Anti-FGFR2b antibody or antigen-binding fragment thereof and use thereof

Abstract

The present disclosure relates to the field of biomedicine technology, and provides an anti-FGFR2b antibody or an antigen-binding fragment thereof and its use, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the CDR combination of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 respectively composed of the amino acid sequences SEQ ID NO: 1, 15, and 3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 respectively composed of the amino acid sequences SEQ ID NO: 6, 7, and 20. The antibody specifically targets FGFR2b, can block cell proliferation induced by the ligand FGF7, has antibody-dependent cellular cytotoxicity (ADCC), and has potential significance in treating diseases related to overexpression of the FGFR2b gene, excessive activation of the ligand action, and poor prognosis.

Description

Technical Field

[0001] The present disclosure relates to an anti-FGFR2b antibody or an antigen-binding fragment thereof and uses thereof, and in particular to a monoclonal antibody that specifically targets FGFR2b, can block cell proliferation induced by the ligand FGF7, and has antibody-dependent cellular cytotoxicity (ADCC) effect, and has potential significance in treating diseases related to FGFR2b gene overexpression, excessive activation of ligand action, and poor prognosis. Background Art

[0002] Fibroblast Growth Factor Receptor 2b (FGFR2b) is a homologous isomer of FGFR2, a member of the receptor tyrosine kinase family. FGFR2b is expressed on the cell membrane like the receptors of the entire FGFR family. The entire receptor is composed of three main elements: (1) FGFR2 extracellular region, which is composed of three Ig domains (about 355aa). The two isoforms FGFR2b or FGFR2c are formed due to selective splicing. The only difference is that the Ig III near the membrane end is about 30aa (also called IIIb or IIIc). The extracellular region is used to bind to the fibroblast growth factor (FGF) ligand; (2) Transmembrane helix: a single transmembrane structure that connects the extracellular domain to the cell; (3) Intracellular tyrosine kinase domain, which plays a key role in signal transduction. After activation, it can phosphorylate tyrosine residues and trigger downstream signaling pathways. After binding to the ligand FGF, FGFRs dimerize, resulting in conformational changes in the structure, activating the intracellular kinase domain, and leading to phosphorylation of the intracellular domain. By recruiting FRS2, SOS, and GRB2 signaling molecules, the downstream RAS and MAPK pathways are activated. The PI3K-mTOR-AKT and STAT3 signaling pathways are also activated by FGFR. FGFR2b is an important signaling receptor involved in multiple cellular processes, including cell proliferation, differentiation, migration, and survival.

[0003] In normal tissues, the expression of FGFR2b supports a variety of physiological processes, including embryonic development and tissue repair. FGFR2b is mainly expressed in cells of epithelial origin, while FGFR2c is mainly expressed in mesenchymal cells. However, dysregulation of FGFR2b expression can be found in various cancers. Amplification, mutation, or overexpression of FGFR2b has been confirmed in a variety of malignancies, including breast cancer, gastric cancer, and lung cancer. Alterations in FGFR2b expression are often associated with enhanced tumor proliferation and poor prognosis. Overexpression of FGFR2b has been reported in a variety of cancers, including breast, endometrial, cervical, lung, esophageal, gastric, pancreatic, and colorectal cancers, but the role of FGFR2b variants between different cancer types is controversial. In patients with esophageal cancer, FGFR2b was expressed in esophageal cancer cells in 41% of patients, and FGFR2b expression was associated with benign cell types in esophageal cancer. Using immunohistochemistry (IHC), it was found that FGFR2b protein was overexpressed in 73 patients (4% of 1974 cases) with gastric cancer, and all tumors with FGFR2b overexpression had FGFR2 amplification detected by fluorescence in situ hybridization. Moreover, the expression of FGFR2b in gastric cancer is particularly significant. In one study, a new FGFR2b primary antibody was used to predict gene amplification by immunohistochemistry, and FGFR2b overexpression was found in 4% of gastric cancers, of which 92% of cases were confirmed as FGFR2 gene amplification by FISH, confirming that this is FGFR2 amplified gastric cancer that mainly expresses the FGFR2b subtype, rather than the FGFR2c subtype. It is worth noting that with the increase in FGFR2b expression levels, a greater survival benefit was observed, indicating that the FGFR2b target may be used as an indicator for predicting tumor efficacy in clinical practice. In 2019, Kim et al. summarized 10 studies from 1996 to 2018, with a total of 4,294 patients included in the pooled analysis of odds ratios (ORs) and 95% confidence intervals (CIs) of pathological characteristics of FGFR2 expression status, as well as the pooled analysis of hazard ratios (HRs) and 95% CIs for overall survival. Compared with tumors with low FGFR2 expression, gastric cancer with FGFR2 overexpression showed deeper invasion depth (pT3-4) (OR = 2.63, 95% CI: 1.70-4.06, p < 0.0001), higher lymph node metastasis rate (OR = 1.87, 95% CI: 1.31-2.67, p < 0.0001) and more advanced stage (III-IV) (OR = 1.78, 95% CI: 1.07-2.96, p = 0.03). In addition, the survival rate of gastric cancer patients with FGFR2 overexpression was significantly lower than that of patients with FGFR2 underexpression tumors (HR=1.40, 95% CI: 1.25-1.58, p<0.00001).In conclusion, this analysis showed that FGFR2 overexpression was associated with adverse pathological features and prognosis in patients with gastric cancer (J Cancer. 2019 Jan 1; 10(1): 20-27).

[0004] In summary, abnormalities in the FGFR2b gene (amplification, mutation, or translocation) lead to gene overexpression and excessive activation of ligand action, which are positively correlated with the occurrence, development, and poor prognosis of gastric cancer. FGFR2 targeted drugs can be divided into four categories based on their mechanism of action: (1) small molecule tyrosine kinase inhibitors (TKIs), including non-selective and selective FGFR inhibitors; (2) antagonistic monoclonal antibodies, which competitively bind to the extracellular region of FGFR and prevent the activation of FGFs–FGFR signaling; (3) FGF ligand traps, which prevent the activity of multiple FGF ligands and ligand-receptor interactions; and (4) antibody-drug conjugates (ADCs), which are composed of FGFR antibodies and cytotoxic loads. Summary of the invention

[0005] On the one hand, the present disclosure provides an antibody or an antigen-binding fragment thereof of fibroblast growth factor receptor 2b (FGFR2b), which comprises a heavy chain variable region and a light chain variable region, wherein the CDR composition of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO:1, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO:15, a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO:3, and a light chain CDR1 consisting of the amino acid sequence SEQ ID NO:6, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO:7, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO:20.

[0006] In some embodiments, the aforementioned antibody or antigen-binding fragment thereof has the following combination of heavy chain variable region and light chain variable region:

[0007] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.21 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical thereto.

[0008] In some embodiments, the aforementioned antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

[0009] In some preferred embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0010] In some preferred embodiments, the light chain comprises an amino acid sequence shown in SEQ ID NO. 23 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0011] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof.

[0012] In some preferred embodiments, the aforementioned nucleic acid has a nucleotide sequence encoding an antibody heavy chain variable region of SEQ ID NO.17 or any variant thereof, and a nucleotide sequence encoding an antibody light chain variable region of SEQ ID NO.22 or any variant thereof. Wherein, any of the variants preferably have a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.17 and / or SEQ ID NO.22.

[0013] In some preferred embodiments, the aforementioned nucleic acid has an antibody heavy chain encoding nucleotide sequence of SEQ ID NO.19 or any variant thereof, and an antibody light chain encoding nucleotide sequence of SEQ ID NO.24 or any variant thereof. Wherein, any of the variants preferably have a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.19 and / or SEQ ID NO.24.

[0014] In another aspect, the present disclosure provides a vector of the aforementioned nucleic acid.

[0015] In another aspect, the present disclosure provides a host cell for the aforementioned nucleic acid vector.

[0016] On the other hand, the present disclosure provides a pharmaceutical composition or pharmaceutical combination comprising the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector and / or cell.

[0017] In some embodiments, the composition or pharmaceutical combination further comprises a second therapeutic agent.

[0018] In some preferred embodiments, the second therapeutic agent is selected from beta-2 adrenergic receptor agonists (SABAs), anticholinergics, adrenergic agonists, glucocorticoids, long-acting beta-2 adrenergic receptor agonists (LABAs), leukotriene antagonists, and mast cell stabilizers.

[0019] In another aspect, the present disclosure provides a method for producing the aforementioned antibody or antigen-binding fragment thereof, the method comprising culturing a host cell containing the aforementioned nucleic acid or expression vector.

[0020] On the other hand, the present disclosure provides the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells or pharmaceutical compositions or drug combinations for preparing drugs or kits for detecting, treating, preventing and / or alleviating diseases associated with the expression of FGFR2b.

[0021] In some preferred embodiments, the disease associated with the expression of FGFR2b is a tumor.

[0022] In some more preferred embodiments, the cancer associated with the expression of FGFR2b is gastric cancer.

[0023] The humanized antibody h21G4 H4K3 provided by the present disclosure can specifically bind to human, cynomolgus monkey and rat FGFR2b, does not bind to human FGFR2c, and has the activity of blocking FGF7-induced cell proliferation, and the molecule itself has ADCC activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the specification and, together with the description, serve to explain the principles of the specification.

[0025] Figure 1 The SDS-PAGE results of prokaryotically expressed human, cynomolgus monkey, rat FGFR2b and human FGFR2c proteins are shown.

[0026] Figure 2 The binding of the chimeric antibody 21G4 to the human FGFR2b protein is shown.

[0027] Figure 3 The chimeric antibody 21G4 and cynomolgus monkey ( Figure 3 -A), rats ( Figure 3 -B) Binding of FGFR2b protein.

[0028] Figure 4 The binding of chimeric antibody 21G4 to cells expressing human FGFR2b is shown.

[0029] Figure 5 The chimeric antibody 21G4 and cynomolgus monkey ( Figure 5 -A), rats ( Figure 5 -B) Binding of FGFR2b cells.

[0030] Figure 6Blockade of FGF7 / FGFR2b binding by the chimeric antibody 21G4 is shown.

[0031] Figure 7 The inhibition of FGF7-induced cell proliferation by the chimeric antibody 21G4 is shown.

[0032] Figure 8 The chimeric antibody 21G4 is shown to be effective for FGF10 / FGFR2b ( Figure 8 -A) and FGF23 / FGFR2c ( Figure 8 -B) The effect of binding.

[0033] Fig. 9 Blockade of FGF10-induced cell proliferation by the chimeric antibody 21G4 is shown.

[0034] Fig.10 The binding specificity of the chimeric antibody 21G4 is shown.

[0035] Fig.11 The affinity of chimeric antibody 21G4 to FGFR2b is shown.

[0036] Fig.12 The humanized antibody h21G4 H4K3 and human FGFR2b antigen ( Fig.12 -A), cynomolgus monkey FGFR2b antigen ( Fig.12 -B), rat FGFR2b antigen ( Fig.12 -C) and human FGFR2c ( Fig.12 -D) Antigen binding.

[0037] Fig.13 The binding of humanized antibody h21G4 H4K3 to cells expressing FGFR2b is shown.

[0038] Fig.14 It is shown that the humanized antibody h21G4 H4K3 blocks FGF7-induced cell proliferation.

[0039] Fig.15 The ADCC activity of the humanized antibody h21G4 H4K3 is shown. DETAILED DESCRIPTION

[0040] I. Definitions

[0041] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0042] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0043] Sequence "identity" or "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Pres, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0044] With respect to the variable domains of antibodies, the term "variable" refers to certain parts of related molecules that have extensive sequence differences between antibodies and are used for specific recognition and binding of a particular antibody to its specific target. However, the variability is not evenly distributed throughout the variable domain of an antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located in the variable domains of light and heavy chains. The more conserved parts of the variable domains are called framework (FR) regions or framework sequences. Each variable domain of a natural heavy chain and light chain includes four FR regions, which mainly adopt a β-pleated configuration, connecting three CDRs, which form loops that connect the β-pleated structure and in some cases form part of the β-pleated structure. The CDRs of each chain are usually linked together in proximity by the FR regions and, with the help of the CDRs from the other chain, contribute to the formation of the antibody target binding site (epitope or determinant) (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. A CDR may have the ability to specifically bind to a cognate epitope.

[0045] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than the full length, but contains at least a portion of the variable region (e.g., one or more CDRs and / or one or more antibody combining sites) of the antibody that binds to an antigen, and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.

[0046] As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in a monoclonal antibody population is identical to other antibody molecules. This property is in contrast to the properties of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0047] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies produced synthetically with the same domains.

[0048] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0049] As used herein, the term "humanized" antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing minimal sequence derived from a non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues in a complementary determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0050] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, the mutations in the CDR are generally no more than one or two. Therefore, the humanized antibodies described in the present disclosure also encompass antibodies comprising 1 or 2 amino acid mutations in the CDR.

[0051] As used herein, the term "CDR" refers to a complementarity-determining region, and each heavy chain and light chain of a known antibody molecule has three CDRs. CDRs are also referred to as hypervariable regions and are present in the variable regions of each heavy chain and light chain of an antibody, with very high variability sites in the primary structure of the CDRs. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the amino terminal of the amino terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the amino terminal of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0052] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0053] As used herein, "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present in a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present in an amount of about or 1×10 7 M -1 or 1×10 8 M -1 or greater affinity constant Ka (or 1×10 -7 M or 1×10 -8M or lower dissociation constant (Kd)) binds to the antigen. Affinity constants can be determined by standard kinetic methods of antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Pat. No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0054] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by a phosphodiester bond. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0055] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. "Isolated" nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture media when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesized. The exemplary isolated nucleic acid molecules provided herein include the isolated nucleic acid molecules of the antibody or Fab provided by encoding.

[0056] As used herein, "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

[0057] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) the exogenous genes into cells.

[0058] As used herein, "expression vector" includes vectors capable of expressing DNA, which is operably linked to regulatory sequences such as promoter regions that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.

[0059] As used herein, the term "bispecific antibody" or "multispecific antibody" refers to the formation of a new antibody construct that binds to two or more different sites and / or targets by functionally linking an antibody (e.g., chemical coupling, gene fusion, non-covalent binding or other methods). Among them, the most commonly used is "bispecific antibody", which specifically refers to an antibody construct that is specific for two different antigens. Typically, a bispecific antibody or multispecific antibody includes at least two antigen binding domains.

[0060] As used herein, the term "chimeric antigen receptor (CAR)" is an engineered receptor that transplants any specificity to immune effector cells. Typically, these receptors are used to transplant the specificity of monoclonal antibodies to T cells; the transfer of their coding sequences is promoted by retroviral or lentiviral vectors or by transposons. CAR-engineered T cells (also abbreviated as CAR-T cells) are genetically engineered T cells equipped with chimeric receptors, whose extracellular recognition units include antibody-derived recognition domains, and whose intracellular regions are derived from lymphocyte stimulation parts. The structure of the prototype CAR is modular and is designed to accommodate various functional domains, so that specificity can be selected and the activation of T cells can be controlled. The preferred antibody-derived recognition unit is a single-chain variable fragment (scFv) of the specificity and binding residues of the heavy and light chain variable regions of the combined monoclonal antibodies. The most common lymphocyte activation part includes a T-cell costimulation (eg, CD28) domain in series with a T-cell trigger (eg, CD3ζ) part. By equipping effector lymphocytes (such as T cells and natural killer cells) with such chimeric receptors, engineered cell redirection has predefined specificity for any desired target antigen, in a non-HLA restricted manner. CAR constructs are introduced in vitro into T cells of peripheral lymphocytes from a given patient using retroviral or lentiviral vectors or transposons. After the resulting CAR engineered T cells are infused back into the patient, they are transported, arrive at their target sites, and when interacting with their target cells or tissues, they undergo activation and play their predefined effector functions. The therapeutic targets of the CAR method include cancer and HIV-infected cells, or autoimmune effector cells.

[0061] As used herein, the term "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the equilibrium dissociation constant (KD). The equilibrium dissociation constant is a function of the dissociation rate constant and the association rate constant (kD, respectively). dis and k on The smaller the KD, the smaller the dissociation, which means the stronger the affinity between the antibody and the antigen. Affinity can be measured by common methods known in the art, for example, using surface plasmon resonance (SPR) to measure KD in a BIACORE instrument. Generally, antibodies are measured with a KD of no more than 1×10 -5 M, for example, less than about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M or 1×10 -10 The molecule dissociates from the antigen with an equilibrium dissociation constant (KD) of M or less.

[0062] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to the binding of antibodies to antigenic epitopes of virus-infected cells or tumor cells, and the binding of their Fc fragments to Fc receptors (FcR) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0063] II. Detailed description of specific implementation scheme

[0064] On the one hand, the present disclosure provides an antibody or an antigen-binding fragment thereof of fibroblast growth factor receptor 2b (FGFR2b), which comprises a heavy chain variable region and a light chain variable region, wherein the CDR composition of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO:1, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO:15, a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO:3, and a light chain CDR1 consisting of the amino acid sequence SEQ ID NO:6, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO:7, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO:20.

[0065] In some embodiments, the aforementioned antibody or antigen-binding fragment thereof has the following combination of heavy chain variable region and light chain variable region:

[0066] The heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.16, and the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.21.

[0067] In some embodiments, the aforementioned antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

[0068] In some preferred embodiments, the aforementioned heavy chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.18.

[0069] In some preferred embodiments, the aforementioned light chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.23.

[0070] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof.

[0071] In some preferred embodiments, the aforementioned nucleic acid has a nucleotide sequence encoding an antibody heavy chain variable region of SEQ ID NO.17 or any variant thereof, and a nucleotide sequence encoding an antibody light chain variable region of SEQ ID NO.22 or any variant thereof. Wherein, any of the variants preferably have a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.17 and / or SEQ ID NO.22.

[0072] In some preferred embodiments, the aforementioned nucleic acid has an antibody heavy chain encoding nucleotide sequence of SEQ ID NO.19 or any variant thereof, and an antibody light chain encoding nucleotide sequence of SEQ ID NO.24 or any variant thereof. Wherein, any of the variants preferably have a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.19 and / or SEQ ID NO.24.

[0073] In another aspect, the present disclosure provides a vector of the aforementioned nucleic acid.

[0074] In another aspect, the present disclosure provides a host cell for the aforementioned nucleic acid vector.

[0075] On the other hand, the present disclosure provides a pharmaceutical composition or pharmaceutical combination comprising the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector and / or cell.

[0076] In another aspect, the present disclosure provides a method for producing the aforementioned antibody or antigen-binding fragment thereof, the method comprising culturing a host cell containing the aforementioned nucleic acid or expression vector.

[0077] On the other hand, the present disclosure provides the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells or pharmaceutical compositions or drug combinations for preparing drugs or kits for detecting, treating, preventing and / or alleviating diseases associated with the expression of FGFR2b.

[0078] In some preferred embodiments, the disease associated with the expression of FGFR2b is a tumor.

[0079] In some more preferred embodiments, the disease associated with the expression of FGFR2b is gastric cancer.

[0080] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0081] Example

[0082] Example 1: Antigen preparation

[0083] Human FGFR2b (SEQ ID NO: 11), cynomolgus monkey FGFR2b (SEQ ID NO: 12), rat FGFR2b (SEQ ID NO: 13) and human FGFR2c (SEQ ID NO: 14) were synthesized, and the sequences were shown in Table 1. The extracellular fragments were cloned into a prokaryotic expression plasmid, and the plasmid was used to transform BL21E.coli. Human, cynomolgus monkey, rat FGFR2b and human FGFR2c proteins were purified, and the SDS-PAGE results were as follows: Figure 1 .

[0084] Table 1 Sequences of human FGFR2b, cynomolgus monkey FGFR2b, rat FGFR2b and human FGFR2c

[0085]

[0086]

[0087]

[0088] Example 2: Generation of anti-FGFR2b antibodies

[0089] (1) Mouse immunization

[0090] Female Balb / C mice aged 4-6 weeks were selected and immunized by antigen, gene gun or combination. The immunization dose was 20 μg of human FGFR2b plasmid per mouse per immunization, with an interval of one week between each immunization. Antigen immunization. The immunization dose was 50 μg of FGFR2b protein per mouse per immunization. The antigen was mixed with TiterMax adjuvant (Sigma-Aldrich) in equal volumes and then subcutaneously and footpad immunized, with an interval of two weeks between each immunization. The antibody binding titer to human FGFR2b in the serum after six immunizations increased significantly, with a titer of about 1:170000-1:200000. At the cellular level, the sera of immunized mice can bind to HEK293-hFGFR2b cells and HEK293-hFGFR2c cells. The titer of the serum binding to the negative sieve protein FGFR2c in the sera of immunized mice was lower than the titer of the FGFR2b antibody.

[0091] (2) Phage screening

[0092] Mouse lymph nodes and spleen cells were taken, and the RNA of the cells was extracted by TRNzol lysis method, and then reverse transcribed to synthesize single-stranded cDNA, which was used as a template to amplify the variable region sequence of the antibody. The FGFR2b immune library was constructed on the self-established Fab antibody fragment phage display platform. Human FGFR2b protein or cells expressing human FGFR2b, human FGFR2c protein and cells expressing human FGFR2c were added to the solid phase immunotube (Maxisorp immunotube) as negative screens. All immune mice were selected for library construction, with a library capacity of 1.5×1010. Dot-Blot detection showed that the heavy chain expression was 100% and the light chain expression was 95%. 10-20 Fabs were randomly selected for sequencing to verify the diversity of the phage library, showing that the heavy chain variable region and the light chain variable region were from different germlines, indicating that the library had good diversity. The screening strategy is antigen and cell combination screening, with human FGFR2b protein or cells as positive screening, human FGFR2c protein as negative screening, and cynomolgus monkey and rat FGFR2b as auxiliary negative screening. The clones that bind to human FGFR2b at the antigen level and clones that bind to human FGFR2b well at the cell level were screened using 96-well plates and sequenced. Combined with the above well plate screening data, the clone 21G4 molecule was finally selected for subsequent activity analysis and evaluation.

[0093] (3) Sequence acquisition of positive clones

[0094] According to the results of phage screening, the variable region sequence of 21G4 positive clones was amplified. The amplified products were sequenced to obtain the heavy chain and light chain variable region sequences of the candidate antibodies. The variable region sequences of the antibody light and heavy chains were cloned into eukaryotic expression vectors by PCR, and the full-length IgG antibody was expressed in CHO-18 cells, and the activity of the full-length antibody was analyzed. The 21G4 sequence is shown in Table 2.

[0095] Table 2 21G4 sequence

[0096]

[0097]

[0098] Example 3: Chimeric Antibody Activity Detection

[0099] (1) Binding of chimeric antibodies to human FGFR2b protein

[0100] 0.5 μg / mL human FGFR2b protein was coated on a 96-well ELISA plate with a carbonate buffer solution at pH 9.6 as the coating solution at 4°C overnight. Wash three times with PBST, and add 200 μl of 2% milk / PBS to each well for blocking for 1 hour. After washing once with PBST, add 100 μl of sample supernatant to each well and incubate at room temperature for 60 minutes; after washing three times with PBST, add 100 μl of HRP-labeled anti-human IgG Fc secondary antibody to each well and incubate at room temperature for 60 minutes. After washing three times with PBST, add 100 μl of TMB substrate to each well and develop at 37°C for 10 minutes. After that, terminate the reaction with 50 μl of 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450 nm. The results show that 21G4 can bind to human FGFR2b protein at the antigen level, EC 50 is 0.02nM, such as Figure 2 shown.

[0101] (2) Binding of chimeric antibodies to cynomolgus monkey and rat FGFR2b

[0102] 0.5 μg / mL of cynomolgus monkey or rat FGFR2b protein was coated on a 96-well ELISA plate with a carbonate buffer solution of pH 9.6 at 4°C overnight. Wash three times with PBST, and add 200 μl of 2% milk / PBS to each well for blocking for 1 hour. After washing once with PBST, add 100 μl of sample supernatant to each well and incubate at room temperature for 60 minutes; after washing three times with PBST, add 100 μl of HRP-labeled anti-human IgG Fc secondary antibody to each well and incubate at room temperature for 60 minutes. After washing three times with PBST, add 100 μl of TMB substrate to each well and develop at 37°C for 10 minutes. After that, stop the reaction with 50 μl of 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450 nm. The results show that the chimeric antibody 21G4 can bind to cynomolgus monkey FGFR2b protein and rat FGFR2b protein at the antigen level, EC 50 Both are 0.02nM, such as Figure 3 shown.

[0103] (3) Binding of chimeric antibodies to cells expressing human FGFR2b

[0104] Human gastric cancer cells KATOIII were collected and added to a 96-well plate, with 2 × 10 4cells, 100μL / well. After centrifugation at 300g for 3 minutes, discard the supernatant, block with 50μL 3% BSA / PBS for 10 minutes, add 50μL of antibody sample to the cells, mix gently and incubate at 4℃ for 30 minutes, wash 3 times with 0.5% BSA / PBS. Add anti-human IgG secondary antibody solution coupled to AF647, mix gently and incubate at 4℃ for 30 minutes, wash 3 times with 0.5% BSA / PBS. Finally, resuspend with PBS containing 5μg / mL PI, mix well and detect with flow cytometer. The results show that the chimeric antibody 21G4 has good binding activity at the cellular level, EC 50 is 2.6nM, such as Figure 4 shown.

[0105] (4) Binding of chimeric antibodies to cynomolgus monkey and rat FGFR2b cells

[0106] The cells were collected and added to a 96-well plate, with 2 × 10 4 cells, 100μL / well. After centrifugation at 300g for 3 minutes, discard the supernatant, block with 50μL 3% BSA / PBS for 10 minutes, add 50μL of antibody sample to the cells, mix gently and incubate at 4°C for 30 minutes, and wash 3 times with 0.5% BSA / PBS. Add anti-human IgG secondary antibody solution conjugated to AF647, mix gently and incubate at 4°C for 30 minutes, and wash 3 times with 0.5% BSA / PBS. Finally, resuspend with PBS containing 5μg / mL PI, mix well and detect by flow cytometry. The results showed that the chimeric antibody 21G4 can bind to cynomolgus monkey FGFR2b cells and rat FGFR2b cells, and bind to the EC of cynomolgus monkey cells. 50 The EC value for binding to rat cells is 0.20 nM. 50 is 0.67nM, such as Figure 5 shown.

[0107] (5) Blockade of FGF7 / FGFR2b binding by chimeric antibodies

[0108] Coat a 96-well ELISA plate with a 50 μg / mL heparin solution in a carbonate buffer solution at pH 9.6 at 4°C overnight. Wash three times with PBST, add 100 μl of a 0.2% μg / mL FGF7 solution to each well and incubate for 1 hour. Wash three times with PBST, incubate with 2% BSA / PBS for another hour, wash three times with PBST, and incubate different concentrations of antibody samples with 0.5 μg / mL human FGFR2b protein (mouse Fc tag) in a blocked U-shaped 96-well plate at 37°C for 15 minutes. Wash the 96-well ELISA plate three times with PBS, add 100 μl of the incubated mixture to each well, and incubate at room temperature for 1 hour. After washing three times with PBST, add 100 μl of HRP-coupled anti-mouse Fc antibody to each well and incubate at room temperature for 1 hour in the dark. After washing three times with PBST, 100 μl TMB substrate was added to each well for color development at 37°C for 10 minutes, and then 50 μl 2M sulfuric acid solution was added to each well to terminate the reaction, and the absorbance was read at a wavelength of 450 nm. The results showed that the chimeric antibody 21G4 could effectively inhibit the binding of FGF7 to human FGFR2b protein at the antigen level. Figure 6 shown.

[0109] (6) Inhibition of FGF7-induced cell proliferation by chimeric antibodies

[0110] Ba / F3-hFGFR2b-1G9 cells were resuspended in IMDM medium containing no mouse IL-3, 2.5 μg / mL heparin and 10% fetal bovine serum, and 3×104 cells were plated at 100 μL / well per well, and starved overnight. The next day, 30 ng / mL of FGF7 was mixed with equal volumes of antibodies of different concentrations and incubated at 37°C for 15 minutes. 100 μL of the incubated FGF7 / antibody mixed solution was added to the cells and cultured in a 5% CO2, 37°C cell culture incubator for 5 days. After the culture was completed, it was detected with Promega's Cell titer Glo (Cat. No. G7570), and the chemiluminescent signal was detected with an enzyme reader. The results showed that the chimeric antibody 21G4 had good proliferation blocking activity, and IC 50 is 1.3nM, such as Figure 7 shown.

[0111] (7) Blockade of FGF10 / FGFR2b and FGF23 / FGFR2c binding by chimeric antibodies

[0112] Coat the 96-well ELISA plate with 50 μg / mL heparin solution, the coating solution is pH 9.6 carbonate buffer, overnight at 4°C. Wash three times with PBST, add 100 μl of 0.2% μg / mL FGF10 / FGF23 solution to each well and incubate for 1 hour. Wash three times with PBST, incubate for another hour with 2% BSA / PBS, wash three times with PBST, and incubate different concentrations of antibody samples with 0.5 μg / mL human FGFR2b protein (mouse Fc tag) in a blocked U-shaped 96-well plate at 37°C for 15 minutes. Wash the 96-well ELISA plate three times with PBS, add 100 μl of the incubated mixture to each well, and incubate at room temperature for 1 hour. After washing three times with PBST, add 100 μl of HRP-coupled anti-mouse Fc antibody to each well and incubate at room temperature for 1 hour in the dark. After washing three times with PBST, 100 μl of TMB substrate was added to each well for color development at 37°C for 10 minutes, and then 50 μl of 2M sulfuric acid solution was added to each well to terminate the reaction, and the absorbance was read at a wavelength of 450 nm. The results showed that the chimeric antibody 21G4 could effectively inhibit the binding of FGF10 to human FGFR2b protein at the antigen level, and the IC 50 The concentration is 5.7 nM, which will not block the binding of negative sieve protein FGFR2c to its ligand FGF23. Figure 8 shown.

[0113] (8) Chimeric antibodies block FGF10-induced cell proliferation

[0114] Ba / F3-hFGFR2b-1G9 cells were resuspended in IMDM medium without mouse IL-3, containing 2.5 μg / mL heparin and 10% fetal bovine serum, and 3×10 cells were added to each well. 4 Cells were plated at 100 μL / well and starved overnight. The next day, 30 ng / mL of FGF10 was mixed with equal volumes of antibodies of different concentrations and incubated at 37°C for 15 minutes. 100 μL of the incubated FGF10 / antibody mixed solution was added to the cells and cultured in a 5% CO2, 37°C cell culture incubator for 5 days. After the culture was completed, Promega's Cell titer Glo (Cat. No. G7570) was used for detection, and the chemiluminescent signal was detected with an enzyme reader. The results showed that the chimeric antibody 21G4 was able to block FGF10-induced cell proliferation, IC 50 is 1.9nM, such as Fig. 9 shown.

[0115] (9) Chimeric Antibody Binding Specificity

[0116] 0.5 μg / mL human FGFR2c protein was coated on a 96-well ELISA plate with a carbonate buffer solution at pH 9.6 as the coating solution at 4°C overnight. Wash three times with PBST, and add 200 μl of 2% milk / PBS to each well for blocking for 1 hour. After washing once with PBST, add 100 μl of sample supernatant to each well and incubate at room temperature for 60 minutes; after washing three times with PBST, add 100 μl of HRP-labeled anti-human IgG Fc secondary antibody to each well and incubate at room temperature for 60 minutes. After washing three times with PBST, add 100 μl of TMB substrate to each well and develop at 37°C for 10 minutes, then terminate the reaction with 50 μl of 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450 nm. The results showed that the chimeric antibody 21G4 did not bind to the human FGFR2c protein, as shown in Fig.10 -A.

[0117] HEK293-hFGFR2c-3D5 negative screening cells were collected and added to a 96-well plate, with 2 × 10 cells per well. 4 cells, 100 μL / well. After centrifugation at 300g for 3 minutes, discard the supernatant, block with 50 μL 3% BSA / PBS for 10 minutes, add 50 μL of antibody sample to the cells, mix gently and incubate at 4°C for 30 minutes, wash 3 times with 0.5% BSA / PBS. Add anti-human IgG secondary antibody solution conjugated to AF647, mix gently and incubate at 4°C for 30 minutes, wash 3 times with 0.5% BSA / PBS. Finally, resuspend with PBS containing 5 μg / mL PI, mix well and detect with flow cytometry. The results showed that the chimeric antibody 21G4 did not bind to cells expressing human FGFR2c, such as Fig.10 -B.

[0118] (10) Chimeric antibody affinity

[0119] The kinetic binding activity of anti-FGFR2b antibodies to human FGFR2b was measured by surface plasmon resonance (SPR) using the BIAcore T200 system. 2 μg / ml of anti-FGFR2b antibodies were immobilized on the sensor chip Protein A, and 1×HBS-EP buffer was used as the running buffer at a flow rate of 10 μl / min for 40 seconds. Different concentrations of human FGFR2b of 0-21.7 nM were injected onto the immobilized anti-FGFR2b antibody surface at a flow rate of 30 μl / min. After each injection cycle, the Protein A chip surface was regenerated at a flow rate of 30 μl / min with a regeneration buffer of 10 mM glycine (pH 2.0). The binding rate constant (ka) and dissociation rate constant (kd) and equilibrium dissociation constant KD were analyzed using background subtraction binding sensorgrams, as shown in Table 3. The resulting data set was fitted to the Langmuir 1:1 kinetics model using BIAevaluation software. The results showed that the chimeric antibody 21G4 binds to human FGFR2b with high affinity. Fig.11 shown.

[0120] Table 3 Affinity determination of chimeric antibody 21G4

[0121] Clone number ka(1 / Ms) kd(1 / s) KD(M) 21G4 4.28E+05 1.20E-04 2.80E-10

[0122] Example 4: Humanization of anti-FGFR2b chimeric antibody

[0123] Humanization was performed based on the chimeric antibody 21G4. The light and heavy chain derivatives were fully synthesized and cloned into a vector containing the antibody κ chain constant region CK or the human IgG1 constant region CH1-CH3. The light and heavy chain derivative plasmids were transfected into CHO cells and expressed for 7-9 days. The supernatant was collected and purified by Protein A column to obtain the humanized molecule h21G4 H4K3. The sequence of h21G4H4K3 is shown in Table 4.

[0124] Table 4 h21G4 H4K3 sequence

[0125]

[0126]

[0127]

[0128] Example 5: Humanized Antibody Activity Detection

[0129] (1) Binding of humanized antibodies to FGFR2b

[0130] 0.5 μg / mL antigen protein was coated on a 96-well ELISA plate, including human FGFR2b, cynomolgus monkey FGFR2b, rat FGFR2b, and human FGFR2c proteins. The coating solution was a carbonate buffer solution at pH 9.6, and the plate was incubated at 4°C overnight. Wash three times with PBST, and add 200 μl of 2% milk / PBS to each well for blocking for 1 hour. After washing once with PBST, add 100 μl of sample supernatant to each well and incubate at room temperature for 60 minutes; after washing three times with PBST, add 100 μl of HRP-labeled anti-human IgG secondary antibody to each well and incubate at room temperature for 60 minutes. After washing three times with PBST, add 100 μl of TMB substrate to each well for color development at 37°C for 10 minutes, terminate the reaction with 50 μl of 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450 nm. The results showed that the humanized antibody h21G4 H4K3 could bind to human FGFR2b, cynomolgus monkey FGFR2b and rat FGFR2b at the antigen level, but did not bind to human FGFR2c protein, as shown in Tables 5 and Fig.12 shown.

[0131] Table 5 Humanized antibody h21G4 H4K3 binding

[0132]

[0133] (2) Binding of humanized antibodies to cells expressing human FGFR2b

[0134] Human gastric cancer cells KATOIII were collected and added to a 96-well plate, with 2 × 10 4 cells, 100 μL / well. After centrifugation at 300g for 3 minutes, discard the supernatant, block with 50 μL 3% BSA / PBS for 10 minutes, add 50 μL of antibody sample to the cells, mix gently and incubate at 4°C for 30 minutes, and wash 3 times with 0.5% BSA / PBS. Add anti-human IgG secondary antibody solution conjugated to AF647, mix gently and incubate at 4°C for 30 minutes, and wash 3 times with 0.5% BSA / PBS. Finally, resuspend with PBS containing 5 μg / mL PI, mix well and detect with flow cytometry. The results showed that the humanized antibody h21G4 H4K3 can bind to human FGFR2b at the cellular level, and EC 50 is 0.97nM, and the results are as follows Fig.13 shown.

[0135] (3) Binding of humanized antibodies to human FGFR2c

[0136] HEK293-hFGFR2c-3D5 cells were collected and added to a 96-well plate, with 2 × 10 cells per well. 4cells, 100 μL / well. After centrifugation at 300g for 3 minutes, discard the supernatant, block with 50 μL 3% BSA / PBS for 10 minutes, add 50 μL of antibody sample to the cells, mix gently and incubate at 4°C for 30 minutes, wash 3 times with 0.5% BSA / PBS. Add anti-human IgG secondary antibody solution conjugated to AF647, mix gently and incubate at 4°C for 30 minutes, wash 3 times with 0.5% BSA / PBS. Finally, resuspend with PBS containing 5 μg / mL PI, mix well and detect by flow cytometry. The results showed that the humanized antibody h21G4 H4K3 could not bind to human FGFR2c cells at the cellular level.

[0137] (4) Blocking effect of humanized antibodies on FGF7-induced cell proliferation

[0138] Ba / F3-hFGFR2b-1G9 cells were resuspended in IMDM medium without mouse IL-3, containing 2.5 μg / mL heparin and 10% fetal bovine serum, and 3×10 cells were added to each well. 4 Cells were plated at 100 μL / well and starved overnight. The next day, 30 ng / mL FGF7 was mixed with different concentrations of antibodies in equal volumes and incubated at 37°C for 15 minutes. 100 μL of the incubated FGF7 / antibody mixed solution was added to the cells and cultured in a 5% CO2, 37°C cell culture incubator for 5 days. After the culture was completed, the cells were tested using Promega's Cell titer Glo. The results showed that the humanized antibody h21G4 H4K3 could block FGF7-induced cell proliferation activity, IC 50 is 3.3nM, such as Fig.14 shown.

[0139] (5) Humanized Antibody ADCC Activity

[0140] Ba / F3-hFGFR2b-1G9 cells were used as target cells and NK92-MI-CD16a cells were used as effector cells. The cells were plated in a 96-well U-shaped plate at a ratio of effector cells to target cells = 2:1. Different concentrations of humanized antibodies were added and incubated at 37°C and 5% CO2 for 4 hours. After incubation, LDH release was detected. Release rate = (experimental well - spontaneous well) ÷ (maximum release well - spontaneous well) × 100%. Fig.15 The humanized antibodies shown all have ADCC activity, EC 50 The EC values ​​of h21G4 and H4K5 were between 0.50 and 1.36 nM. 50 is 0.50nM.

Claims

1. An antibody or antigen-binding fragment thereof for fibroblast growth factor receptor 2b (FGFR2b), comprising a heavy chain variable region and a light chain variable region, wherein the CDR combination of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 15, a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3, wherein the amino acid residue represented by SEQ ID NO: 3 is WGY; and a light chain CDR1 consisting of the amino acid sequence SEQ ID NO: 6, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 7, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO:

20.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

21.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

21. 4 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 type antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 18, and the light chain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

23.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

23.

7. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the nucleic acid has an antibody heavy chain variable region encoding nucleotide sequence that is at least 80% identical to SEQ ID NO: 17, and an antibody light chain variable region encoding nucleotide sequence that is at least 80% identical to SEQ ID NO:

22.

8. The nucleic acid according to claim 7, wherein the nucleic acid has a nucleotide sequence encoding an antibody heavy chain variable region as shown in SEQ ID NO: 17, and a nucleotide sequence encoding an antibody light chain variable region as shown in SEQ ID NO:

22.

9. The nucleic acid of claim 7 or 8, wherein the nucleic acid has an antibody heavy chain encoding nucleotide sequence that is at least 80% identical to SEQ ID NO: 19, and an antibody light chain encoding nucleotide sequence that is at least 80% identical to SEQ ID NO:

24.

10. The nucleic acid according to any one of claims 7 to 9, wherein the nucleic acid has a nucleotide sequence encoding an antibody heavy chain as shown in SEQ ID NO: 19, and a nucleotide sequence encoding an antibody light chain as shown in SEQ ID NO:

24.

11. A vector comprising the nucleic acid of any one of claims 7-10.

12. A host cell comprising the nucleic acid of any one of claims 7 to 10 or the vector of claim 11.

13. A pharmaceutical composition or drug combination comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

14. The pharmaceutical composition or pharmaceutical combination according to claim 13, wherein the composition or pharmaceutical combination further comprises a second therapeutic agent.

15. The pharmaceutical composition or pharmaceutical combination according to claim 14, wherein the second therapeutic agent is selected from beta-2 adrenergic receptor agonists (SABA), anticholinergics, adrenergic agonists, glucocorticoids, long-acting beta-2 adrenergic receptor agonists (LABA), leukotriene antagonists and mast cell stabilizers.

16. A method for producing the antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising culturing the host cell of claim 12 containing the nucleic acid encoding any one of claims 7-10 or the vector of claim 11.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or the pharmaceutical composition or pharmaceutical combination according to any one of claims 13 to 15 in the preparation of a medicament or kit for detecting, treating, and / or alleviating a disease associated with the expression of FGFR2b, wherein the disease associated with the expression of FGFR2b is gastric cancer.

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