hinge region or a partial IgG1 hinge region.

[0276] Preferably, the Fc region of an antibody of the present invention comprises, from N-terminus to C-terminus, CH2-CH3, or, from N-terminus to C-terminus, hinge region-CH2-CH3. In some embodiments, the Fc region suitable for use in an antibody or bispecific antibody of the present invention is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3, or human IgG4 Fc.

[0277] In some embodiments, the Fc constituting the multispecific binding molecule may comprise a mutation replacing lysine (eg, with alanine) at the C-terminus, thereby preventing the fragment (eg, the target binding region) fused to its C-terminus from being cleaved.

[0278] In one embodiment, the Fc region is from human IgG1 Fc, e.g., comprising or consisting of the amino acid sequence shown in SEQ ID NO: 93, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto.

[0279] In one embodiment, the Fc region is from human IgG1 Fc, e.g., comprising or consisting of the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence that is at least 90% identical thereto, e.g., 95%, 96%, 97%, 99% or more identical thereto, e.g., it comprises a substitution of lysine to alanine at the C-terminus.

[0280] Bispecific binding molecules

[0281] In some preferred embodiments, the present invention provides a bispecific binding molecule comprising a first target binding region that specifically binds to FGFR2 and a second target binding region that specifically binds to VEGF (eg, VEGFA, eg, VEGF165), and optionally an Fc region.

[0282] In some embodiments, the present invention provides a bispecific binding molecule comprising a first target binding region, a second target binding region, and an Fc dimer, wherein the first target binding region is a Fab fragment that specifically binds to FGFR2, and the second target binding region is a VEGF receptor or a functional fragment thereof, such as an extracellular domain, that specifically binds to VEGF.

[0283] In some embodiments, the bispecific binding molecules of the invention comprise one or two first target binding regions and one or two second target binding regions.

[0284] In some embodiments, the bispecific binding molecules of the invention comprise a first target binding region and a second target binding region. In some embodiments, the bispecific binding molecules of the invention comprise two first target binding regions and two second target binding regions. In some embodiments, the two first target binding regions are identical. In some embodiments, the two second target binding regions are identical. In some embodiments, the bispecific binding molecules of the invention comprise two identical first target binding regions and two identical second target binding regions.

[0285] In one embodiment, the bispecific binding molecule of the invention is a fusion protein.

[0286] In some embodiments, the bispecific binding molecules of the invention specifically bind to VEGF, eg, human VEGFA, eg, human VEGF165 protein, eg, with high affinity.

[0287] In some embodiments, the bispecific binding molecules of the invention or fragments thereof bind with high affinity to FGFR2 (eg, human FGFR2) and bind with higher affinity to VEGF, eg, human VEGFA, eg, human VEGF165 protein.

[0288] In some embodiments, the bispecific binding molecules of the invention have an equilibrium dissociation constant (K D ) binds to human VEGF165 protein, the K D Less than or equal to about 5 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.15 nM.

[0289] In some embodiments, the bispecific binding molecules of the invention, or fragments thereof, have an equilibrium dissociation constant (KD ) binds to human FGFR2, the K D Less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM or 5 nM.

[0290] In some embodiments, the bispecific binding molecules of the invention or fragments thereof are capable of blocking VEGF signaling.

[0291] In some embodiments, the bispecific binding molecules of the invention or fragments thereof are capable of blocking the binding of VEGF165 to its receptor VEGFR2.

[0292] In some embodiments, the bispecific binding molecules of the invention or fragments thereof mediate internalization of FGFR2 and induce cytotoxicity of effector cells.

[0293] In some embodiments, the bispecific binding molecules of the invention or fragments thereof are capable of killing tumor cells.

[0294] In some embodiments, the molecules of the invention or fragments thereof are effective in treating tumors.

[0295] In one embodiment, the first target binding region is a Fab fragment of an anti-FGFR2 antibody. In one embodiment, the second target binding region is a VEGFR receptor, such as VEGFR1, or a functional fragment thereof, such as a fragment comprising or consisting of an extracellular domain or a fragment thereof (e.g., a functional fragment such as extracellular domain 2). In one embodiment, one or more second target binding regions are respectively linked to the N-terminus of the heavy chain of the Fab fragment of the first target binding region.

[0296] In one embodiment, a bispecific binding molecule of the invention comprises a first target binding region, a second target binding region and an Fc region.

[0297] The first target binding region is a Fab fragment of an anti-FGFR2 antibody, wherein the Fab fragment comprises a Fab heavy chain VH-CH1 and a Fab light chain VL-CL,

[0298] The second target binding region comprises or consists of a VEGF receptor, such as VEGFR1, or a functional fragment thereof (the fragment comprises or consists of an extracellular domain or a fragment thereof, such as extracellular domain 2);

[0299] wherein the C-terminus of CH1 of the heavy chain of the Fab fragment is fused to the N-terminus of the Fc region, and the N-terminus of the extracellular domain of the VEGF receptor is fused to the C-terminus of the Fc region to constitute the heavy chain;

[0300] and the light chain of the Fab fragment constitutes a light chain;

[0301] Optionally, the bispecific binding molecule comprises two heavy chains and two light chains, wherein the two heavy chains may be the same or different, or the two light chains may be the same or different.

[0302] In some embodiments, the Fab fragment comprising the first target binding region is a Fab fragment of an anti-FGFR2 antibody of the present invention. In the case where the binding molecule comprises two first target binding regions, the two Fab fragments can be the same or different, for example, binding to different FGFR2 epitopes, or binding to the same FGFR2 epitope but differing in sequence.

[0303] In some embodiments, the VEGF receptor is VEGFR1. In some embodiments, the functional fragment comprises or consists of the extracellular domain of VEGFR1 or a fragment thereof. In some embodiments, the fragment of the extracellular domain comprises or consists of ECD2.

[0304] In some embodiments, the C-terminal lysine of the Fc region is replaced with an alanine. Thus, in some embodiments, the Fc region suitable for constructing the bispecific binding molecules of the present invention comprises the amino acid sequence shown in SEQ ID NO: 97, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity thereto and comprising an alanine at the C-terminus, or consists of said amino acid sequence.

[0305] In some embodiments, the fusion is direct or through a linker or connecting peptide. In some embodiments, the connecting peptide is selected from (GGGGS)n, wherein n=1, 2, 3 or 4.

[0306] In some embodiments, the heavy chain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO:89, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence;

[0307] and / or

[0308] The light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0309] IV. Modification of Anti-FGFR2 Antibodies or Fragments thereof or Multispecific Binding Molecules

[0310] The molecules of the invention (eg, anti-FGFR2 antibody molecules or fragments thereof or multispecific binding molecules) can be mutated to obtain desired properties.

[0311] For example, the FR regions contained in the variable regions of the molecules of the present invention can be modified to reduce their immunogenicity or to remove T cell epitopes.

[0312] The molecules of the invention can also be engineered to introduce one or more modifications in the constant region, e.g., the heavy chain constant region, e.g., the Fc region, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, the molecules of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody), or modified to alter its glycosylation, thereby again altering one or more functional properties of the molecule.

[0313] In one embodiment, cysteine ​​residues may be added or removed, for example to facilitate assembly of the different chains of the molecule or to increase or decrease the stability of the molecule.

[0314] In some embodiments, the heavy chain constant region or Fc region contained in the molecule of the present invention can be modified to adjust one or more properties of the antibody, such as changing its effector function, such as ADCC and / or CDC activity and / or changing its in vivo half-life, such as enhancing ADCC activity, enhancing CDC activity and / or enhancing ADCC and CDC activity, or extending the in vivo half-life of the antibody. In some embodiments, the modification is one or more amino acid substitutions.

[0315] In some preferred embodiments, the one or more amino acid substitutions occur in the heavy chain constant region sequence at positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 3 01, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439. In some preferred embodiments, the one or more amino acid substitutions occur in one or more of the heavy chain constant region sequence positions L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396 according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from one or more of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from one or more of N297A substitution, N297Q substitution, L235A substitution together with L237A substitution, L234A substitution together with L235A substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, D265A substitution, A327Q substitution, and P329A substitution according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions occur at one or more of positions 235, 239, 243, 292, 300, 330, 332, and 396 of the heavy chain constant region sequence according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from at least one of S239D, L235V, F243L, R292P, Y300L, A330L, I332E, and P396L according to the EU numbering system.

[0316] In some preferred embodiments, the heavy chain constant region has one or more sets of mutations occurring simultaneously at a combination of positions selected from the group consisting of: (1) L235 / F243 / R292 / Y300 / P396, (2) F243 / R292 / Y300 / V305 / P396, (3) D270 / K326 / A330 / K334, (4) S 239 / A330 / I332, (5) S298 / E333 / K334, (6) L234 / L235 / G236 / S239 / H268 / D270 / S298, (7) M252 / S254 / T256, (8) L234 / L235 / D265, (9) G236 / S239 / I332 and (10) S239 / I332.

[0317] In some preferred embodiments, the heavy chain constant region has one or more mutations selected from the following combinations of mutations according to the EU numbering system: (1) L235V / F243L / R292P / Y300L / P396L, (2) F243L / R292P / Y300L / V305I / P396L, (3) D270E / K326D / A330M / K334E, (4) S239D / A330L / I332E, (5) S298A / E333A / K334A, (6)L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, (7)M252Y / S254T / T256 E, (8)L234A / L235A / D265A, (9)L234F / L235E / D265A, (10)G236A / S239D / I332E and (11)S239D / I332E.

[0318] In some embodiments, the molecules of the invention may be post-translationally modified to alter the effector function of the antibody, such as by changing the glycosylation pattern of the molecules of the invention.

[0319] In some embodiments, the glycosylation of the molecules of the present invention can be modified. For example, an aglycosylated antibody or binding molecule can be prepared (i.e., the antibody or binding molecule lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the molecules of the present invention for an antigen. Such carbohydrate modifications can be achieved by, for example, altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0320] Additionally or alternatively, antibodies or binding molecules with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies or fusion proteins with increased bisecting GlcNac structures. Such altered glycosylation has been shown to increase the ADCC ability of the antibody or binding molecule. Such carbohydrate modifications can be achieved, for example, by expressing the antibody or binding molecule in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing the molecules of the present invention, thereby producing antibodies or binding molecules with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), and therefore, antibodies or binding molecules expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. Ms704, Ms705, and Ms709 FUT8 - / -Cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene (encoding a fucosyltransferase) such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating enzymes involved in α-1,6 linkages. EP 1,176,195 also describes cell lines with low or no activity for the enzyme that adds fucose to N-acetylglucosamine bound to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, thereby also resulting in hypofucosylation of antibodies expressed in this host cell (see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies or binding molecules with modified glycosylation profiles can also be produced in eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies or binding molecules with modified glycosylation profiles can be produced in plant cells, such as duckweed. Methods for producing antibodies in plant systems are disclosed in U.S. patent application filed August 11, 2006, corresponding to Alston & Bird LLP attorney docket number 040989 / 314911. PCT publication WO 99 / 54342 describes a cell line engineered to express glycosyltransferases that modify glycoproteins, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), whereby antibodies or binding molecules expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody or binding molecule (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, fucosidases can be used to cleave off fucose residues of antibodies; for example, fucosidase α-L-fucosidase removes fucosyl residues from antibodies or binding molecules (Tarentino et al. (1975) Biochem. 14: 5516-23).

[0321] Another modification of the molecules herein that the present invention contemplates is pegylation. Antibodies or binding molecules can be pegylated to, for example, extend the biological (e.g., serum) half-life of the antibodies or binding molecules. In order to pegylate antibodies or binding molecules, typically under conditions wherein one or more PEG groups are attached to the antibodies or binding molecules, the antibodies or binding molecules are reacted with polyethylene glycol (PEG), such as reactive esters or aldehyde derivatives of PEG. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any one of the forms of PEG that have been used to derivatize other proteins, such as single (C1-C10) alkoxypolyethylene glycol or aryloxypolyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody or binding molecule to be pegylated is an antibody or binding molecule without glycosylation. Methods for pegylating proteins are known in the art and can be applied to molecules of the present invention. See, for example, EPO 154 316 and EP 0 401 384.

[0322] Therefore, the present invention relates to an ADCC-enhanced antibody or fragment thereof or multispecific binding molecule. In some embodiments, the ADCC-enhanced antibody or fragment thereof or multispecific binding molecule is an antibody or fragment thereof or multispecific binding molecule with an altered glycosylation type, such as an antibody or fragment thereof or multispecific binding molecule with low or no fucosylation, or an antibody or fragment thereof or multispecific binding molecule with increased bisecting GlcNac structures.

[0323] As described herein, this can be achieved by modifying the antibody or fragment thereof or the multispecific binding molecule, or by expressing it in a host cell with altered glycosylation machinery.

[0324] V. Encoding nucleic acids and host cells containing the same

[0325] In one aspect, the invention provides nucleic acids encoding any of the above anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules or any one chain thereof.

[0326] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31, 32, 43, 44, 48, 49, 53, 54, 57, 58, 62, 63, 68-79, 80, 81, 85, 86, 87, 88, and 104-107, or a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: A nucleic acid having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in any one of NOs: 1, 2, 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31, 32, 43, 44, 48, 49, 53, 54, 57, 58, 62, 63, 68-79, 80, 81, 85, 86, 87, 88 and 104-107.

[0327] As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding molecules of the present invention can be produced by methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0328] On the one hand, the present invention provides the nucleic acid of any antibody or any antibody chain of coding above.When expressing from suitable expression vector, the polypeptide by described nucleic acid encoding can show human and / or monkey (for example cynomolgus monkey)) FGFR2 antigen binding ability.

[0329] In yet another aspect, the present invention provides nucleic acids encoding any of the above bispecific binding molecules or fusion proteins. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can display human or monkey (e.g., cynomolgus monkey) FGFR2 antigen binding ability.

[0330] In one embodiment, the nucleic acids encoding each chain of an antibody, fragment thereof, or multispecific binding molecule of the present invention can be in the same vector or in different vectors. In yet another embodiment, the nucleic acids encoding each chain of an antibody, fragment thereof, or multispecific binding molecule of the present invention can be introduced into the same or different host cells for expression. Thus, in some embodiments, the method for producing an antibody, fragment thereof, or multispecific binding molecule of the present invention comprises the steps of culturing a host cell containing nucleic acids encoding each chain under conditions suitable for expression of each chain of the molecule, thereby producing the antibody, fragment thereof, or multispecific binding molecule of the present invention.

[0331] In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0332] In one embodiment, a host cell comprising the nucleic acid or the vector is provided, for example, a vector for cloning or expressing an anti-FGFR2 antibody or fragment thereof or a multispecific binding molecule. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells such as CHO cells (e.g., CHO-S, such as ExpiCHO-S) or 293 cells (e.g., 293F or HEK293 cells) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0333] In some embodiments, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Mammalian host cell lines suitable for producing antibodies are known in the art.

[0334] The host cell may also be a cell with an altered glycosylation machinery as described herein, such as a cell in which the FUT8 gene is completely or partially knocked out or the function of the FUT8 gene or protein is completely or partially disrupted, such as a FUT8 knockout CHO cell.

[0335] VI. Production and Purification of Anti-FGFR2 Antibodies or Fragments thereof or Multispecific Binding Molecules of the Invention

[0336] In one embodiment, a method for preparing an anti-FGFR2 antibody or fragment thereof or a multispecific binding molecule of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-FGFR2 antibody or fragment thereof or multispecific binding molecule (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for expression of the anti-FGFR2 antibody or fragment thereof or multispecific binding molecule or its chains, and optionally recovering the anti-FGFR2 antibody or fragment thereof or multispecific binding molecule from the host cell (or host cell culture medium).

[0337] The polynucleotide encoding the polypeptide chains of the anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector containing one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this goal, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0338] Anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0339] The purity of the anti-FGFR2 antibodies or fragments thereof, or multispecific binding molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0340] VII. Assays for Anti-FGFR2 Antibodies or Fragments thereof or Multispecific Binding Molecules

[0341] The anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0342] In one aspect, the anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules of the invention are tested for their target (e.g., antigen, e.g., free antigen or antigen expressed on a cell) binding activity, e.g., by known methods such as thin-layer interferometry, ELISA, flow cytometry, etc. Binding to VEGF and / or FGFR2 (or VEGF and / or FGFR2 expressed on a cell) can be determined using methods known in the art, exemplary methods are disclosed herein, e.g., as described in Examples 4 or 8. In some embodiments, radioimmunoassay (RIA), thin-layer interferometry (BLI), electrochemiluminescence (ECL), surface plasmon resonance (SPR), or flow cytometry (FACS) is used for measurement.

[0343] The present invention also provides assays for identifying the biological activity of an anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule. The biological activity is selected from the properties of the anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule of the present invention.

[0344] For example, the binding activity of the molecules of the present invention to cells expressing VEGF and / or FGFR2 can be determined by methods known in the art, such as fluorescent reporter molecules and flow cytometry, or the exemplary methods disclosed in the Examples herein, such as by determining the binding of the antibody molecules of the present invention to VEGF and / or FGFR2 expressed on cells by the methods shown in Examples 3 or 7.

[0345] For example, the ability of the multispecific binding molecules of the present invention to block the binding of FGFR2 to its ligand can be determined by methods known in the art, such as the method described in Example 9.

[0346] For example, the ability of the multispecific binding molecules of the present invention to block the binding of VEGF (eg, VEGF165) to its receptor (eg, VEGFR2) can be determined by methods known in the art, such as the method described in Example 13.

[0347] For example, receptor internalization and cytotoxicity mediated by the molecules of the present invention can be determined by methods known in the art, such as the method described in Example 14.

[0348] For example, the tumor inhibitory activity or structural safety of the molecules of the present invention can be determined by methods known in the art, such as tumor cell killing experiments (such as ADCC effect), or in vivo efficacy experiments performed on tumor models, such as the methods shown in Example 15 or 16.

[0349] Cells used in any of the above in vitro assays are primary cells or cell lines, including cells that naturally express or overexpress VEGF (e.g., human or monkey (e.g., cynomolgus monkey)) or FGFR2 (e.g., human or monkey (e.g., cynomolgus monkey) FGFR2), such as cells that overexpress FGFR2, such as 293 cells or CHO cells or SNU-16 cells.

[0350] It will be appreciated that any of the above assays can be performed using a combination of the antibodies of the invention and additional active agents.

[0351] VIII. Immunoconjugates, pharmaceutical compositions, pharmaceutical combination products, and kits of anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules of the present invention

[0352] In some embodiments, the present invention provides immunoconjugates comprising any of the anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules described herein. Preferably, the immunoconjugate comprises one or more other therapeutic agents (e.g., cytotoxins or small molecule compounds) or markers. In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC).

[0353] In some embodiments, the present invention provides a composition, medicament or formulation comprising any of the anti-FGFR2 antibodies or fragments thereof, or multispecific binding molecules or immunoconjugates described herein, preferably the composition is a pharmaceutical composition.

[0354] In one embodiment, a composition, e.g., a pharmaceutical composition, comprises an anti-FGFR2 antibody or fragment thereof or a multispecific binding molecule or immunoconjugate of the invention in combination with one or more additional therapeutic agents.

[0355] The composition, medicament or preparation of the present invention may further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents and absorption delaying agents, etc.

[0356] For the use of pharmaceutical excipients and their applications, see also "Hand book of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0357] The compositions, medicaments or preparations of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injections or eye drops), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0358] Medicaments or formulations comprising the anti-FGFR2 antibodies, fragments thereof, or multispecific binding molecules described herein can be prepared by mixing an anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule or immunoconjugate of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, for example, in the form of a lyophilized formulation or aqueous solution.

[0359] The compositions or medicaments or formulations of the present invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.

[0360] The present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-FGFR2 antibody or fragment thereof or a multispecific binding molecule or immunoconjugate of the present invention, and one or more other therapeutic agents.

[0361] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0362] - a first container containing a pharmaceutical composition comprising an anti-FGFR2 antibody or fragment thereof, or a multispecific binding molecule or immunoconjugate of the present invention;

[0363] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0364] In some embodiments, the additional therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0365] IX. Uses of anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules and methods of using the same

[0366] In one aspect, the present invention relates to an anti-FGFR2 antibody or fragment thereof or a multispecific binding molecule of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) for use in therapy.

[0367] In some embodiments, the present invention relates to an anti-FGFR2 antibody or fragment thereof or multispecific binding molecule of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) for use as a medicament.

[0368] In another aspect, the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule of the present invention, or an immunoconjugate, composition, or medicament or formulation comprising the same. In some embodiments, the present invention provides a method for specifically activating T cells in a subject, comprising administering to the subject an anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule of the present invention, or an immunoconjugate, composition, or medicament or formulation comprising the same.

[0369] In some embodiments, the disease is a FGFR2-related disease, such as a disease of abnormal expression (e.g., high expression) of FGFR2. In some embodiments, the disease is a tumor, such as cancer. Cancer can be in the early, middle, or late stages or be a metastatic cancer. In some embodiments, the cancer can be a solid tumor or a hematologic tumor. In some embodiments, the cancer is gastric cancer, lung cancer such as non-small cell lung cancer, esophageal cancer, bile duct cancer (e.g., unresectable or metastatic bile duct cancer), breast cancer, pancreatic cancer, or colorectal cancer.

[0370] In some embodiments, the disease treatment would benefit from blocking the VEGF, eg, VEGFA, signaling pathway.

[0371] In some embodiments, the cancer is a FGFR2 positive cancer.In some embodiments, the cancer is characterized as in the patient (for example, in patient cancer tissue or cell) have or have the protein level and / or nucleic acid level of the FGFR2 raised (for example, compared with the protein level and / or nucleic acid level of the FGFR2 in the same tissue of healthy individuals, or compared with the protein level and / or nucleic acid level of the FGFR2 in the adjacent healthy tissue of the patient), for example, the tumor cell of the cancer has or have the protein level and / or nucleic acid level of the FGFR2 raised (for example, compared with the protein level and / or nucleic acid level of the FGFR2 in the cell of the same tissue of healthy individuals, or compared with the protein level and / or nucleic acid level of the FGFR2 in the healthy cells of the same tissue of the patient or the cell of adjacent healthy tissue).

[0372] The anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration, intrapulmonary administration and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion. Depending on whether the medication is short-term or long-term, the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0373] For the prevention or treatment of disease, the appropriate dosage of the anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0374] In other aspects, the present invention provides an anti-FGFR2 antibody or fragment thereof or a multispecific binding molecule of the present invention, or an immunoconjugate or composition or combination product comprising the same, for use as described herein, such as for preventing or treating the relevant diseases or disorders mentioned herein.

[0375] In other aspects, the present invention provides use of an anti-FGFR2 antibody or fragment thereof or a multispecific binding molecule of the present invention, or an immunoconjugate or composition or combination product comprising the same, in the production or preparation of a medicament for the uses described herein, e.g., for preventing or treating the relevant diseases or conditions mentioned herein.

[0376] In some embodiments, the anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0377] In some embodiments, the treatment modality is, for example, surgery or radiation therapy.

[0378] In some embodiments, the additional therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0379] X. Diagnosis and testing

[0380] In one aspect, the present invention also relates to methods for diagnosis and detection (eg, for diagnostic or non-diagnostic purposes) of anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules and compositions for diagnosis and detection comprising the same.

[0381] In certain embodiments, the anti-FGFR2 antibodies provided herein can be used to detect the presence of FGFR2 in a biological sample.

[0382] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid, such as blood, serum, or plasma.

[0383] In certain embodiments, the method comprises contacting a biological sample with an anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule as described herein under conditions that allow binding to FGFR2, and detecting whether a complex is formed between the anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule and FGFR2. The formation of a complex indicates the presence of FGFR2. The method can be an in vitro or in vivo method.

[0384] In certain embodiments, labeled anti-FGFR2 antibodies or fragments thereof or multispecific binding molecules are provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected moieties, such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction. In some embodiments, the label is, for example, a marker such as biotin or hFc.

[0385] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-FGFR2 antibody, fragment thereof, or multispecific binding molecule of the invention. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during or after treatment with another therapy.

[0386] In some embodiments, FGFR2 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0387] XI. Specific Implementation Plan

[0388] In one embodiment, the present invention relates to the following specific embodiments:

[0389] 1. An anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising

[0390] i. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 63, 69, 71, 73, 75, 77 or 79, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 81, 86 or 88;

[0391] ii. three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 2, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 7;

[0392] iii. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 22, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 81;

[0393] iv. three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 37, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 41;

[0394] v. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 27, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 32;

[0395] vi. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 12, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 17;

[0396] vii. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 44 or 104, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 49 or 105;

[0397] viii. three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 54, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 58; or

[0398] ix. The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 22, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 81.

[0399] 2. An anti-FGFR2 antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein

[0400] i. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 65 or 66; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 67; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 82; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 83; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 84;

[0401] ii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 5; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10;

[0402] iii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 23; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 24; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 25; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 82; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 83; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 84;

[0403] iv. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 38; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 39; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10;

[0404] v. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 28; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 29; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 30; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 33; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 34; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 35;

[0405] vi. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20;

[0406] vii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 45; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 46; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 47; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 50; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 51; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 52; or

[0407] viii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 55; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 56; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 59; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 60; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 61.

[0408] 3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region

[0409] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104; or

[0410] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104.

[0411] 4. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, comprising a light chain variable region VL, wherein the light chain variable region

[0412] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105; or

[0413] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105.

[0414] 5. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0415] i. the VH comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 63, 69, 71, 73, 75, 77 or 79, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 81, 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0416] ii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0417] iii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0418] iv. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0419] v. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0420] vi. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0421] vii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0422] viii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0423] ix. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0424] x. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0425] xi. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0426] xii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0427] xiii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0428] xiv. the VH comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 44 or 104, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 49 or 105, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; the VH comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: The amino acid sequence of NO:49, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence;

[0429] xv. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0430] xvi. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0431] 6. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0432] i. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 63, 69, 71, 73, 75, 77 or 79, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81, 86 or 88;

[0433] ii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81;

[0434] iii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 69, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88;

[0435] iv. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 71, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88;

[0436] v. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 73, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88;

[0437] vi. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88;

[0438] vii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 77, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88;

[0439] viii. the VH comprises the amino acid sequence shown in SEQ ID NO: 79 or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO86 or 88 or consists of the amino acid sequence;

[0440] ix. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 7;

[0441] x. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81;

[0442] xi. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 41;

[0443] xii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 32;

[0444] xiii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0445] xiv. The VH comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 104, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 49 or 105; the VH comprises or consists of the amino acid sequence of SEQ ID NO: 44, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 49;

[0446] xv. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 104, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 105; or

[0447] xvi. The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 54, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 58.

[0448] 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-6, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1.

[0449] 8. The antibody or antigen-binding fragment thereof of embodiment 7, wherein the heavy chain constant region

[0450] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 91; or

[0451] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 91.

[0452] 9. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-8, which comprises a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

[0453] 10. The antibody or antigen-binding fragment thereof according to embodiment 9, wherein the light chain constant region

[0454] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 92; or

[0455] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 92.

[0456] 11. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 10, wherein the antibody comprises a heavy chain, wherein the heavy chain

[0457] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, 11, 21, 26, 36, 43, 53, 62, 68, 70, 72, 74, 76, 78 or 106; or

[0458] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 1, 11, 21, 26, 36, 43, 53, 62, 68, 70, 72, 74, 76, 78 or 106; or

[0459] (iii) The heavy chain of (ii), wherein the lysine at the C-terminus of the heavy chain is substituted with alanine.

[0460] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the antibody comprises a light chain, wherein the light chain

[0461] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, 16, 31, 40, 48, 57, 80, 85, 87 or 107; or

[0462] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 6, 16, 31, 40, 48, 57, 80, 85, 87 or 107.

[0463] 13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-12, comprising a heavy chain and a light chain, wherein

[0464] i. the heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 62, 68, 70, 72, 74, 76 or 78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 80, 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0465] ii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0466] iii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0467] iv. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0468] v. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0469] vi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0470] vii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0471] viii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 85 or 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0472] ix. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0473] x. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:21, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:80, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0474] xi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:36, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0475] xii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:26, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:31, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0476] xiii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:11, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0477] xiv. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:43 or 106, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:48 or 107, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0478] xv. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:43, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0479] xvi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0480] xvii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:53, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:57, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0481] xviii. The heavy chain and light chain of any one of i-xvii, wherein the lysine at the C-terminus of the heavy chain is substituted with alanine.

[0482] 14. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-13, wherein the antibody is a humanized antibody.

[0483] 15. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-14, wherein the antibody is a monoclonal antibody.

[0484] 16. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-15, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as heavy chain variable region VHH, dAb (domain antibody), heavy chain antibody or linear antibody.

[0485] 17. The antibody or antigen-binding fragment thereof according to embodiment 16, wherein the single-chain antibody is scFv.

[0486] 18. A multispecific binding molecule comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1-17.

[0487] 19. The multispecific binding molecule according to embodiment 18 is a bispecific binding molecule.

[0488] 20. The multispecific binding molecule of embodiment 19, which is a bispecific binding molecule, such as a fusion protein, that specifically binds to FGFR2 and VEGF, such as VEGFA, such as VEGF165.

[0489] 21. The multispecific binding molecule of embodiment 20, comprising a first target binding region that specifically binds to FGFR2, and a second target binding region that specifically binds to VEGFA, wherein the first target binding region comprises the antibody or antigen-binding fragment thereof of any one of embodiments 1-17.

[0490] 22. The multispecific binding molecule of embodiment 21, wherein the second target binding region comprises a VEGF receptor or a functional fragment thereof or an antibody or an antigen-binding fragment thereof that specifically binds to VEGFR2.

[0491] 23. The multispecific binding molecule of embodiment 22, wherein the VEGF receptor is VEGFR1, and / or the functional fragment comprises or consists of:

[0492] The extracellular domain of a VEGF receptor or a fragment thereof, for example, the fragment of the extracellular domain comprises or consists of extracellular domain 2.

[0493] 24. The multispecific binding molecule of embodiment 23, wherein

[0494] The extracellular domain of VEGFR1 comprises or consists of the following amino acid sequence:

[0495] i. the amino acid sequence shown in SEQ ID NO: 102,

[0496] ii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 102; or

[0497] iii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 102 and comprises the amino acid sequence of SEQ ID NO: 100;

[0498] Or the extracellular domain 2 of VEGFR1 comprises or consists of the following amino acid sequence:

[0499] i. the amino acid sequence shown in SEQ ID NO: 100,

[0500] ii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 100; or

[0501] iii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 100 and comprises the amino acid sequence shown in SEQ ID NO: 100.

[0502] 25. The multispecific binding molecule of any one of embodiments 18-24, wherein the first target binding region is a Fab fragment of an anti-FGFR2 antibody as defined in any one of embodiments 1-17.

[0503] 26. The multispecific binding molecule of embodiment 25, wherein the Fab comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

[0504] 27. The multispecific binding molecule of embodiment 26, wherein the CH1

[0505] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 96; or

[0506] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 96.

[0507] 28. The multispecific binding molecule of any one of embodiments 18-27, wherein the multispecific binding molecule comprises an Fc region, such as an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different.

[0508] 29. The multispecific binding molecule of embodiment 28, wherein the Fc region is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably from the Fc region of IgG1.

[0509] 30. The multispecific binding molecule of embodiment 29, wherein the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 93 or an amino acid sequence that is at least 90% identical thereto, e.g., 95%, 96%, 97%, 99% or more identical thereto.

[0510] 31. The multispecific binding molecule of embodiment 29, wherein the Fc region comprises a C-terminal lysine replaced by an alanine.

[0511] 32. The multispecific binding molecule of embodiment 31, wherein the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 97 or an amino acid sequence that is at least 90% identical thereto, e.g., 95%, 96%, 97%, 99% or more identical thereto.

[0512] 33. The multispecific binding molecule of any one of embodiments 28-32, which is a bispecific binding molecule and which comprises a first target binding region, a second target binding region and an Fc region,

[0513] The first target binding region is a Fab fragment of an anti-FGFR2 antibody, wherein the Fab fragment comprises a Fab heavy chain VH-CH1 and a Fab light chain VL-CL,

[0514] The second target binding region comprises or consists of the extracellular domain of a VEGF receptor or a fragment thereof (e.g., which comprises or consists of ECD2);

[0515] wherein the C-terminus of CH1 of the heavy chain of the Fab fragment is fused to the N-terminus of the Fc region, and the N-terminus of the extracellular domain of the VEGF receptor is fused to the C-terminus of the Fc region to constitute the heavy chain;

[0516] And the light chain of the Fab fragment constitutes the light chain.

[0517] 34. The multispecific binding molecule of embodiment 33, wherein the bispecific binding molecule comprises two heavy chains and two light chains, wherein the two heavy chains may be the same or different, or the two light chains may be the same or different.

[0518] 35. The multispecific binding molecule of embodiment 33 or 34, wherein the N-terminus of the extracellular domain of the VEGF receptor is fused to the C-terminus of the Fc region via a linker, for example, the linker comprises or consists of (GGGGS)n, wherein n=1, 2, 3 or 4.

[0519] 36. The multispecific binding molecule of embodiment 35, wherein

[0520] The heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;

[0521] and / or

[0522] The light chain comprises the amino acid sequence shown in SEQ ID NO:85, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

[0523] 37. The antibody or antigen-binding fragment thereof of any one of embodiments 1-17, or the multispecific binding molecule of any one of embodiments 18-36, wherein the antibody or antigen-binding fragment thereof or the multispecific binding molecule is ADCC-enhanced, for example, it has an altered glycosylation type, for example, is hypofucosylated or afucosylated or has increased bisecting GlcNac structures.

[0524] 38. A nucleic acid molecule encoding or consisting of any one chain of the antibody or antigen-binding fragment thereof of any one of embodiments 1-17, or any one chain of the multispecific binding molecule of any one of embodiments 18-36.

[0525] 39. An expression vector comprising the nucleic acid molecule of embodiment 37. Preferably, the expression vector is pCDNA, such as pCDNA3.1.

[0526] 40. A host cell comprising the nucleic acid molecule of embodiment 37 or the expression vector of embodiment 38. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells.

[0527] 41. The host cell of claim 40, wherein the host cell is a host cell with an altered glycosylation mechanism, such as a cell in which the FUT8 gene is completely or partially knocked out or the function of the FUT8 gene or protein is completely or partially disrupted, such as a FUT8 gene knockout CHO cell.

[0528] 42. A method for preparing the antibody or antigen-binding fragment thereof of any one of embodiments 1-17 and 37 or the multispecific binding molecule of any one of embodiments 18-37, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 38 or the expression vector of embodiment 39 or the host cell of embodiment 40 or 41 under conditions suitable for expression of the antibody chains, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0529] 43. The antibody or antigen-binding fragment thereof or multispecific binding molecule prepared by the method of claim 42.

[0530] 44. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-17 and 37 or the multispecific binding molecule of any one of embodiments 18-37.

[0531] 45. A pharmaceutical composition, medicament or formulation comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-17 and 37 or the multispecific binding molecule of any one of embodiments 18-37, or the immunoconjugate of embodiment 44, and optionally a pharmaceutically acceptable excipient.

[0532] 46. ​​A pharmaceutical combination product comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-17 and 37 or the multispecific binding molecule of any one of embodiments 18-37, or the immunoconjugate of embodiment 44, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents).

[0533] 47. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 1-17 and 37, or the multispecific binding molecule of any one of embodiments 18-37, or the immunoconjugate of embodiment 44, or the pharmaceutical composition or formulation of embodiment 45; or the pharmaceutical combination product of embodiment 46.

[0534] 48. The method of embodiment 47, wherein the cancer is an FGFR2-positive cancer, e.g., the cancer is characterized by having or having elevated protein levels and / or nucleic acid levels of FGFR2 in tumor cells of the cancer (e.g., compared to the protein levels and / or nucleic acid levels of FGFR2 in cells of the same tissue of a healthy individual, or compared to the protein levels and / or nucleic acid levels of FGFR2 in healthy cells of the same tissue or cells of adjacent healthy tissue of the patient).

[0535] 49. The method of embodiment 47 or 48, wherein the cancer is a solid tumor or a hematological tumor, such as gastric cancer, lung cancer such as non-small cell lung cancer, esophageal cancer, bile duct cancer (such as unresectable or metastatic bile duct cancer), or breast cancer, pancreatic cancer or colorectal cancer.

[0536] 50. The method of any one of embodiments 47-49, wherein the method further comprises administering the drug in combination with other therapies, such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint molecule inhibitors or agonists).

[0537] 51. A method for detecting the presence of FGFR2 in a biological sample, comprising:

[0538] (i) contacting a biological sample with the antibody or antigen-binding fragment thereof of any one of embodiments 1-17 and 37, or the multispecific binding molecule of any one of embodiments 18-37, under conditions that allow binding to FGFR2,

[0539] (ii) detecting whether a complex is formed between the antibody or multispecific binding molecule and FGFR2,

[0540] The formation of the complex indicates the presence of FGFR2.

[0541] Example

[0542] Example 1. Generation of anti-human FGFR2 antibodies

[0543] Hybridomas of the anti-human FGFR2 antibodies of the present invention can be prepared by the method described below. However, the method for preparing the anti-human FGFR2 antibodies of the present invention is not limited to this method, and can also be prepared by other methods known in the art.

[0544] 1.1 Antigen expression

[0545] The extracellular region gene sequences of human FGFR2 protein (NP_001138385.1) and cynomolgus macaque FGFR2b protein (XP_028682763.1) were codon-optimized and synthesized by Universal Biosystems (Anhui) Co., Ltd. After adding a 6× His tag, they were inserted into the expression vector pcDNA3.1(+) to obtain plasmids encoding huFGFR2-his (SEQ ID NO: 94) and cynoFGFR2-his (SEQ ID NO: 95). The two plasmids were then transfected into ExpiCHO-S cells.

[0546] Specifically, use ExpiCHO according to the manufacturer's product manual. TM ExpiCHO-S cells were transfected with the plasmids to express human FGFR2-his and cynomolgus macaque FGFR2-his proteins using an expression system (ThermoFisher, Cat# A29133). Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the expressed histidine-tagged antigen was purified using a NiSepharose High Performance / NiSepharose Excel affinity chromatography system (Cytiva, 71502810-EH). The purified antigen was concentrated and sterile filtered. The antigen purity was determined by SDS-PAGE and size exclusion, indicating a purity >90% and suitable for use in further experiments.

[0547] 1.2 Animal immunization

[0548] Adopt people's above-mentioned human FGFR2-his as immunogen, immunize 5 BALB / c mice (Weitong Lihua)+5 SJL mice (Shanghai Slake). 10 mice are carried out biweekly, a total of four immune injections (Table 1). Respectively 7 days after the second immunization and 7 days after the third immunization, mice are carried out venous blood sampling, and the antibody titer in the serum of immune animals is detected by ELISA. After observing that the antibody titer fully rises, the last booster immunization is carried out. Finally, two immune mice are selected for obtaining cells that produce antibodies. Four days after the last booster immunization, the spleen of immune mice is taken, and the lymphocytes separated from the spleen are fused with myeloma cells to produce hybridomas.

[0549] Table 1. Immunization schedule for BALB / c mice

[0550] 1.2 Screening of target hybridomas

[0551] Cell culture supernatants from the 4,800 hybridomas from Example 1.1 were screened using an ELISA assay. Following the ELISA assay, 198 positive supernatants were selected for flow cytometry analysis against the KATO-III cell line (a human gastric cancer cell line). Hybridoma clones corresponding to positive supernatants that bound to KATO-III cells were confirmed by a subsequent ELISA assay. Finally, these ELISA-positive hybridoma clones were selected for the next subcloning stage.

[0552] 1.3 Subcloning of parental hybridomas

[0553] The selected hybridoma cell line (parent clone) was subcloned to ensure monoclonality. Subcloning was performed by re-plating the parent clone using a single-step cloning system. The positive subclones from Example 1.2 were transferred to 96-well cell culture plates. The subclones were screened by ELISA using human FGFR2-his as an antigen to obtain hybridomas that produced antibodies that bound to human FGFR2. The antibodies produced were named: mouse antibody F3F8, mouse antibody G11B8, mouse antibody H3F7-a, mouse antibody E3A1, mouse antibody D12D8, mouse antibody F6B6, mouse antibody A1B2, and mouse antibody A5H12.

[0554] 1.4 Sequencing of anti-FGFR2 antibodies produced by hybridomas

[0555] Antibody sequencing was completed by GenScript NGS sequencing. The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) are shown in Table 2.

[0556] Table 2: Amino acid sequences of heavy and light chain variable regions of anti-FGFR2 antibodies (SEQ ID NOs are represented by numbers)

[0557] Example 2: Construction, expression and purification of anti-FGFR2 chimeric antibodies

[0558] The DNA sequences of the murine antibodies in Table 2 were codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. The genes encoding the VH regions of each antibody were inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 91) to obtain plasmids encoding the heavy chain of each anti-FGFR2 antibody; the genes encoding the VL regions of each antibody were inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human κ light chain constant region (SEQ ID NO: 92) to obtain plasmids encoding the light chain of each anti-FGFR2 antibody.

[0559] Plasmids encoding the heavy and light chains of each anti-FGFR2 antibody were co-transfected into ExpiCHO-S cells to express the murine chimeric antibodies shown in Table 2.

[0560] Specifically, use ExpiCHO according to the manufacturer's product manual. TM ExpiCHO-S cells were co-transfected with plasmids encoding the heavy and light chains of the anti-FGFR2 antibodies shown in Table 2 using the Expression System (ThermoFisher, Cat# A29133) to express the anti-FGFR2 chimeric antibodies, as well as a control antibody. Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the antibodies expressed and secreted in the supernatant were purified using the MabSelectSure Protein A affinity chromatography system (GE Healthcare). The purified antibodies were concentrated and sterile filtered. The purity of the antibody protein was determined by SDS-PAGE and size exclusion. The results showed that the antibody purity was >90% and was suitable for use in the next step of the experiment.

[0561] Bemarituzumab (a fully human monoclonal antibody against FGFR2) (heavy chain amino acid sequence is SEQ ID NO: 98, light chain amino acid sequence is SEQ ID NO: 99) was used as a control antibody, and the sequences were from PCT Publication No. WO2018213304A1. The heavy and light chains were constructed into the same expression vector to obtain plasmids encoding the heavy and light chains of the antibody. The plasmid was transfected into CHO-K1 (ATCC, CCL-61) cells with the Fut-8 gene knocked out. TM) cell lines, and the obtained control antibody is hereinafter referred to as Bemarituzumab.

[0562] Example 3 Anti-FGFR2 chimeric antibody binds to human tumor cells

[0563] Cell binding was detected by flow cytometry to determine whether the anti-FGFR2 chimeric antibody of the present invention could bind to tumor cells expressing human FGFR2 protein (eg, human gastric cancer cell line KATO-III).

[0564] The semi-adherent KATO-Ⅲ (Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu229) cells were enzymatically digested to obtain a single cell suspension. The suspension was centrifuged at 300×g for 4 minutes at room temperature and the culture medium was discarded. The cell pellet was resuspended in PBS and 1×10 5 Cells were seeded into 96-well V-bottom plates (Nice, Catalog No. 701211) and centrifuged at 300×g for 4 minutes at room temperature, after which the supernatant was discarded. The cell pellet was serially diluted and resuspended with the resulting mouse chimeric anti-FGFR2 antibody (initial concentration: 25μg / mL, followed by 4-fold serial dilutions, totaling 8 concentrations) and incubated at 4°C for 30 minutes. The cells were washed once with PBS and centrifuged at 300×g for 4 minutes, after which the supernatant was discarded. The cell pellet was resuspended in a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific (Jackson ImmunoResearch, Catalog No. 109-116-098), and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS and resuspended, and the fluorescence signal in the PE channel was detected using a flow cytometer (Beckman CytoFlex). The results are shown in Figures 1 and 2.

[0565] Figure 1 shows that murine chimeric anti-FGFR2 antibodies (i.e., antibodies E3A1, D12D8, F6B6, A1B2, and A5H12) can all bind to KATO-III cells expressing human FGFR2, and the EC50s for antibody binding to cells are 8.321 nM, 7.373 nM, 14.43 nM, 0.3364 nM, and 2.299 nM, respectively.

[0566] Figure 2 shows that murine chimeric anti-FGFR2b antibodies (i.e., antibodies F3F8, G11B8, and H3F7-a) can bind to KATO-III cells expressing human FGFR2, and the EC50s for antibody binding to cells are 3.969 nM, 0.9494 nM, and 1.086 nM, respectively.

[0567] Example 4 Binding of anti-FGFR2 antibodies to the FGFR family

[0568] The ELISA method was used to detect the binding ability of the obtained anti-FGFR2 antibodies to FGFR family members.

[0569] Human FGFR1 protein (ACROBiosystems, Catalog No.: FG1-H5223), human FGFR3 protein (SinoBiological, Catalog No.: 16486-H08H), and human FGFR4 protein (SinoBiological, Catalog No.: 10538-H08H) were immobilized on 96-well plates by incubation overnight at 4°C. The 96-well plates were then blocked by incubation with 1% BSA in PBS at 37°C for 1 hour. After blocking, antibodies (i.e., F3F8, D12D8, G11B8, A1B2, H3F7-a, E3A1, A5H12, F6B6, and Bemarituzumab) were incubated with each FGFR family protein immobilized on the 96-well plates at 37°C for 1 hour. After incubation, the 96-well plates were washed three times with PBST, incubated with peroxidase-labeled goat anti-human FcIgG (Jackson ImmunoResearch, Cat. No. 109-035-098) diluted 1 / 15,000 in binding buffer at 37°C for 1 hour, washed again, developed with TMB and stopped using 1 M H2SO4.

[0570] Representative binding curves of F3F8, D12D8, G11B8, A1B2, H3F7-a, E3A1, A5H12, F6B6, and Bemarituzumab to human FGFR family proteins are shown in Figures 3 , 4 , 5 , 6 , 7 , and 8 , respectively.

[0571] As can be seen from Figures 3, 5, 6 and 8, F3F8, D12D8, G11B8, A1B2, H3F7-a, E3A1, A5H12, F6B6 and Bemarituzumab do not bind to FGFR1 and FGFR4.

[0572] As can be seen from Figures 4 and 7 , except for A5H12 and E3A1 which have a certain binding to FGFR3, the other antibodies do not bind to the same family protein FGFR3.

[0573] Example 5: Binding of anti-FGFR2 antibodies to human FGFR2c protein

[0574] The binding performance of the FGFR2 antibody obtained in Example 2 to the human FGFR2IIIc protein was detected using the ELISA method.

[0575] Human FGFR2IIIc (KACTUS, Catalog No.: FGR-HM2CD) was fixed to a 96-well plate by incubation overnight at 4°C. The 96-well plate was then blocked by incubation with 1% BSA in PBS at 37°C for 1 hour. After blocking, antibodies (i.e., G11B8, A1B2, H3F7-a, A5H12, and Bemarituzumab) were added and incubated with each FGFR family protein fixed to the 96-well plate at 37°C for 1 hour. After incubation, the 96-well plate was washed three times with PBST, incubated with a peroxidase-labeled goat anti-human IgG Fab secondary antibody (Jackson Immuno Research, Catalog No.: 109-035-097) diluted 1 / 15,000 in binding buffer at 37°C for 1 hour, washed again, developed with TMB, and stopped using 1M H2SO4.

[0576] Representative binding curves of G11B8, A1B2, H3F7-a, A5H12, and Bemarituzumab binding to FGFR2c protein are shown in FIG9 .

[0577] As shown in FIG9 , A1B2 can bind to FGFR2-IIIc, while other antibodies do not bind to FGFR2c.

[0578] Example 6: Humanization and Characterization of Murine Anti-FGFR2 Antibodies

[0579] 6.1 Humanization of mouse anti-FGFR2 antibody

[0580] The obtained antibody G11B8 was humanized. Specifically, the sequence of antibody G11B8 was searched and compared in the IMGT database, and the human germline gene sequence IGHV1-69-2*01 with high homology to the heavy chain variable region of antibody G11B8 was obtained as the humanized framework for the heavy chain variable region, and the human germline gene sequence IGKV1-33*01 with high homology to the light chain variable region was obtained as the humanized framework for the light chain variable region. The CDRs in the heavy and light chain variable regions of antibody G11B8 (the boundaries of the three CDRs in the sequence were defined by IMGT) were transplanted into the corresponding humanized frameworks to form the humanized antibody G11B8 variable region. In order to maintain the affinity of the humanized antibody for G11B8, the obtained humanized antibody variable region needs to be backmutated.

[0581] The sequences of the humanized heavy chain variable region and humanized light chain variable region were obtained. The variable region amino acid sequences were sent to Universal Biosystems (Anhui) Co., Ltd. for codon optimization and gene synthesis. The gene encoding the humanized VH region was inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 91) to obtain a plasmid encoding the full-length heavy chain of the anti-FGFR2 humanized antibody. The gene encoding the humanized VL region was inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human kappa light chain constant region (SEQ ID NO: 92) to obtain a plasmid encoding the full-length light chain of the anti-FGFR2 humanized antibody. Similarly, A1B2 was humanized and the corresponding expression plasmids were constructed. Table 4 lists the names and heavy chain variable region sequences of each heavy chain, as well as the names and light chain variable region sequences of each light chain after humanization of the murine antibodies G11B8 and A1B2.

[0582] Table 3. Names of heavy and light chains and variable region sequences of humanized antibodies

[0583] 6.2 Expression and Purification of Humanized Anti-FGFR2 Antibodies

[0584] Similar to Example 2, plasmids encoding the full-length heavy chain of the anti-FGFR2 humanized antibody and plasmids encoding the full-length light chain of the anti-FGFR2 humanized antibody were combined (Table 4) and co-transfected into ExpiCHO-S cells to express each anti-FGFR2 humanized antibody, and the corresponding purification was performed as described in Example 2. The purified anti-FGFR2 humanized antibody was concentrated and sterile filtered, and the purity of the anti-FGFR2 humanized antibody was detected by SDS-PAGE and size exclusion chromatography (SEC).

[0585] Table 4: Humanized antibody expression combination (SEQ ID NO is represented by numbers)

[0586] 6.3 Physicochemical Analysis of Humanized Anti-FGFR2 Antibodies

[0587] The purity of the obtained humanized anti-FGFR2 antibodies listed in Table 4 was confirmed by size exclusion chromatography. Specifically, 20 μg of each humanized anti-FGFR2 antibody sample listed in Table 4 was injected onto a TSKG3000SWXL column using 100 mM sodium phosphate + 100 mM Na₂SO₄ (pH 7.0) as the running buffer. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data was analyzed using OpenLAB software. The results demonstrated that the purity of each humanized anti-FGFR2 antibody listed in Table 5 was >90%, and therefore suitable for use in the next step of the experiment.

[0588] Example 7 Binding of humanized anti-FGFR2 antibodies to tumor cell lines

[0589] Cell binding was detected by flow cytometry to determine whether the anti-FGFR2 humanized antibody of the present invention could bind to tumor cells expressing human FGFR2b protein.

[0590] The semi-adherent KATO-Ⅲ (Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu229) cells were enzymatically digested to obtain a single cell suspension. The suspension was centrifuged at 300×g for 4 minutes at room temperature and the culture medium was discarded. The cell pellet was resuspended in PBS and 1×10 5 Cells were seeded into 96-well V-bottom plates (Nice, Catalog No. 701211) and centrifuged at 300 × g for 4 minutes at room temperature, after which the supernatant was discarded. The cell pellet was resuspended in a serial dilution of a humanized anti-FGFR2 antibody (initial concentration: 20 μg / mL or 25 μg / mL, followed by 4-fold dilutions for a total of 8 concentrations) and incubated at 4°C for 30 minutes. A chimeric antibody or bemarituzumab was used as the positive antibody, following the same dilution schedule, and the cell pellet was resuspended and incubated. The cells were washed once with PBS, centrifuged at 300 × g for 4 minutes, and the supernatant discarded. The cell pellet was resuspended in a 1:200 dilution of a fluorescent secondary antibody, R-PE-conjugated Affini Pure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson Immuno Research, Catalog No. 109-116-098), and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS, then resuspended and the fluorescence signal in the PE channel was detected using a flow cytometer (Beckman CytoFlex). The results are shown in Figures 10 and 11. The results show that all anti-FGFR2 humanized antibodies can bind to KATO-III cells, and the binding ability of the humanized antibodies is comparable to that of the chimeric antibody and the positive control antibody.

[0591] Example 8: Affinity detection of humanized monoclonal antibodies to human FGFR2 and cynomolgus monkey FGFR2

[0592] In this experiment, the binding affinity of huG11B8-5 humanized monoclonal antibody to human FGFR2-his protein (SEQ ID NO: 94) and cynomolgus monkey FGFR2-his protein (SEQ ID NO.: 95) was detected using ForteBio Octet RED96e according to the manufacturer's instructions.

[0593] Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was placed in running buffer (1× PBS, XiGene, Catalog No. XG3650, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate according to the following steps:

[0594] a) Equilibrate the baseline with running buffer for 180 s;

[0595] b) Add humanized protein diluted in running buffer to a final concentration of 5 μg / mL and solidify for 200 s;

[0596] c) Equilibrate the baseline with running buffer for 300 s;

[0597] d) Add the following concentrations of human FGFR2-his protein or cynomolgus monkey FGFR2-his protein diluted in running buffer to each well: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM. Bind for 200 s and dissociate for 600 s.

[0598] e) Regeneration solution (0.01M Gly-HCl, pH 1.5) for 30 seconds. The experimental data were fitted and calculated using the 1:1 binding model using FortebioDataAnalysis software;

[0599] Table 5 summarizes the binding affinity of huG11B8-5 humanized mAb to human FGFR2 extracellular protein and cynomolgus monkey FGFR2 extracellular protein.

[0600] Table 5 Binding affinity of huG11B8-5 humanized mAb to human FGFR2 extracellular protein and cynomolgus monkey FGFR2 extracellular protein

[0601] Table 5 shows that the humanized antibodies of the present invention can specifically bind to human FGFR2 extracellular protein or cynomolgus monkey FGFR2 extracellular protein with high affinity.

[0602] Example 9 Humanized monoclonal antibody ligand blocking

[0603] The ELISA method was used to detect the ability of the obtained huG11B8-5 humanized monoclonal antibody to block the binding of FGFR2 to the ligand.

[0604] Human FGF7 protein (SinoBiological, Catalog No.: 10210-H07E), human FGF10 protein (SinoBiological, Catalog No.: 10573-HNAE), and human FGF22 protein (R&D Systems, Catalog No.: 3867-FG-035) were immobilized onto 96-well plates by incubation overnight at 4°C. The 96-well plates were then blocked by incubation with 1% BSA in PBS at 37°C for 1 hour. After blocking, equal volumes of huG11B8-5 and FGFR2-his were mixed and immobilized onto the 96-well plates and incubated at 37°C for 1 hour. After incubation, the 96-well plate was washed three times with PBST, incubated with peroxidase-labeled mouse anti-His-Tag IgG (Chemistry, Cat. No. CW0285M) diluted 1 / 10000 in binding buffer at 37°C for 1 hour, washed again, developed with TMB and the reaction was terminated using 1M H2SO4.

[0605] The curves of huG11B8-5 blocking the binding of FGFR2 protein to the ligand are shown in Figures 12, 13 and 14, respectively.

[0606] As shown in Figures 12, 13 and 14, huG11B8-5 can block the binding of FGFR2 to the ligands FGF7, FGF10 and FGF22.

[0607] Example 10 Classification and Identification of Binding Epitopes of Humanized Monoclonal Antibodies to Human FGFR2 Protein

[0608] In this experiment, according to the manufacturer's instructions, ForteBio Octet RED96e was used to detect whether huG11B8-5 humanized mAb, huA1B2-3 humanized mAb, and Bemarituzumab bind to the same epitope as human FGFR2-his protein (Y210630, SEQ ID NO: 94).

[0609] Briefly, a HISK sensor (ForteBio, Catalog No. 18-5120) was placed in running buffer (1X PBS, XiGene, Catalog No. XG3650, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate according to the following steps:

[0610] a) Equilibrate the baseline with running buffer for 180 s;

[0611] b) adding human FGFR2-his protein diluted with running buffer to a final concentration of 5 μg / mL and solidifying to 0.3 nM;

[0612] c) Equilibrate the baseline with running buffer for 30 seconds;

[0613] d) Humanized mAbs huG11B8-5, huA1B2-3, and Bemarituzumab diluted to 200 nM in running buffer were added to each well as the first binding antibody, and allowed to bind for 180 s and equilibrate to the baseline for 30 s.

[0614] e) Humanized mAbs huG11B8-5, huA1B2-3, and Bemarituzumab were diluted to 200 nM in running buffer as the second binding antibody, and the buffer wells were set as reference wells for subtraction.

[0615] e) Regeneration solution (0.01 M Gly-HCl, pH 1.5) for 30 seconds. Experimental data were analyzed using process epitope;

[0616] Table 6 analyzes the binding epitope overlap between humanized mAb huG11B8-5, humanized mAb huA1B2-3 and Bemarituzumab and human FGFR2-his protein.

[0617] Table 6 Classification of binding epitopes of humanized mAbs huG11B8-5, huA1B2-3 and Bemarituzumab to human FGFR2-his protein

[0618] Table 6 shows that the epitopes bound to human FGFR2-his protein by the humanized antibodies huG11B8-5, huA1B2-3 and Bemarituzumab of the present invention can be divided into two categories: huG11B8-5 and Bemarituzumab belong to the same category, and huA1B2-3 belongs to another category.

[0619] Example 11 Construction of Antibody-Fusion Protein

[0620] This application uses standard construction methods to obtain an antibody fusion protein with the structure shown in Figure 22:

[0621] huG11B8-VEGFR1 heavy chain: The nucleotide sequence encoding VEGFR1 domain 2 (SEQ ID NO: 100) was synthesized and ligated to the C-terminus of the huG11B8H-3 sequence (with a linker between the fragments: GGGGSGGGGSGGGGS (SEQ ID NO: 90)). To prevent breakage and substitution, the terminal lysine of the Fc residue was replaced with alanine to obtain an expression vector for the anti-FGFR2-VEGFR1 fusion protein heavy chain. The anti-FGFR2-VEGFR1 fusion protein heavy chain comprises, from N-terminus to C-terminus, an anti-FGFR2 heavy chain (with the terminal K mutated to A) and VEGFR1.

[0622] huG11B8-VEGFR1 light chain: huG11B8L-1 (SEQ ID NO: 85)

[0623] Table 7 Antibody fusion protein

[0624] The obtained heavy and light chain expression vectors (see Table 6) were co-transfected into ExpiCHO-S cells and expressed under appropriate conditions to obtain a bispecific antibody protein. The expression, purification, and preliminary analysis procedures were the same as in Example 2. The resulting fusion protein is hereinafter referred to as huG11B8-VEGFR1. Similarly, VEGFR1D2-ISO (heavy chain: SEQ ID NO: 110; light chain: SEQ ID NO: 111) was expressed as described above.

[0625] In addition, ADCC-enhanced huG11B8-VEGFR1 and G11B8 antibodies, Bevacizumab analogs (heavy chain: SEQ ID NO: 108; light chain: SEQ ID NO: 109), referred to herein as huG11B8-VEGFR1-F and G11B8-F, were also expressed. The specific preparation method was the same as Bemarituzumab in Example 2.

[0626] Example 12 Detection of affinity between antibody-fusion protein and FGFR2 and VEGF165

[0627] In this experiment, the binding affinity of huG11B8-VEGFR1-F antibody fusion protein to human FGFR2-his protein (SEQ ID NO: 94) and human VEGF165 protein (ACROBiosystems, Cat. No. VE5-H5248) was detected using ForteBioOctet RED96e according to the manufacturer's instructions.

[0628] Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was placed in running buffer (1x PBS, XiGene, Catalog No. XG3650, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate according to the following steps:

[0629] a) Equilibrate the baseline with running buffer for 180 s;

[0630] b) Add antibody-fusion protein diluted in running buffer to a final concentration of 5 μg / mL and solidify for 200 s;

[0631] c) Equilibrate the baseline with running buffer for 300 s;

[0632] d) Add the following concentrations of human FGFR2-his protein or human VEGF165 protein diluted in running buffer to each well: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM. Bind for 200 s and dissociate for 600 s.

[0633] e) Regeneration solution (0.01M Gly-HCl, pH 1.5) for 30 seconds. The experimental data were fitted and calculated using the 1:1 binding model using FortebioDataAnalysis software;

[0634] Table 8 summarizes the binding affinity of huG11B8-VEGFR1-F antibody-fusion protein to FGFR2-his and VEGF165 proteins.

[0635] Table 8: Binding affinity of huG11B8-VEGFR1-F antibody-fusion protein to human FGFR2 and VEGF165 protein

[0636] Table 8 shows that the antibody-fusion protein of the present invention can specifically bind to human FGFR2 protein or human VEGF165 protein with high affinity.

[0637] Example 13 huG11B8-VEGFR1 dual antibody inhibits the binding of VEGF165 to VEGFR2

[0638] The eukaryotic expression vector encoding the human VEGFR2 (Uniprot: P35968) gene and the NFAT-luc2P reporter gene element (Promega, Cat#E8481) were transfected into 293T cells using Lipofectamine™ 2000 (Invitrogen, Cat#11668019) transfection reagent according to the manufacturer's instructions. 48 hours after transfection, the cells were screened with 0.3 μg / mL puromycin (Invitrogen, Cat#A1113802) and 85 μg / mL hygromycin B (Invitrogen, Cat#10687010) to obtain a 293T polyclonal stable cell line that highly expresses human VEGFR2 and NFAT-luc2P. After limiting dilution of the polyclonal cells, the monoclonal cell line 293T / NFAT / VEGFR2 that stably and highly expresses NFAT-luc2P and human VEGFR2 was obtained.

[0639] 293T / NFAT / VEGFR2 cells were harvested by digestion and centrifugation. The supernatant was discarded and the cell pellet was resuspended in assay medium (90% DMEM + 10% fetal bovine serum). Counted using a Countstar cell counter, the cell suspension was diluted to 8×10⁵ / mL in assay medium. The test fusion protein was prepared at 3× the final assay concentration using assay medium containing 6 ng / mL VEGF165. The test antibody (huG11B8-VEGFR1, bevacizumab analog) was diluted to a final assay concentration of 5 nM using a two-fold serial dilution series for a total of nine concentrations.

[0640] Inoculate the 293T / NFAT / VEGFR2 cell suspension into a 384-well plate at 25 μL / well. Add the gradient antibody dilution solution at 25 μL / well. Centrifuge the entire plate at 600 rpm for 30 seconds to remove any liquid from the well walls. Incubate in a 37°C, 5% CO2, saturated humidity incubator for 6 hours. Thaw Bio-lite Buffer (Brand: Novezan; Catalog Number: DD1201-03) at room temperature in advance and set aside. Remove the test plate from the incubator and equilibrate at room temperature for 10-15 minutes. Add 25 μL Bio-lite Buffer to each well and mix thoroughly. Lyse at room temperature for 3-5 minutes. Use the fluorescence detection module of the multi-function microplate reader to select the 384-well white plate and then detect the luminescence signal reading. Import data into Graphpad Prism software, with the x-axis representing the logarithmic value of molar concentration. Fit the data using the "Dose-Response-Stimulation → Log (inhibitor) vs. Response - Variable Slope (four parameters)" method. Record the IC50 value.

[0641] The experimental results are shown in FIG15 . The huG11B8-VEGFR1 fusion protein can inhibit the binding of VEGF165 to VEGFR2 in a dose-dependent manner, with an IC50 value of 0.06252 nM. The inhibitory activity is significantly stronger than that of the control antibody.

[0642] Example 14 Antibody-mediated FGFR2 internalization and ADCT effects on tumor cell surfaces

[0643] To detect receptor internalization and cytotoxicity mediated by FGFR2 antibodies and their fusion proteins, a primary PBMC-dependent cytotoxicity experimental system was established. SNU-16 cells (Kebai, CBP60502) endogenously expressing human FGFR2 were used as target cells, and human peripheral blood mononuclear cells (PBMCs) were used as effector cells. The specific experiments are as follows.

[0644] PBMC cells were revived and the cell density was adjusted to 1×10 cells using RPMI-1640 (Gibco, 22400) + 10% FBS (Giboco, 10099) complete medium. 6 / mL, 100 μL per well (i.e., 100,000 cells per well) were plated into a 96-well plate (Corning, 7007). Cultured target SNU-16 cells were collected and centrifuged at 200g for 5 minutes. The supernatant was discarded and the cells were resuspended in PBS. The cells were centrifuged at 200g for 5 minutes, the supernatant was discarded, the cells were resuspended in PBS and counted. CFSE (Merck, SCT110) was added at a dilution of 1:5000 and the cells were incubated at 37°C for 15 minutes. FBS was added and the cells were incubated at 37°C for 5 minutes to terminate the staining. The cells were centrifuged at 200g for 5 minutes, the supernatant was discarded, and 3 mL of complete medium was added to wash the cells. The cells were resuspended in complete medium, counted, and the target cell density was adjusted to 2×10 5 / ml, and plated 50 μL per well (i.e., 10,000 cells per well) into the aforementioned 96-well plate (Effector:Target = 10:1). Prepare test antibodies at 4x the final assay concentration. The highest assay concentration for test antibodies IgG1 (SinoBiological, Catalog No. HG1K), Bemarituzumab, huG11B8-5, and fusion protein (huG11B8-VEGFR1-F) is 100 nM. Perform a 10-fold serial dilution to obtain two concentration points, and add 50 μL to each well. Blank (target cells plus complete medium) and antibody (target cells plus antibody) controls were also included. The final total volume was 200 μL, and the cells were incubated in a cell culture incubator for a further 72 hours. After completion of the incubation, remove the assay plate, transfer the entire cell suspension to a 96-well V-bottom plate (NEST, 701211), centrifuge at 400g for 3 minutes, discard the supernatant, and wash once with PBS. A 1:100 suspension of FGFR2 polyclonal antibody (Thermo, PA5-14651) was prepared in PBS. 100 μL of the antibody suspension was added to each well to resuspend the cells and incubated at 4°C for 30 min. The cells were washed twice with PBS. Donkey anti-rabbit secondary antibody (Jackson, 711-116-152, 1:200) and a dead-alive dye (Biolegend, 423114, 1:1000) were added to each well and incubated at 4°C for 30 min. The cells were washed twice with PBS, and the cell pellet was resuspended in 100 μL of PBS for analysis by flow cytometry.

[0645] The results are shown in Figure 16, which show that FGFR2 antibodies can induce downregulation of FGFR2 receptors on the surface of SNU-16 cells. In addition, FGFR2 antibodies and their fusion proteins can further downregulate FGFR2 receptors on the surface of SNU-16 cells through PBMC cytotoxicity.

[0646] Example 15 Antibody-mediated ADCC

[0647] To test the ADCC activity of FGFR2 antibodies and their bispecific fusion proteins, an ADCC system dependent on the NK92MI-human CD16aV engineered cell line was established. Ba / F3 cells engineered to express human FGFR2b (Kangyuan Broad Biotechnology Co., Ltd., Cat. No. KC1654) were used as target cells for the experiments. The details are as follows:

[0648] NK92MI (Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP61113) was cultured with N500 NK cell serum-free medium (Dakoway, catalog number: 6113031) and then engineered. Specifically, the nucleotide sequence encoding the human CD16AV-FcR1γ sequence (SEQ ID NO: 103) was cloned into a stable expression vector (PiggyBac Dual promoter (SBI, catalog number: PB513-B1), and the human CD16AV-FcR1γ sequence was transfected into NK92MI cells using a Bio-Rad electroporator (BIO-RAD, model: Gene Pul Ser X Cell™). NK92MI cells were then screened with 1 μg / mL puromycin to obtain NK92MI cells that highly expressed human CD16a (also referred to as "NK92MI-huCD16aV" cells).

[0649] After collecting the normally cultured NK92MI-huCD16aV, centrifuge at 200× for 5 minutes and discard the supernatant. Resuspend the cell pellet in fresh culture medium and adjust the cell density to 3×10 5 100 μL per well (i.e., 30,000 cells per well) of the test antibody was plated at 4x the final assay concentration. The highest assay concentration for the test antibodies IgG1 control (SinoBiological, Catalog No. HG1K), Bemarituzumab, huG11B8-5, huG11B8-VEGFR1-F, and huG11B8-VEGFR1 was 400 nM. A five-fold serial dilution was performed to obtain a total of eight concentration points. 50 μL of the sample was added to each well and mixed with the effector cells, followed by incubation at 37°C for 30 minutes. Ba / F3 cells expressing human FGFR2b were cultured in 1640 complete medium supplemented with 5 μg / mL puromycin, 8 ng / mL mouse IL-3 (Suzhou Nearshore Protein Technology Co., Ltd., Catalog No. CP39), and 10% fetal bovine serum.

[0650] After collecting the target cells, centrifuge at 200 × g and discard the supernatant. Resuspend the cell pellet in PBS and count. Add CFSE (Merck, catalog number: SCT110) at a dilution of 1:5000 and place at 37°C for 15 minutes. Add fetal bovine serum and place at 37°C for 5 minutes to terminate the staining. Centrifuge at 200 × g for 5 minutes and discard the supernatant. Resuspend the cell pellet in 1640 complete medium containing 32 ng / mL IL-3 and 10% fetal bovine serum. Adjust the target cell density to 2 × 10 5 / mL, 50 μL per well (i.e., 10,000 cells per well) was plated into the above-mentioned 96-well plate (Effector cell: target cell = 3:1). Blank (target cells plus complete medium) and antibody (target cells plus antibody) controls were also set up in the experiment. The final total volume was 200 μL, and then the cells were incubated in a cell culture incubator for another 4 hours. After the incubation, centrifuge at 400 × g for 3 minutes, and discard 20 μL of supernatant from each well. Prepare propidium iodide (Biyuntian, product number: ST511) dilution (1:25 dilution) with PBS, add 20 μL to each well, and incubate at room temperature for 10 minutes. The apoptosis rate of target cells was detected by flow cytometry.

[0651] The results are shown in Figure 17, which show that both the FGFR2 antibody and its bispecific fusion protein can induce NK92MI-CD16aV to kill Ba / F3 cells expressing FGFR2b compared with the IgG1 control.

[0652] Example 16 In vivo efficacy of antibody fusion protein in tumor models

[0653] The in vivo antitumor effect of huG11B8-VEGFR1-F was investigated in the SNU16 tumor model in CB17-SCID mice.

[0654] CB17-SCID mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Mouse gastric cancer SNU16 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. Cells were cultured in a 37°C, 5% CO2 incubator in RPMI-1640 medium containing 10% inactivated fetal bovine serum.

[0655] CB17-SCID mice were subcutaneously implanted with 10 × 10 6 SNU16 cells / mouse. When the average tumor volume reached 160 mm 3 Around 60 days post-inoculation, mice were selected based on tumor volume and body weight and randomly assigned to four experimental groups, with six mice in each group. On days 0, 4, 7, 11, and 14 after grouping, mice were intraperitoneally administered with blank control (PBS) (G1), Bemarituzumab (10 mg / kg) (G2), G11B8-F (10 mg / kg) (G3), and fusion protein huG11B8-VEGFR1-F (10 mg / kg) (G4), respectively. The maximum long axis (L) and maximum wide axis (W) of the tumors in the tumor-bearing mice were measured twice weekly with a vernier caliper during the experimental period, and tumor volume was calculated using the following formula: V = L × W2 / 2.

[0656] Treatment with G11B8-F and huG11B8-VEGFR1-F resulted in significant tumor growth inhibition compared to the PBS group, with huG11B8-VEGFR1-F showing a stronger tumor inhibition effect, demonstrating a 127.14% tumor growth inhibition rate (TGI%) at a dose of 10 mg / kg. The results are shown in Figure 18 and Table 9 below. Figure 18 shows changes in tumor volume in tumor-bearing mice after administration of the antibody fusion protein huG11B8-VEGFR1.

[0657] Table 9. Inhibition of SNU16 tumor growth by the test article Note: a. Mean ± standard error;

[0658] b. TGI = [1-(tumor volume change in the treatment group / tumor volume change in the control group)] × 100%;

[0659] c. Statistical analysis (One-way ANOVA) of the tumor volumes of the drug-treated groups and the PBS control group on day 18 of group administration was performed. *P<0.05, ****P<0.0001.

[0660] Throughout the experiment, the animals were active and fed well during the dosing period. Animals in groups G1-G4 all experienced some weight gain, indicating good tolerance to the test article. Weight changes for all animals are shown in Figure 19 and Table 10. Figure 19 shows weight changes in tumor-bearing mice following administration of the antibody fusion protein huG11B8-VEGFR1-F.

[0661] Table 10. Effects of the test articles on the body weight of mice

[0662] Note: a. Mean ± standard error; b. Body weights of the drug-treated group and the PBS control group were statistically analyzed on the 18th day after group administration (One-way ANOVA).

[0663] In addition, 10 × 10 6 SNU16 cells / mouse. When the average tumor volume reached 160 mm 3Around 6:00 p.m., mice were selected based on tumor volume and body weight and randomly assigned to 6 experimental groups, with 6 mice in each group. On days 0, 3, 7, 10, 14, 17, 21, and 24 after grouping, mice were intraperitoneally administered with blank control (PBS) (G1), VEGFR1D2-ISO (5 mg / kg) (G2), huG11B8-VEGFR1-F (1 mg / kg) (G3), huG11B8-VEGFR1-F (5 mg / kg) (G4), huG11B8-VEGFR1 (1 mg / kg) (G5), and huG11B8-VEGFR1 (5 mg / kg) (G6), respectively. The maximum long axis (L) and maximum wide axis (W) of tumors in tumor-bearing mice were measured twice weekly with a vernier caliper during the experimental period, and tumor volume was calculated using the following formula: V = L × W2 / 2.

[0664] On day 27 of tumor growth, VEGFR1D2-ISO significantly inhibited tumor growth at a 5 mg / kg dose compared to the PBS group, with a tumor growth inhibition rate (TGI%) of 84.59%. At 1 mg / kg, huG11B8-VEGFR1-F exhibited a stronger tumor inhibitory effect than huG11B8-VEGFR1, with a TGI of 53.97% versus 26.36%. At 5 mg / kg, huG11B8-VEGFR1-F and huG11B8-VEGFR1 exhibited comparable tumor inhibition. The results are shown in Figure 20 and Table 11 below.

[0665] Table 11. Inhibition of SNU16 tumor growth by the test article Note: a. Mean ± standard error;

[0666] b. TGI = [1-(tumor volume change in the treatment group / tumor volume change in the control group)] × 100%;

[0667] c. Statistical analysis (One-way ANOVA) of the tumor volumes of the drug-treated groups and the PBS control group on day 27 of group administration showed: *P<0.05, **P<0.01, ****P<0.0001.

[0668] Throughout the experiment, the animals were active and ate well during the dosing period. The weight changes of all animals are shown in Figure 21 and Table 12.

[0669] Table 12. Effects of the test articles on the body weight of mice Note: a. Mean ± standard error; b. Statistical analysis (One-way ANOVA) of body weights in the drug-treated group and the PBS control group on day 27 of administration. *P<0.05, **P<0.01.

[0670] Sequence Listing:

Claims

1. An anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising i. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 63, 69, 71, 73, 75, 77 or 79, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 81, 86 or 88; ii. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 2, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 7; iii. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 22, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 81; iv. three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 37, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 41; v. three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 27, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 32; vi. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 12, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 17; vii. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 44 or 104, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 49 or 105; viii. the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO: 54, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO: 58; or ix. The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region VH as shown in SEQ ID NO:22, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region VL as shown in SEQ ID NO:

81.

2. An anti-FGFR2 antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3, wherein i. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 65 or 66; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 67; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 82; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 83; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 84; ii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:10; iii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:23; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:24; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:25; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:82; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:83; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:84; iv. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:38; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:39; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:42; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:10; v. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:28; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:29; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:30; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:33; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:34; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:35; vi. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20; vii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:45; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:46; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:50; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:51; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:52; or viii. HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:3; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:55; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:56; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:59; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:60; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

61.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 2, 12, 22, 27, 37, 44, 54, 63, 69, 71, 73, 75, 77, 79 or 104.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a light chain variable region VL, wherein the light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7, 17, 32, 41, 49, 58, 81, 86, 88 or 105.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein i. the VH comprises or consists of an amino acid sequence as shown in SEQ ID NO:63, 69, 71, 73, 75, 77 or 79, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as shown in SEQ ID NO:81, 86 or 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; ii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:63, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:81, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; iii. the VH comprises or consists of an amino acid sequence as shown in SEQ ID NO: 69, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as shown in SEQ ID NO: 86 or 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; iv. the VH comprises or consists of an amino acid sequence as shown in SEQ ID NO:71, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as shown in SEQ ID NO86 or 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; v. the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO:73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO:86 or 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto. or consisting of the amino acid sequence; vi. the VH comprises or consists of an amino acid sequence as shown in SEQ ID NO:75, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as shown in SEQ ID NO86 or 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; vii. the VH comprises or consists of an amino acid sequence as shown in SEQ ID NO:77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as shown in SEQ ID NO86 or 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; viii. the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO:79, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO86 or 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; ix. the VH comprises or consists of the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; x. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:22, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:81, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xi. the VH comprises or consists of the amino acid sequence of SEQ ID NO:37, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of the amino acid sequence of SEQ ID NO:41, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; xii. the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:32, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xiii. the VH comprises or consists of the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of the amino acid sequence of SEQ ID NO:17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; xiv. the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO:44 or 104, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO:49 or 105, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO:44, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO:49 or 105, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto The amino acid sequence shown in NO:49, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting of said amino acid sequence; xv. the VH comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 104, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 105, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or xvi. the VH comprises the amino acid sequence shown in SEQ ID NO:54, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO:58, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein i . the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 63, 69, 71, 73, 75, 77 or 79, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81, 86 or 88; ii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81; iii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 69, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88; iv. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 71, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88; v. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 73, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88; vi. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88; vii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 77, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88; viii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 79, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO 86 or 88; ix. the VH comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of the amino acid sequence, and The VL contains the amino acid sequence shown in SEQ ID NO:7 or consists of the amino acid sequence; x. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 81; xi. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 41; xii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 32; xiii. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; xiv. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 44 or 104, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or 105; the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 44, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 49; xv. the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 104, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 105; or xvi. The VH comprises or consists of the amino acid sequence shown in SEQ ID NO:54, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO:

58.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 91; or (ii) comprises the amino acid sequence of SEQ ID NO: 91 or consists of the amino acid sequence.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 92; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:

92.

11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the antibody comprises a heavy chain, the heavy chain (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, 11, 21, 26, 36, 43, 53, 62, 68, 70, 72, 74, 76, 78 or 106; or (ii) comprises or consists of an amino acid sequence of SEQ ID NO: 1, 11, 21, 26, 36, 43, 53, 62, 68, 70, 72, 74, 76, 78 or 106; or (iii) The heavy chain of (ii), wherein the lysine at the C-terminus of the heavy chain is substituted with alanine.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the antibody comprises a light chain (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, 16, 31, 40, 48, 57, 80, 85, 87 or 107; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 6, 16, 31, 40, 48, 57, 80, 85, 87 or 107.

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, comprising a heavy chain and a light chain, wherein i. the heavy chain comprises or consists of an amino acid sequence as shown in SEQ ID NO:62, 68, 70, 72, 74, 76 or 78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of an amino acid sequence as shown in SEQ ID NO:80, 85 or 87, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; ii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; iii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:68, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; iv. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:70, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; v. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; vi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:74, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; vii. The heavy chain contains the amino acid sequence shown in SEQ ID NO: 76 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, or consisting of, an amino acid sequence having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:85 or 87, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; viii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:85 or 87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; ix. the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; x. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xii. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:26, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xiii. the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:11, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xiv. the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:43 or 106, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:48 or 107, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xv. The heavy chain comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or is composed of the amino acid sequence The light chain comprises or consists of an amino acid sequence as shown in SEQ ID NO:48 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; xvi. the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; xvii. the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:53, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or xviii. The heavy chain and light chain of any one of i-xvii, wherein the lysine at the C-terminus of the heavy chain is substituted with alanine.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody is a humanized antibody.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is a monoclonal antibody.

16. The antibody or antigen-binding fragment thereof of any one of claims 1 to 15, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody, (Fab')2, single-domain antibody such as heavy chain variable region VHH, dAb (domain antibody), heavy chain antibody or linear antibody.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the single-chain antibody is a scFv.

18. A multispecific binding molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

19. The multispecific binding molecule of claim 18, which is a bispecific binding molecule.

20. The multispecific binding molecule of claim 19, which is a bispecific binding molecule such as a fusion protein that specifically binds to FGFR2 and VEGF, such as VEGFA, such as VEGF165.

21. The multispecific binding molecule of claim 20, comprising a first target binding region that specifically binds FGFR2, and a second target binding region that specifically binds VEGFA, wherein the first target binding region comprises the antibody or antigen-binding fragment thereof of any one of claims 1-17.

22. The multispecific binding molecule of claim 21, wherein the second target binding region comprises a VEGF receptor or a functional fragment thereof or an antibody or an antigen-binding fragment thereof that specifically binds to VEGFR2.

23. The multispecific binding molecule of claim 22, wherein the VEGF receptor is VEGFR1, and / or the functional fragment comprises or consists of: The extracellular domain of a VEGF receptor or a fragment thereof, for example, the fragment of the extracellular domain comprises or consists of extracellular domain 2.

24. The multispecific binding molecule of claim 23, wherein The extracellular domain of VEGFR1 comprises or consists of the following amino acid sequence: i. the amino acid sequence shown in SEQ ID NO: 102, ii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 102; or iii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 102 and comprises the amino acid sequence shown in SEQ ID NO: 100; Or the extracellular domain 2 of VEGFR1 comprises or consists of the following amino acid sequence: i. the amino acid sequence shown in SEQ ID NO: 100, ii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 100; or iii. an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 100 and comprises the amino acid sequence shown in SEQ ID NO:

100.

25. The multispecific binding molecule of any one of claims 18-24, wherein the first target binding region is a Fab fragment of an anti-FGFR2 antibody as defined in any one of claims 1-17.

26. The multispecific binding molecule of claim 25, wherein the Fab comprises a CH1, wherein the CH1 is a CH1 from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 from IgG1.

27. The multispecific binding molecule of claim 26, wherein the CH1 (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 96; or (ii) comprises the amino acid sequence of SEQ ID NO: 96 or consists of the amino acid sequence.

28. The multispecific binding molecule of any one of claims 18-27, wherein the multispecific binding molecule comprises an Fc region, such as an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different.

29. The multispecific binding molecule of claim 28, wherein the Fc region is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably the Fc region from IgG1.

30. The multispecific binding molecule of claim 29, wherein the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO:93, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical thereto.

31. The multispecific binding molecule of claim 29, wherein the Fc region comprises a C-terminal lysine replaced with an alanine.

32. The multispecific binding molecule of claim 31, wherein the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO:97 or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical thereto.

33. The multispecific binding molecule of any one of claims 28 to 32, which is a bispecific binding molecule and which comprises a first target binding region, a second target binding region and an Fc region, The first target binding region is a Fab fragment of an anti-FGFR2 antibody, wherein the Fab fragment comprises a Fab heavy chain VH-CH1 and a Fab light chain VL-CL, The second target binding region comprises or consists of the extracellular domain of the VEGF receptor or a fragment thereof (e.g., it comprises or consists of ECD2); wherein the C-terminus of CH1 of the heavy chain of the Fab fragment is fused to the N-terminus of the Fc region, and the N-terminus of the extracellular domain of the VEGF receptor is fused to the C-terminus of the Fc region to constitute the heavy chain; And the light chain of the Fab fragment constitutes the light chain.

34. The multispecific binding molecule of claim 33, wherein the bispecific binding molecule comprises two heavy chains and two light chains, wherein the two heavy chains may be identical or different, or the two light chains may be identical or different.

35. The multispecific binding molecule of claim 33 or 34, wherein the N-terminus of the extracellular domain of the VEGF receptor is fused to the C-terminus of the Fc region via a linker, e.g., the linker comprises or consists of (GGGGS)n, wherein n=1, 2, 3 or 4.

36. The multispecific binding molecule of claim 35, wherein The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:89, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence; and / or The light chain comprises the amino acid sequence shown in SEQ ID NO:85, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

37. The antibody or antigen-binding fragment thereof of any one of claims 1-17, or the multispecific binding molecule of any one of claims 18-36, wherein the antibody or antigen-binding fragment thereof or the multispecific binding molecule is ADCC enhanced, e.g., it has an altered glycosylation type, e.g., it is hypofucosylated or afucosylated or has increased bisecting GlcNac structures.

38. A nucleic acid molecule encoding any one chain of the antibody or antigen-binding fragment thereof of any one of claims 1 to 17, or any one chain of the multispecific binding molecule of any one of claims 18 to 36, or consisting of said nucleic acid sequence.

39. An expression vector comprising the nucleic acid molecule of claim 37, preferably, the expression vector is pCDNA, such as pCDNA3.

1.

40. A host cell comprising the nucleic acid molecule of claim 37 or the expression vector of claim 38, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells.

41. The host cell of claim 40, wherein the host cell is a host cell with an altered glycosylation machinery, such as a cell in which the FUT8 gene is completely or partially knocked out or the function of the FUT8 gene or protein is completely or partially destroyed, such as a FUT8 gene knocked out CHO cell.

42. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1-17 and 37 or the multispecific binding molecule of any one of claims 18-37, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 38 or the expression vector of claim 39 or the host cell of claim 40 or 41 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

43. An antibody or antigen-binding fragment thereof or a multispecific binding molecule prepared by the method of claim 42.

44. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17 and 37 or the multispecific binding molecule of any one of claims 18-37.

45. A pharmaceutical composition or medicament or formulation comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17 and 37 or the multispecific binding molecule of any one of claims 18-37, or the immunoconjugate of claim 44, and optionally a pharmaceutically acceptable excipient.

46. ​​A pharmaceutical combination product comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17 and 37 or the multispecific binding molecule of any one of claims 18-37, or the immunoconjugate of claim 44, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

47. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-17 and 37 or the multispecific binding molecule of any one of claims 18-37, or the immunoconjugate of claim 44, or the pharmaceutical composition or formulation of claim 45; or the pharmaceutical combination product of claim 46.

48. The method of claim 47, wherein the cancer is a FGFR2-positive cancer, e.g., the cancer is characterized by having or having elevated protein levels and / or nucleic acid levels of FGFR2 in tumor cells of the cancer (e.g., compared to the protein levels and / or nucleic acid levels of FGFR2 in cells of the same tissue of a healthy individual, or compared to the protein levels and / or nucleic acid levels of FGFR2 in healthy cells of the same tissue or cells of adjacent healthy tissue of the patient).

49. The method of claim 47 or 48, wherein the cancer is a solid tumor or a hematological tumor, such as gastric cancer, lung cancer, such as non-small cell lung cancer, esophageal cancer, bile duct cancer (e.g., unresectable or metastatic bile duct cancer), or breast cancer, pancreatic cancer, or colorectal cancer.

50. The method of any one of claims 47-49, wherein the method further comprises administering in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint molecule inhibitors or agonists).

51. A method for detecting the presence of FGFR2 in a biological sample, comprising (i) contacting a biological sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 17 and 37 or the multispecific binding molecule of any one of claims 18 to 37 under conditions that allow binding to FGFR2, (ii) detecting whether a complex is formed between the antibody or multispecific binding molecule and FGFR2, The formation of the complex indicates the presence of FGFR2.

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