Anti-FGFR2 / PD-1 dual-specific antibody

Abstract

This invention relates to an anti-FGFR2 / PD-1 bispecific antibody. The anti-FGFR2 / PD-1 bispecific antibody of the present invention comprises a first antigen-binding domain D1 and a second antigen-binding domain D2, where D1 is an anti-FGFR2 antibody or its antigen-binding fragment, and D2 is an anti-PD-1 antibody or its antigen-binding fragment. The anti-FGFR2 / PD-1 bispecific antibody of the present invention exerts an antitumor effect by suppressing the proliferation of FGFR2 target cells, killing target cells through ADCC activity, and simultaneously inhibiting the binding of PD-1 to its ligand, thereby releasing the inhibitory state of T cells.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody pharmaceutical technology, and more particularly to anti-FGFR2 / PD-1 bispecific antibodies. [Background technology]

[0002] Fibroblast growth factor receptors (FGFRs) are highly conserved receptor tyrosine kinases, generally comprising four typical structures, FGFR1-4, and FGFR5, which lacks an intracellular tyrosine kinase domain. The extracellular domain of FGFRs contains three immunoglobulin-like domains, of which the third Ig domain undergoes carboxyl group selective isomerization to produce the IIIb or IIIc isoforms of FGFR1-FGFR3. FGFR2-IIIb, expressed in epithelial cells, are activated by FGFs and form a ternary complex with heparan sulfate proteoglycans. This complex mediates the nasal pathway through signaling pathways such as RAS-RAF-MAPK, PI3K-AKT, signaling factors, transcription activators, and phospholiase Cγ, and is involved in physiological processes such as angiogenesis, cell proliferation and migration, organ development, and wound healing. Abnormalities in FGFR2 signaling, such as overexpression of FGFR2 and its ligands, mutations in the FGFR2 receptor, and isoform switching, can induce cancer in normal cells. Examples include FGFR2 single nucleotide polymorphisms in breast cancer cells and overexpression of FGFR2-IIIb and its ligands in pancreatic cancer cells. Currently, approved drugs targeting this area include Johnson & Johnson's erdafitinib, Incyte's pemigatinib, and Bridge Bio-Pharma's infiglatinib, all of which are small molecule inhibitors, with relatively few high molecular weight antibody drugs under development. For example, bemarituzumab monoclonal antibody, an FGFR2-targeted therapy for HER2-negative locally advanced or metastatic gastric cancer and gastroesophageal junction cancer, is still in clinical development.

[0003] Programmed cell death protein 1 (PD-1), also known as CD279, is a type I membrane protein consisting of 288 amino acids and belongs to the immunoglobulin superfamily. It is a member of the amplified CD28 / CTLA-4 family of T cell regulators, and its structure consists of an extracellular IgV domain, a transmembrane domain, and an intracellular terminal. The intracellular terminal has two phosphorylation sites for immune receptor tyrosine: an inhibitory motif and an activating motif, allowing it to bind to SHP-1 and SHP-2 phosphatases. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages. When expressed on the surface of activated immune cells, PD-1 protein interacts with its ligands, PD-L1 and PD-L2, upregulating the E3 ubiquitin ligases CBL-b and c-CBL, causing downregulation of immune cell receptors, inhibiting their activation and cytokine release, or completely initiating programmed cell death. High expression of PD-L1 and PD-L2 has been observed in various solid tumors and hematopoietic malignancies, suggesting that tumor cells acquire immune evasion by utilizing immunosuppressive responses mediated by PD-1 and its ligands. In 2018, nivolumab, the world's first PD-1 inhibitor, was approved in China as a second-line treatment for advanced non-small cell lung cancer with wild-type EGFR / ALK driver genes. Subsequently, cintirimab, the first domestically produced PD-1 antibody drug, was also approved for the treatment of Hodgkin lymphoma. With the deepening of clinical research on PD-1, PD-1 inhibitors have made significant progress in the field of cancer treatment and have become one of the most noteworthy cancer immunotherapies in recent years. Globally approved indications include melanoma, lymphoma, lung cancer, gastric cancer, liver cancer, and colorectal cancer.

[0004] Bispecific antibodies (BsAbs), also known as bifunctional antibodies, are specific drugs that simultaneously target two different antigens, or different epitopes of the same antigen. BsAbs can act on tumor cells by mediated by immune cells or viral molecules, enhancing their ability to kill target cells. Furthermore, by simultaneously binding to different antigens on tumor cells, they can increase their binding specificity and reduce off-target effects. Bispecific antibodies have expanded the application fields of antibody drugs and provided a new research approach to tumor immunology.

[0005] However, at present, there are no satisfactory bispecific antibodies targeting both FGFR2 and PD-1. Therefore, the development of new anti-FGFR2 and PD-1 bispecific antibodies is an urgent need in this field. [Overview of the project]

[0006] The objective of the present invention is to provide an anti-FGFR2 / PD-1 bispecific antibody.

[0007] The bispecific antibody provided by the present invention specifically binds to FGFR2 and PD-1, suppressing the proliferation of FGFR2 target cells and killing target cells through ADCC activity, while simultaneously protecting T cells from exerting normal immune function and eliminating target cells. Furthermore, the object of the present invention is to provide a polynucleotide molecule encoding the bispecific antibody, an expression vector containing the molecule, a host cell containing the expression vector, a method for preparing the bispecific antibody, a pharmaceutical composition containing the bispecific antibody, an immune complex containing the bispecific antibody, the use of the bispecific antibody or the pharmaceutical composition in the preparation of therapeutic agents for cancer or tumors, and a method for treating cancer or tumors using the fusion protein, the pharmaceutical composition, or the immune complex.

[0008] In a first aspect of the present invention, a bispecific antibody is provided, the bispecific antibody is The first antigen-binding domain D1 and, the second antigen-binding domain D2, wherein said D1 specifically binds to the target molecule FGFR2 protein, and said D2 specifically binds to the target molecule PD-1 protein.

[0009] In another preferred embodiment, said D1 is an anti-FGFR2 antibody or an antigen-binding fragment thereof.

[0010] In another preferred embodiment, said D2 is an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0011] In another preferred embodiment, said antibody includes an animal-derived antibody (e.g., a mouse antibody), a chimeric antibody, and a humanized antibody.

[0012] In another preferred embodiment, said antibody fragment includes a heavy chain variable region and a light chain variable region.

[0013] In another preferred embodiment, said antibody fragment includes a single-chain variable region fragment (scFv) and a double-chain variable region fragment (dcFv).

[0014] In another preferred embodiment, said D1 and said D2 are linked by a linker.

[0015] In another preferred embodiment, D1 is an antibody, and said linker is (G4S)n, preferably (G4S)n, where n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6), preferably n = 3 or 4, and more preferably, said linker is shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0016] In another preferred embodiment, D1 is an anti-FGFR2 antibody, and D2 is bound to a region of D1 selected from the group consisting of a heavy chain variable region, a heavy chain constant region, a light chain variable region, or a combination thereof via a linker. In another preferred embodiment, D1 is an anti-FGFR2 antibody, and D2 is bound to the end of the heavy chain constant region of D1 via a linker.

[0017] In another preferred embodiment, the bispecific antibody is a homodimer or a heterodimer.

[0018] In another preferred embodiment, the bispecific antibody comprises a monomer and has a structure represented by formula I or formula II from the N-terminus to the C-terminus. [ka] Here, VL A This indicates the light chain variable region of an anti-FGFR2 antibody or its antigen-binding fragment. VH A This indicates the heavy chain variable region of the anti-FGFR2 antibody or its antigen-binding fragment. VL B This indicates the light chain variable region of an anti-PD-1 antibody or its antigen-binding fragment. VH B This indicates the heavy chain variable region of the anti-PD-1 antibody or its antigen-binding fragment. CH exhibits a heavy chain steady region. CL represents the light chain constant region. L1 and L2 are, independently, couplings or linkers. "~" indicates a disulfide bond or covalent bond. The hyphen "-" indicates a peptide bond. Here, the bispecific antibody has the activity to bind simultaneously to FGFR2 and PD-1.

[0019] In another preferred embodiment, the anti-FGFR2 or anti-PD-1 antigen binding fragment is selected from Fab, F(ab'), F(ab')2, Fv, or single-chain Fv(scFv), and the anti-FGFR2 or anti-PD-1 antibody is an IgG antibody.

[0020] In another preferred embodiment, the anti-FGFR2 or anti-PD-1 antibody is selected from a chimeric antibody, a mouse antibody, or a humanized antibody. In another preferred embodiment, the IgG antibody comprises a heavy chain constant region and a light chain constant region. More preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3, or human IgG4, and the light chain constant region is selected from human κ (Kappa) or human λ (Lambda).

[0021] In another preferred embodiment, the constant region of the IgG antibody contains at least one amino acid mutation.

[0022] In another preferred embodiment, D1 is an IgG antibody and D2 is an scFv. More preferably, D2 is ligated to the N-terminus or C-terminus of D1, and even more preferably, D2 is ligated to the heavy chain of D1.

[0023] In another preferred embodiment, the D2 includes one, two, three, or more anti-PD-1 scFvs.

[0024] In another preferred embodiment, the scFv includes a VH-L1-VL structure or a VL-L1-VH structure from the N-terminus to the C-terminus.

[0025] In another preferred embodiment, the amino acid sequences of linkers L1 and L2 are independently (G4S) n And n is chosen from 1, 2, 3, 4, 5, 6.

[0026] In another preferred embodiment, the amino acid sequence of linker L1 is shown in SEQ ID NO: 17.

[0027] In another preferred embodiment, the amino acid sequence of linker L2 is shown in SEQ ID NO: 18.

[0028] In another preferred embodiment, the bispecific antibody is a homodimer comprising an anti-FGFR2 IgG antibody and two anti-PD-1 scFvs, each scFv comprising a variable region VH and a variable region VL, where VH and VL are linked via a linker L1 to form a VL-L1-VH structure, and each anti-PD-1 scFv is linked via a linker L2 to the C-terminus of the anti-FGFR2 immunoglobulin antibody IgG.

[0029] In another preferred embodiment, the bispecific antibody comprises a heavy chain and a light chain, the amino acid sequences of the heavy chain and the light chain are selected from the following group. a) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 29, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 28. b) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 30, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 28, or c) A polypeptide having both anti-FGFR2 activity and anti-PD-1 activity, formed by the substitution, deletion, or addition of one or more amino acid residues to the amino acid sequence of a) or b), and derived from a) or b).

[0030] In another preferred embodiment, the bispecific antibody comprises an active fragment and / or derivative thereof, the active fragment and / or derivative thereof retaining 70-100% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) of the anti-FGFR2 activity and 70-100% of the anti-PD-1 activity of the bispecific antibody.

[0031] In another preferred embodiment, the derivative of the bispecific antibody is a polypeptide of the bispecific antibody that has undergone one or more amino acid mutations (deletions, insertions, and / or substitutions of amino acids) and maintains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity.

[0032] In another preferred embodiment, the amino acid mutation is a conserved amino acid substitution.

[0033] In another preferred embodiment, the amino acid mutation is located in a framework region or a constant region.

[0034] In another preferred embodiment, the anti-FGFR2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three heavy chain complementarity determining regions: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3. The aforementioned light chain variable region includes three light chain complementarity determination regions: LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6.

[0035] In another preferred embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three heavy chain complementarity determining regions: HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9. The light chain variable region includes three light chain complementarity determination regions: LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12.

[0036] In another preferred embodiment, the anti-FGFR2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region being shown in SEQ ID NO: 14.

[0037] In another preferred embodiment, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region being shown in SEQ ID NO: 16.

[0038] In another preferred embodiment, the heavy chain constant region comprises an Fc fragment, the Fc fragment derived from IgG1 or IgG4.

[0039] In another preferred embodiment, the Fc fragment is derived from an Fc fragment of IgG1.

[0040] In another preferred embodiment, the Fc fragment comprises at least one amino acid mutation.

[0041] In another preferred embodiment, the mutation is used to enhance the ADCC activity of a bispecific antibody.

[0042] In another preferred embodiment, the Fc fragment derived from IgG1 is It has a mutation selected from the group consisting of S239D, I332E, A330L, G236A, preferably S239D and I332E.

[0043] In another preferred embodiment, the heavy chain steady region includes CH1, CH2, and CH3.

[0044] In another preferred embodiment, the CH1, CH2, and CH3 amino acid sequences are shown independently in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0045] In another preferred embodiment, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 23.

[0046] In another preferred embodiment, the heavy chain constant region includes an amino acid mutation selected from the group consisting of S239D and I332E.

[0047] In another preferred embodiment, the anti-FGFR2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, where the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14, and / or the anti-PD1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, where the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16, Preferably, the variable region contains at least one amino acid mutation.

[0048] In another preferred embodiment, the heavy chain constant region of the anti-FGFR antibody includes amino acid mutations selected from the following group. a) The heavy chain constant region has a mutation at the 239th amino acid, preferably S293D, and / or b) The heavy chain constant region has a mutation at the 332nd amino acid, preferably I332E.

[0049] In another preferred embodiment, the sequence of the heavy chain constant region of the anti-FGFR antibody is shown in SEQ ID NO: 23, or the sequence of the heavy chain constant region of the anti-FGFR antibody is shown in SEQ ID NO: 24.

[0050] In another preferred embodiment, the anti-FGFR or anti-PD1 antigen binding fragment is selected from Fab, F(ab'), F(ab')2, Fv, or single-chain Fv(scFv), and the anti-FGFR or anti-PD1 antibody is an IgG antibody.

[0051] In another preferred embodiment, D1 is an IgG antibody and D2 is an scFv. Preferably, D2 is ligated to the N-terminus or C-terminus of D1, and more preferably, D2 is ligated to the heavy chain of D1.

[0052] In a second embodiment of the present invention, a polynucleotide molecule encoding a bispecific antibody provided in the first embodiment of the present invention is provided.

[0053] In a third embodiment of the present invention, an expression vector comprising the polynucleotide molecule described in the second embodiment of the present invention is provided.

[0054] In a fourth embodiment of the present invention, a host cell containing the expression vector described in the third embodiment of the present invention is provided.

[0055] In a fifth embodiment of the present invention, a) A step of culturing host cells according to the fourth aspect of the present invention under expression conditions to express a bispecific antibody, b) A step of separating and purifying the bispecific antibody described in step a), The present invention provides a method for preparing a bispecific antibody, which includes the above. A sixth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of a bispecific antibody provided in the first aspect of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0056] In a seventh aspect of the present invention, the use of a bispecific antibody provided in the first aspect of the present invention or a pharmaceutical composition described in the sixth aspect of the present invention is provided in the preparation of a therapeutic agent for cancer or tumor.

[0057] Preferably, the cancer or tumor is an FGFR2-positive cancer or tumor.

[0058] In another preferred embodiment, The aforementioned cancers or tumors include lung cancer, bone cancer, gastric cancer, pancreatic adenocarcinoma, prostate cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, prostate cancer, pancreatic adenocarcinoma, brain tumor, testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, soft tissue sarcoma, pediatric solid tumors, lymphoid lymphoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, pituitary adenoma, melanoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia, or combination thereof. Selected from primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, melanoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic and acute leukemia, or a combination thereof.

[0059] In an eighth aspect of the present invention, a) A bispecific antibody according to any one of the first aspects of the present invention, b) Provide an immune complex comprising a complex portion selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0060] In another preferred embodiment, the complex portion is selected from a fluorescent or luminescent marker, a radiomarker, a contrast agent for magnetic resonance imaging or computed tomography (CT), or an enzyme, radionuclide, biotoxin, or cytokine capable of producing a detectable product.

[0061] In another preferred embodiment, the immune complex comprises an antibody-drug conjugate. In another preferred embodiment, the immune complex is a pharmaceutical composition for treating tumors or cancer.

[0062] A ninth aspect of the present invention provides a method for treating cancer or tumor, comprising administering to a subject who requires a bispecific antibody according to the first aspect of the present invention, a pharmaceutical composition according to the sixth aspect of the present invention, or an immune complex according to the eighth aspect of the present invention.

[0063] In another preferred embodiment, the method further includes administration in combination with other antitumor agents.

[0064] It should be understood that each of the above technical features of the present invention and each of the technical features specifically described below (for example, in the examples) can be combined with each other within the scope of the present invention to constitute novel or preferred technical solutions. Due to space limitations, each will not be explained in detail here. [Brief explanation of the drawing]

[0065] [Figure 1A] This shows a schematic diagram of the structure of anti-FGFR2 / PD-1 bispecific antibody a or b. [Figure 1B] This shows a schematic diagram of the structure of the anti-FGFR2 / PD-1 bispecific antibody c. [Figure 1C] This shows a schematic diagram of the structure of the anti-FGFR2 / PD-1 bispecific antibody d. [Figure 2A] This shows the UPLC-SEC spectrum of the anti-FGFR2 / PD-1 bispecific antibody a. [Figure 2B] This shows the UPLC-SEC spectrum of the anti-FGFR2 / PD-1 bispecific antibody b. [Figure 2C] This shows the UPLC-SEC spectrum of the anti-FGFR2 / PD-1 bispecific antibody c. [Figure 2D] This shows the UPLC-SEC spectrum of the anti-FGFR2 / PD-1 bispecific antibody d. [Figure 3A] This shows the de-N-glycosylated complete molecular weight mass spectrum of the anti-FGFR2 / PD-1 bispecific antibody a. [Figure 3B]This shows the de-N-glycosylated complete molecular weight mass spectrum of the anti-FGFR2 / PD-1 bispecific antibody b. [Figure 4A] This shows the DSC spectrum of anti-FGFR2 / PD-1 bispecific antibody a. [Figure 4B] This shows the DSC spectrum of anti-FGFR2 / PD-1 bispecific antibody b. [Figure 5A] This shows the binding of anti-FGFR2 / PD-1 bispecific antibodies a and b to FGFR2 by ELISA. [Figure 5B] This shows the binding of anti-FGFR2 / PD-1 bispecific antibodies c and d to FGFR2 by ELISA. [Figure 5C] This shows the binding of anti-FGFR2 / PD-1 bispecific antibodies a and b to PD-1 by ELISA. [Figure 5D] This shows the binding of anti-FGFR2 / PD-1 bispecific antibodies a and d to PD-1 by ELISA. [Figure 6A] This demonstrates the binding activity of anti-FGFR2 / PD-1 bispecific antibodies a and b to Jurkat-PD-1-NFAT-luc cells. [Figure 6B] This demonstrates the PD-1 / PD-L1 binding inhibitory activity of anti-FGFR2 / PD-1 bispecific antibodies a and b. [Figure 7A] This shows the ADCC activity of FGFR2 / PD-1 bispecific antibodies a and b against SNU-16 cells. [Figure 7B] This shows the ADCC activity of anti-FGFR2 / PD-1 bispecific antibodies a and b against T cells. [Figure 8] This demonstrates the inhibitory effect of anti-FGFR2 / PD-1 bispecific antibodies a and b on FGF1-induced Ba / F3-FGFR2IIIb cell proliferation. [Figure 9A] This demonstrates the antitumor effect of anti-FGFR2 / PD-1 bispecific antibody b in an MC38 transplanted tumor model. [Figure 9B] This shows the toxic side effects of anti-FGFR2 / PD-1 bispecific antibody b in a mouse MC38 transplanted tumor model. [Figure 10A] This shows the antitumor effects of anti-FGFR2 / PD-1 bispecific antibodies a and b in a SNU-16 transplanted tumor model. [Figure 10B] This shows the toxic side effects of FGFR2 / PD-1 bispecific antibodies a and b in a mouse SNU-16 transplanted tumor model. Specific Embodiments

[0066] Through extensive and meticulous research, the inventors have obtained a novel bispecific antibody targeting the tumor cell surface molecule FGFR2 and the immune cell surface molecule PD-1. This antibody can simultaneously bind to both the FGFR2 antigen and the PD-1 antigen while maintaining antibody activity at both ends. The bispecific antibody of the present invention can achieve a T cell-mediated immune response while simultaneously killing tumor cells expressing FGFR2 through ADCC activity. Therefore, the bispecific antibody of the present invention is expected to be developed as an antitumor drug with excellent therapeutic effects. Based on this, the present invention was completed.

[0067] term

[0068] To facilitate understanding of this disclosure, we first define certain terms. In this application, unless otherwise specified, the following terms shall have the meanings set forth below.

[0069] The term "approximately" refers to a value or composition within a tolerance range for a particular value or composition as determined by those skilled in the art, which may depend on the measurement method, measurement value, or composition.

[0070] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetrameric glycoproteins having identical structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain by one covalent disulfide bond, and the number of disulfides between heavy chains of different immunoglobulin isotypes differs. Each heavy and light chain also has intrachain disulfide bonds at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by a constant region, the heavy chain constant region consisting of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region containing one domain CL. The constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant region does not directly participate in antibody-antigen binding, but exerts various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region contains IgG1, IgG2, IgG3, and IgG4 subtypes, while the light chain constant region contains κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of an antibody are covalently linked by an interpolypeptide disulfide bond formed between the hinge regions.

[0071] The "immunoglobulin antibody IgG" described in this invention is a molecule of approximately 150 kDa, composed of four peptide chains, containing two identical γ heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thereby having a tetrameric quaternary structure. The two heavy chains are linked to each other by disulfide bonds, and each is bound to one light chain. The resulting tetramer consists of two identical halves that form a fork-like or Y-shaped structure, with identical antigen-binding sites at both ends of the fork. IgG antibodies are classified into several subclasses (e.g., IgG1, 2, 3, 4) based on minute differences in the amino acid sequence in the constant region of the heavy chain.

[0072] In this invention, the term "bispecific antibody (or biantibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes. Based on symmetry, bispecific antibodies are classified into molecules with symmetric or asymmetric structures. Depending on the number of binding sites, bispecific antibodies are classified into divalent, trivalent, tetravalent, and polyvalent molecules.

[0073] In this invention, the term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., an individual antibody within a cluster is identical except for a few possible spontaneous variations. Monoclonal antibodies exhibit high specificity for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations (usually mixtures containing different antibodies for different antigenic determinants), each monoclonal antibody is directed towards a single determinant cluster on an antigen. In addition to its specificity, monoclonal antibodies have the advantage of being able to be synthesized by hybridoma culture and not being contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring antibody production by a specific method.

[0074] In this specification, the term “antigen-binding fragment” refers to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments, etc., that possess antigen-binding activity. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fv fragments, or (iv) single-chain Fv(scFv). In this specification, the term “antigen-binding fragment” also includes domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, bisome antibodies, trisome antibodies, tetrasome antibodies, microantibodies, nanobodies (e.g., monovalent and bivalent nanobodies), modular immunotherapies, and other modified molecules such as shark variable IgNAR domains.

[0075] In this invention, the terms "Fab" and "Fc" refer to the fact that papain can cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of the light chain. The Fc fragment is a crystallizable fragment (Fc) and consists of the CH2 and CH3 domains of the antibody. The Fc fragment lacks antigen-binding activity and is the interaction site between the antibody and effector molecules or cells. The term "F(ab')2" fragment antibody refers to an antibody obtained by digesting the entire IgG antibody with pepsin, removing most of the Fc region while retaining some hinge region, and having two antigen-binding F(ab') regions linked by disulfide bonds.

[0076] In this invention, the terms "scFv" or "single-chain variable region fragment scFv" refer to a single-chain antibody fragment (scFv) which is a fusion protein containing the variable regions of the immunoglobulin heavy chain VH and light chain VL, wherein VH and VL are linked by a linker containing 15 to 25 amino acids, and the fusion protein retains the same antigen specificity as a complete immunoglobulin.

[0077] In this invention, the Fv antibody includes a variable region of the antibody heavy chain and a variable region of the light chain, but does not include a constant region, and contains the smallest antibody fragment having all antigen-binding sites. Generally, the Fv fragment also includes a polypeptide linker between the VH domain and the VL domain, which can form the structure necessary for antigen binding.

[0078] In this invention, the term "variable" refers to a difference in a portion of the sequence within the variable region of an antibody, thereby shaping the binding affinity and specificity of various specific antibodies to specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three fragments within the hypervariable region, or region, called the complementarity-determining region (CDR), in both the heavy and light chain variable regions. The conserved portion of the variable region is called the frame region (FR). The four FR regions in the native heavy and light chain variable regions basically exhibit a β-sheet structure connected by three CDRs that form connecting loops, and can sometimes form a partial β-sheet structure. The CDRs of each chain are held in close proximity to each other via the FR region, and together with the CDR of the other chain, they form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0079] In this specification, the term “framework region FR” refers to the amino acid sequence inserted between CDRs, i.e., the relatively conserved portion of the variable regions of the light and heavy chains of immunoglobulins among different immunoglobulins of a single species. The light and heavy chains of immunoglobulins each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Thus, the light chain variable domain can be called (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be called FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, the derivative of which is substantially identical to a naturally occurring human antibody FR, i.e., having 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology. Knowing the amino acid sequence of the CDR, a person skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H. In this specification, the term “human framework region” refers to a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the framework region of a naturally occurring human antibody.

[0080] In this specification, the term "linker" refers to one or more amino acid residues that are inserted into an immunoglobulin domain and provide sufficient mobility for the light chain and heavy chain domains to fold into an interchangeable bivariable-region immunoglobulin.

[0081] In the present invention, preferred linkers refer to linker L1 and L2, where L1 is linked to VH and VL of a single-chain antibody (scFv), and L2 is linked between CH1 and CH2 of the antibody heavy chain or directly linked to the heavy chain of the antibody (e.g., the N-terminus or C-terminus of the antibody heavy chain) via L2. Examples of suitable linkers include glycine (Gly) or serine (Ser) residues, and the labeling and sequence of amino acid residues in the linker may vary depending on the type of secondary structure element to be achieved in the linker.

[0082] In the present invention, the terms "anti-", "bind", and "specifically bind" refer to non-random binding reactions between two molecules, such as the reaction between an antibody and its target antigen. Generally, an antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10 -7 M, for example, about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction for representing the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the relative binding affinity between an antibody and an antigen can be measured using the surface plasmon resonance (SPR) method in a BIACORE apparatus or the ELISA method for the relative binding affinity of antibody-antigen binding.

[0083] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody or the region in an antigen that binds to the antibody.

[0084] As used herein, the term "amino acid mutation in the heavy chain constant region" refers to an amino acid mutation at the corresponding position in naturally occurring human IgG1, for example, the 239th or 332nd amino acid mutation of human IgG1.

[0085] In this specification, the bispecific antibodies of the present invention also include their conserved variants, in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced with amino acids having similar or analogous properties, compared to the amino acid sequence of the antibody of the present invention, thereby forming a polypeptide. These conserved variant polypeptides are preferably produced by amino acid substitutions according to Table A. [Table A]

[0086] bispecific antibody The bispecific antibody of the present invention is a bispecific antibody that can specifically bind to FGFR2 and PD-1, and comprises an anti-FGFR2 antibody moiety and an anti-PD-1 antibody moiety. The bispecific antibodies of the present invention may be dimers, trimers, or polymers, and are preferably homodimers or heterodimers. The anti-FGFR2 antibody portion or the anti-PD-1 antibody portion of the bispecific antibodies of the present invention may contain one or more antibodies or their antigen-binding fragments, and are preferably 1, 2, 3, 4, 5, or 6. The sequences of the present invention employ the numbering scheme of the Kabat system.

[0087] In constructing the bispecific antibody of the present invention, problems related to the chemical and physical stability of the bispecific antibody have also been resolved. Specifically, these include the expression of physically stable molecules, improved heat and salt-dependent stability, reduced aggregation, improved solubility at high concentrations, and maintenance of affinity for the two antigens FGFR2 and PD-1.

[0088] Encoding nucleic acids and expression vectors In another aspect of the present invention, a polynucleotide molecule encoding the bispecific antibody described above is provided. The polynucleotide of the present invention may be in the form of DNA or RNA. Examples of DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. Once the desired sequence is obtained, it can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, transplanting it into cells, and then isolating the desired sequence from the proliferated host cells by conventional methods.

[0089] The method for preparing polynucleotides described in the present invention is a conventional method in the art and preferably includes the following preparation methods: obtaining a polynucleotide molecule encoding the monoclonal antibody by gene cloning technology such as PCR, or obtaining a polynucleotide molecule encoding the monoclonal antibody by artificial whole sequence synthesis.

[0090] Those skilled in the art will understand that a polynucleotide homolog can be obtained by appropriately substituting, deleting, modifying, inserting, or adding to the nucleotide sequence encoding the amino acid sequence of the bispecific antibody. The polynucleotide homolog of the present invention can be prepared by substituting, deleting, or adding one or more bases in the gene encoding the bispecific antibody while maintaining antibody activity.

[0091] In another embodiment of the present invention, an expression vector comprising the aforementioned polynucleotide molecule is provided. These vectors can be used to transform suitable host cells into which proteins can be expressed. The expression vectors described herein are conventional expression vectors in the art and refer to expression vectors comprising a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a suitable regulatory sequence such as a marker gene and / or sequence, and other suitable sequences. The expression vector may be a virus such as a suitable bacteriophage or phagemide, or a plasmid. For more technical details, see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, edited by Sambrook et al. For many known techniques and protocols used in nucleic acid manipulation, see, for example, Current Protocols in Molecular Biology, 2nd edition, edited by Ausubel et al. The expression vector described in the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.4, pcDNA3.1 / ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF, or pCHO 1.0, and more preferably pcDNA3.4.

[0092] The present invention further provides a host cell containing the expression vector. The host cells described in the present invention may be any conventional host cells in the art, as long as they can stably replicate the recombinant expression vector and efficiently express the nucleotides it carries. Here, the host cells include prokaryotic expression cells and eukaryotic expression cells, and the host cells preferably include COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21(DE3), or TG1, and more preferably include E. coli TG1, BL21(DE3) cells (expressing single-chain antibodies or Fab antibodies), or CHO-K1 cells (expressing full-length IgG antibodies). The preferred recombinant expression transformants of the present invention are obtained by transforming the host cells with the expression vector. Here, the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method, or an electroporation method.

[0093] Preparation method The host cell culture method and the antibody isolation and purification method described in the present invention are common methods in the art, and for specific operating procedures, please refer to the corresponding cell culture technology manual and antibody isolation and purification technology manual. The method for producing the anti-FGFR2 / PD-1 bispecific antibody disclosed in the present invention includes the following steps: culturing the host cells under expression conditions to express a bispecific antibody that can specifically bind to FGFR2 and PD-1; and isolating and purifying the bispecific antibody. By using the above method, recombinant proteins can be purified into substantially homogeneous substances.

[0094] The anti-FGFR2 / PD-1 bispecific antibody disclosed in this invention can be isolated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, the anti-FGFR2 / PD-1 bispecific antibody bound to the affinity column can be eluted using conventional methods such as high-salt buffer or pH adjustment. The inventors conducted detection experiments using the obtained anti-FGFR2 / PD-1 bispecific antibody and found that it binds well to target cells and antigens and has high affinity.

[0095] Pharmaceutical composition In another embodiment of the present invention, a composition that is a pharmaceutical composition is provided. In the present invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition that exhibits a more stable therapeutic effect by combining the bispecific antibody of the present invention with a pharmaceutically acceptable carrier. These formulations ensure the structural integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while simultaneously protecting the polyfunctional groups of the protein from degradation (including, but not limited to, aggregation, deamination, and oxidation). Typically, these substances may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous vector medium, in which case the pH may typically be about 5.0 to 8.0, preferably about 6.0 to 8.0, and the pH may vary depending on the properties of the substances being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes of administration, including (but not limited to) intravenous injection, intravenous drip infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal injection), intracranial injection, or intracavitary injection. Liquid formulations are usually stable for at least one year at 2 to 8°C, and lyophilized formulations are stable for at least six months at 30°C. The bispecific antibody formulations may be suspensions, injectables, lyophilized formulations, etc., which are widely used in the pharmaceutical industry.

[0096] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the bispecific antibody (or its complex) described in the present invention and a pharmaceutically acceptable vector or excipient. These vectors include, but are not limited to, saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. The formulation needs to be adapted to the dosage form. The pharmaceutical composition of the present invention can be prepared, for example, by conventional methods as an aqueous solution containing physiological saline or glucose and other excipients, and can be made into an injectable preparation. Pharmaceutical compositions such as injectable preparations and solutions should preferably be prepared under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 10 μg / kg body weight to about 5 mg / kg body weight per day. Furthermore, the bispecific antibody of the present invention can also be used in combination with other therapeutic agents.

[0097] When using a pharmaceutical composition, a safe and effective amount of bispecific antibody or its immune complex is administered to a mammal. Here, the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 5 mg / kg body weight, preferably about 10 μg / kg body weight to 10 mg / kg body weight. Of course, the specific dosage should take into account further factors such as the mode of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0098] use In another embodiment of the present invention, the use of a bispecific antibody or pharmaceutical composition that can specifically bind to FGFR2 and PD-1 is provided for the preparation of a drug used for the treatment of cancer or tumors.

[0099] In the present invention, a therapeutic agent for cancer or tumors refers to an agent that suppresses and / or treats a tumor, and may include delaying the onset of tumor-related symptoms and / or reducing the severity of these symptoms, as well as reducing existing tumor-related symptoms, preventing the onset of other symptoms, and reducing or preventing tumor metastasis.

[0100] Preferably, the agents described in the present invention target the following tumors, but are not limited to these. Lung cancer, bone cancer, stomach cancer, pancreatic adenocarcinoma, prostate cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, penile cancer, prostate cancer, pancreatic adenocarcinoma, brain tumor, testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, kidney cancer or ureteral cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, soft tissue sarcoma, pediatric solid tumors, lymphoid lymphoma, central nervous system tumors, primary central nervous system lymphoma, spinal cord tumors, brainstem glioma, pituitary adenoma, melanoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia or combination thereof. Primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, melanoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia, or a combination thereof.

[0101] When administering the bispecific antibody and its composition to animals, including humans, the dosage will vary depending on the patient's age, weight, the characteristics and severity of the disease, the route of administration, and other factors. The dosage should be determined based on animal experiment results and other various factors, but the total dosage should not exceed a certain range. Specifically, the dosage for intravenous injection is 1 to 1800 mg / day.

[0102] The bispecific antibodies and compositions of the present invention can also be administered in combination with other antitumor drugs to achieve more effective tumor treatment. These antitumor drugs include, but are not limited to, the following: 1. Cytotoxic agents: 1) Agents that act on the chemical structure of nucleic acids: alkylating agents such as mechloretamine, nitrosourea, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxaliplatin; and Adriamycin / Doxorubicin and Dactinomycin D. D) Antibiotics such as daunorubicin, epirubicin, and mithramycin; 2) Drugs that affect nucleic acid metabolism: dihydrofolate reductase inhibitors such as methotrexate and pemetrexed; thymidine synthase inhibitors such as fluorouracil (5-fluorouracil, capecitabine); purine nucleoside synthase inhibitors such as mercaptopurine; nucleotide reductase inhibitors such as hydroxycarbamide; DNA polymerase inhibitors such as cytosine albinoside and gemcitabine Drugs; 3) Drugs that act on tubulin: docetaxel, vincristine, vinorelbine, podophyllotoxin, homohalingotonin, etc.; 2. Hormonal agents: anti-estrogen agents such as tamoxifen, droloxifene, and exemestane; aromatase inhibitors such as aminoglutethimide, formestane, letrozle, and anastrozole; anti-androgen agents: flutamiflu RH-LH agonists / antagonists: goserelin acetate, leuprorelin acetate, etc.; 3.Biological reaction modifiers that exert antitumor effects by regulating the body's immune function: such as interferon, interleukin-2, and thymosin; 4. Monoclonal antibody drugs: such as trastuzumab, rituximab, cetuximab, and bevacizumab. The bispecific antibodies and compositions disclosed in this invention can be administered in combination with one or more of the aforementioned antitumor drugs.

[0103] The main advantages of this invention are as follows: The efficacy and inventiveness of the anti-FGFR2 / PD-1 bispecific antibody of the present invention are as follows. (1) It can bind to the T cell surface molecule PD-1, regulate the immune activity of T cells, specifically bind to tumor cells expressing FGFR2, and induce immune cells to target and kill FGFR2-positive tumor cells. In particular, it shows a significant inhibitory effect on the proliferation of FGFR2-positive tumor cells, and its ADCC activity is also relatively significant. (2) Has high specificity and good safety (3) Has a high expression level. (4) Having structural stability

[0104] The present invention will be described in more detail below, in accordance with specific embodiments. It should be understood that this is used solely to illustrate the present invention and not to limit its scope. In the following embodiments, experimental methods not specifically indicated follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or manufacturer-recommended conditions. Unless otherwise specified, percentages and quantities are on a weight basis.

[0105] The experimental materials used in the following examples, their suppliers, and the methods for preparing the experimental reagents are described in detail below.

[0106] Experimental materials: Transformation recipient cells: Shengong brand, product number B528412. 293F cells: GIBCO brand, product number R79007. hPBMC: Ausiells brand, product number FPB004F-C-MLR. 0.45 μm filter: Merck Millipore brand, product number SLHV013SL. SNU-16 cells: Nanjing Kebai brand, product number CBP60502. Jurkat-PD1-NFAT-luc cells: Promega brand, product number J1252. CHO-K1-PD-L1 cells: Promega brand, product number J1252. Ba / F3 cells: Saili brand, product number SLB-CL0019A. 293FT cells: GIBCO brand, product number R70007. Hitrap Mabselect Sure affinity chromatography column: Cytiva brand, product number 11003493. Waters MassPREP TM Micro Desalting Column: Waters brand, model number 186004032.

[0107] Experimental reagents: Endotoxin-free plasmid large-scale kit: Tiangen brand, product number DP117. DNA purification and recovery kit: Tiangen brand, product number DP214. ClonExpressTM II One Step Cloning Kit: Vazyme brand, product number C112. PrimeSTAR® HS DNA Polymerase: takara brand, product number R010B. Coating buffer: Dissolve 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate in secondary distilled water and dilute to 1 L. PBST: PBS + 0.05% Tween 20. Tween 20: Arading brand, product number T104863. ELISA blocking buffer: PBST + 1% BSA. BSA: Seiko brand, product number A60332. Human-FGFR2-his protein: Kactus brand, catalog number FGR-HM1BD. HRP-labeled goat anti-human Fc antibody: Biodragon brand, catalog number BF03031. TMB:BD Biosciences brand, part number 555214. Stop solution: 2M sulfuric acid solution. 0.25% Pancreatin-EDTA: GIBCO brand, catalog number 25200-072. 1640 Complete Medium: RPMI 1640 medium + 10% FBS + 1% Pen Strep + 1% sodium pyruvate. RPMI1640 culture medium: GIBCO brand, catalog number 22400089. FBS: GIBCO brand, item number 10099-141. Pen Strep: GIBCO brand, part number 1514022. Sodium pyruvate: GIBCO brand, part number 11360-070. DMEM complete medium: DMEM high glucose medium + 10% FBS + 1% Pen Strep + 1% GLUMAX. DMEM high glucose medium: GIBCO brand, catalog number 11965-092. GlutaMAX: GIBCO brand, part number 35050-061. CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay: Promega brand, part number G7572. Goat anti-human Fab-FITC: Thermo Fisher brand, part number MA1-10379. Glycosidase F: NEB brand, product number P0704L.

[0108] Experimental equipment: Mastercycler nexus PCR machine: Purchased from Eppendorf. Hitrap Mabselect Sure column: Purchased from Cytiva. HiLoad 26 / 600 Superdex 200 pg column: Purchased from Cytiva. SpectraMax i3x microplate reader: Purchased from Molecular Devices. SpectraMax M5 microplate reader: Purchased from Molecular Devices. Coulter CytoFLEX flow cytometer: Purchased from Beckman. FD-0007 Fully Automatic Capillary Differential Scanning Calorimeter: Purchased from Malvern. LC / MS liquid chromatograph mass spectrometer: Purchased from Waters.

[0109] The arrangement of the present invention is shown in Table B below. [Table B] TIFF2026521030000005.tif238169TIFF2026521030000006.tif226169TIFF2026521030000007.tif184170

[0110] Example 1: Construction of an anti-FGFR2 / PD-1 bispecific antibody molecule This invention constructs an anti-FGFR2 / PD-1 bispecific antibody by employing a tandem conjugation configuration of an anti-FGFR2 monoclonal antibody IgG and an anti-PD-1 monoclonal antibody scFv. Its structure is shown in Figure 1A.

[0111] Of these, the anti-FGFR2 monoclonal antibody sequence in the anti-FGFR2 / PD-1 bispecific antibody is derived from the humanized monoclonal antibody disclosed in patent CN102905723A and is used as a positive control for the anti-FGFR2 monoclonal antibody. The anti-PD-1 monoclonal antibody sequence in the anti-FGFR2 / PD-1 bispecific antibody is derived from the humanized monoclonal antibody disclosed in patent CN201710054783.5 and is used as a positive control for the anti-PD-1 monoclonal antibody.

[0112] The anti-FGFR2 / PD-1 bispecific antibody scFv-VLVH was converted to VL-L1-VH, or PD-1-scFv-VLVH (SEQ ID NO: 27), by linking the anti-PD-1 light chain variable region VL (SEQ ID NO: 16) and the anti-PD-1 heavy chain variable region VH (SEQ ID NO: 15) via the peptide linker L1 (SEQ ID NO: 17).

[0113] The C-terminus of the anti-FGFR2 monoclonal antibody heavy chain (SEQ ID NO: 25) and the N-terminus of the variable region fragment PD-1-scFv-VLVH (SEQ ID NO: 27) of the anti-PD-1 monoclonal antibody were linked via peptide linker L2 (SEQ ID NO: 18) to obtain the heavy chain of anti-FGFR2 / PD-1 bispecific antibody a (SEQ ID NO: 29), while the light chain of the anti-FGFR2 monoclonal antibody (SEQ ID NO: 28) was retained.

[0114] We selected a corresponding site in human IgG1 and introduced a mutation into the constant region of the heavy chain of an anti-FGFR2 monoclonal antibody. Through screening, we selected S239D and I332E, obtaining the anti-FGFR2 monoclonal antibody heavy chain HC-S239D-I332E.

[0115] The N-terminus of the anti-FGFR2 monoclonal antibody heavy chain HC-S239D-I332E (SEQ ID NO: 26) and the variable region fragment PD-1-scFv-VLVH (SEQ ID NO: 27) of the anti-PD-1 monoclonal antibody were linked via peptide linker L2 (SEQ ID NO: 18) to obtain the heavy chain of anti-FGFR2 / PD-1 bispecific antibody b (SEQ ID NO: 30), while the light chain of the anti-FGFR2 monoclonal antibody (SEQ ID NO: 28) was retained.

[0116] On the other hand, while constructing anti-FGFR2 / PD-1 bispecific antibody molecules, we designed various linking methods and configurations for linking anti-FGFR2 monoclonal antibody IgG or its scFv with anti-PD-1 monoclonal antibody IgG or its scFv, thereby forming different forms of anti-FGFR2 / PD-1 bispecific antibody molecules.

[0117] By linking the anti-FGFR2 heavy chain variable region VH (SEQ ID NO: 13) and the anti-FGFR2 light chain variable region VH (SEQ ID NO: 14) via peptide linker L1 (SEQ ID NO: 17), VH-L1-VL, i.e., FGFR2-scFv-VHVL, was obtained. By linking the C-terminus of the anti-PD-1 monoclonal antibody heavy chain to the N-terminus of the anti-FGFR2 monoclonal antibody variable region fragment FGFR2-scFv-VHVL via peptide linker L2 (SEQ ID NO: 18), the heavy chain of the anti-FGFR2 / PD-1 bispecific antibody c was obtained, while the light chain of the anti-PD-1 monoclonal antibody was retained. The structure is shown in Figure 1B.

[0118] The anti-PD-1 heavy chain variable region VH (SEQ ID NO: 15) and the anti-PD-1 light chain variable region VL (SEQ ID NO: 16) were linked via peptide linker L1 (SEQ ID NO: 17) to obtain VH-L1-VL, i.e., PD-1-scFv-VHVL. The C-terminus of the anti-FGFR2 monoclonal antibody heavy chain (SEQ ID NO: 25) and the N-terminus of the variable region fragment PD-1-scFv-VHVL of the anti-PD-1 monoclonal antibody were linked via peptide linker L2 (SEQ ID NO: 18) to obtain the heavy chain of the anti-FGFR2 / PD-1 bispecific antibody d, while the light chain of the anti-FGFR2 monoclonal antibody (SEQ ID NO: 28) was retained. The structure is shown in Figure 1C.

[0119] To improve the expression efficiency of antibody molecules in CHO cells, codon optimization was performed on the nucleic acid sequence of the anti-FGFR2-PD1 bispecific antibody molecule. Considering codon bias, GC content, mRNA secondary structure, and repeat sequences, the synthesis of the antibody nucleic acid sequence was commissioned to Bioengineering Co., Ltd. Please refer to the sequence listing for amino acid and nucleic acid sequences.

[0120] Example 2 Expression and purification of anti-FGFR2 / PD-1 bispecific antibody The heavy and light chain DNA fragments of anti-FGFR2 / PD-1 bispecific antibodies a and b were subcloned into vector pcDNA3.4, respectively. Recombinant plasmids were extracted and co-transfected into 293F cells and / or CHO cells. After culturing the cells for 5–7 days, the culture medium was rapidly centrifuged, filtered through a 0.22 μm filter, and injected into a Hitrap Mabselect Sure affinity chromatography column. Proteins were eluted in bulk with 100 mM citrate, pH 3.5. The target sample was collected and dialyzed and replaced with PBS at pH 7.4. The purified protein was measured by ULC-SEC.

[0121] The measurement results for anti-FGFR2 / PD-1 bispecific antibodies a, b, c, and d are shown in Figures 2A, 2B, 2C, and 2D. The antibody proteins were homogeneous, with monomer purity of anti-FGFR2 / PD-1 bispecific antibody a reaching over 93%, monomer purity of anti-FGFR2 / PD-1 bispecific antibody b being slightly higher than bispecific antibody a, reaching over 96%, and purity of anti-FGFR2 / PD-1 bispecific antibodies c and d being slightly lower than bispecific antibody a, at over 86% and 88%, respectively.

[0122] Example 3: De-N-glycosylation complete molecular weight measurement of anti-FGFR2 / PD-1 bispecific antibody One mg each of anti-FGFR2 / PD-1 bispecific antibodies a and b were collected, desalted in ultrapure water by ultrafiltration, and then concentrated to over 20 mg / ml. A 60 μl sample was taken, 59 μl of ultrapure water was added, and then 1 μl of glycosidase F was added. After incubation in a 37°C water bath for 2 hours, the samples were analyzed using Waters MassPREP. TM The deglycosylated total molecular weight of anti-FGFR2 / PD-1 bispecific antibody samples a and b was measured using LC / MS liquid chromatography-mass spectrometry with a Micro Desalting Column.

[0123] The experimental results are shown in Figures 3A and 3B. The theoretical deglycosylated molecular weight of anti-FGFR2 / PD-1 bispecific antibody a was 196892.05 Da, and the measured molecular weight after mass spectrometry was 196896.98 Da. The theoretical deglycosylated molecular weight of anti-FGFR2 / PD-1 bispecific antibody b was 196979.98 Da, and the measured molecular weight after mass spectrometry was 196986.28 Da. The theoretical molecular weights of anti-FGFR2 / PD-1 bispecific antibodies a and b are in close agreement with the measured molecular weights.

[0124] Example 4: Measurement of thermal stability of anti-FGFR2 / PD-1 bispecific antibody The thermal stability of anti-FGFR2 / PD-1 bispecific antibodies was evaluated by measuring the change in molar heat capacity with temperature using differential scanning calorimeter (DSC). Protein solutions of anti-FGFR2 / PD-1 bispecific antibodies a and b were diluted to 0.5 mg / mL using 1×PBS (pH 7.4) buffer, and 500 μL of these solutions were measured using a fully automated capillary differential scanning calorimeter to obtain the DSC spectra shown in Figures 4A and 4B.

[0125] Experimental results showed that as the temperature increased, Tm1 and Tm2 of FGFR2 / PD-1 bispecific antibody a showed endothermic peaks at 62.74°C and 79.08°C, respectively, while Tm1 and Tm2 of FGFR2 / PD-1 bispecific antibody b showed endothermic peaks at 60.56°C and 81.01°C, respectively. Both anti-FGFR2-PD-1 bispecific antibodies a and b showed their first endothermic peak at approximately 60°C and their second endothermic peak at approximately 80°C. This suggests that the thermal stability of anti-FGFR2 / PD-1 bispecific antibody proteins a and b is equivalent.

[0126] Example 5: Measurement of antigen affinity of bispecific antibodies by enzyme-linked immunosorbent assay (ELISA) 5.1 Affinity measurement of anti-FGFR2 / PD-1 bispecific antibodies against FGFR2 Recombinant Human-FGFR2-His protein was diluted to 200 ng / ml using coating buffer, added to a microplate at 100 μl / well, and left at room temperature for 2 hours or overnight at 4°C. The coating buffer (residual droplets removed with absorbent paper) was removed, and blocking buffer was added to the microplate at 200 μl / well and left at room temperature for 1-2 hours. The blocking buffer (residual droplets removed with absorbent paper) was removed, and anti-FGFR2 / PD-1 bispecific antibodies a and b, as well as anti-FGFR2 monoclonal antibody, were diluted to 10 μg / ml with blocking buffer. A 12-step concentration gradient (maximum concentration 10 μg / ml, minimum concentration 0.002 ng / ml) was created by 4-fold dilution and sequentially added to a blocked microplate at 100 μl / well, and left at room temperature for 1 hour. The plate was washed three times with PBST (removing residual droplets with absorbent paper), HRP-labeled goat anti-human Fc antibody was diluted 1:3000 with blocking buffer, added to the microplate at 100 μl / well, and left at room temperature for 30 minutes. The plate was washed three times with PBST (removing residual droplets with absorbent paper), TMB chromogenic reagent was added at 100 μl / well, left at room temperature in the dark for 5 minutes, and the substrate colorimetric reaction was stopped by adding 70 μl / well of stop solution. The OD value at 450 nm was read using a microplate reader, and data analysis and plotting were performed using GraphPad. 50 The result was calculated.

[0127] The experimental results are shown in Figure 5A. EC binding of anti-FGFR2 / PD-1 bispecific antibody molecules a and b, as well as the positive control anti-FGFR2 monoclonal antibody, to Human-FGFR2-His. 50 The values ​​(in nM) are 0.0568, 0.0512, and 0.0392, respectively, indicating that the affinity of the three is equivalent.

[0128] The affinity of anti-FGFR2 / PD-1 bispecific antibody molecules c and d, as well as the positive control anti-FGFR2 monoclonal antibody, for Human-FGFR2-His was measured using the same method. The experimental results are shown in Figure 5B. EC 50The values ​​(in nM) were 0.6244, 0.0615, and 0.0297, respectively. The affinity of anti-FGFR2 / PD-1 bispecific antibody d was similar to that of the positive control anti-FGFR2 monoclonal antibody. However, the affinity of anti-FGFR2 / PD-1 bispecific antibody c was significantly lower than that of the positive control anti-FGFR2 monoclonal antibody.

[0129] The structures of anti-FGFR2 / PD-1 bispecific antibodies a, b, and d are similar, all employing a tandem linkage of anti-FGFR2 monoclonal antibody IgG and anti-PD-1 monoclonal antibody scFv. On the other hand, anti-FGFR2 / PD-1 bispecific antibody c employs a tandem linkage of anti-PD-1 monoclonal antibody IgG and anti-FGFR2 monoclonal antibody scFv. Of these, bispecific antibodies a and b showed higher affinity than bispecific antibody c. This indicates that the bispecific antibody molecule formed by the tandem linkage of anti-FGFR2 monoclonal antibody IgG and anti-PD-1 monoclonal antibody scFv has a high binding affinity to the FGFR2 antigen.

[0130] 5.2 Affinity measurement of anti-FGFR2 / PD-1 bispecific antibodies against PD-1 Recombinant Human-PD-1-His protein was diluted to 200 ng / ml using coating buffer, added to a microplate at 100 μl / well, and left at room temperature for 2 hours or overnight at 4°C. The coating buffer (residual droplets removed with absorbent paper) was removed, and blocking buffer was added to the microplate at 200 μl / well, and left at room temperature for 1-2 hours. The blocking buffer (residual droplets removed with absorbent paper) was removed, and anti-FGFR2 / PD-1 bispecific antibodies a and b, as well as anti-PD-1 monoclonal antibody, were diluted to 10 μg / ml with blocking buffer. A 12-step concentration gradient (maximum concentration 10 μg / ml, minimum concentration 0.002 ng / ml) was created by 4-fold dilution and sequentially added to a blocked microplate at 100 μl / well, and left at room temperature for 1 hour. The plate was washed three times with PBST (removing residual droplets with absorbent paper), HRP-labeled goat anti-human FC antibody was diluted 1:3000 with blocking buffer, added to the microplate at 100 μl / well, and left at room temperature for 30 minutes. The plate was washed three times with PBST (removing residual droplets with absorbent paper), TMB was added at 100 μl / well, left at room temperature in the dark for 5 minutes, and stopped by adding 70 μl / well of stop solution to stop the substrate color reaction. The OD value at 450 nm was read using a microplate reader, and data analysis and plotting were performed using GraphPad. 50 The result was calculated.

[0131] The experimental results are shown in Figure 5C. EC molecules a and b of the anti-FGFR2 / PD-1 bispecific antibody, as well as the positive control anti-PD-1 monoclonal antibody, bind to Human-PD-1-His. 50 The values ​​(in nM) were 0.1216, 0.1411, and 0.0586, respectively. The lower affinity of bispecific antibodies a and b compared to the monoclonal antibody indicates that the bispecific antibody, formed by conjugating the variable region of the anti-PD-1 monoclonal antibody to the VL-VH region and then to the heavy chain of the anti-FGFR2 monoclonal antibody, exhibits slightly reduced binding ability to the PD-1 antigen.

[0132] The affinity of anti-FGFR2 / PD-1 bispecific antibody molecules a and d, as well as the positive control anti-PD-1 monoclonal antibody, for Human-FGFR2-His was measured using the same method. The experimental results are shown in Figure 5D. EC 50 The values ​​(in nM) were 0.1599, 0.3564, and 0.0856, respectively, indicating that the Human-PD-1-His affinity of anti-FGFR2 / PD-1 bispecific antibody a was slightly higher than that of FGFR2 / PD-1 bispecific antibody d. This suggests that bispecific antibody molecules formed by tandem binding of the variable region of a PD-1 monoclonal antibody (VL-VH conjugation) and the scFv of an anti-FGFR2 monoclonal antibody have a high binding affinity to the PD-1 antigen.

[0133] Example 6: PD-1 / PD-L1 binding inhibitory activity of anti-FGFR2 / PD-1 bispecific antibody Jurkat-PD-1-NFAT-luc cells, which express PD-1 on their cell surface, were used as target cells, and the binding affinity of an anti-FGFR2 / PD-1 bispecific antibody to these cells was measured using a flow cytometer. Cell density was measured using DPBS containing 1% BSA at 1.5 × 10⁶ 6The cells were adjusted to the required concentration (cells / mL) and added to a 96-well plate at 100 μL / well. Using DPBS containing 1% BSA, the anti-FGFR2 / PD-1 bispecific antibodies a and b, as well as the positive control anti-PD-1 monoclonal antibody, were diluted to 200 nM, then diluted fourfold to create a 12-step concentration gradient. These were added to a 96-well plate at 100 μL / well and thoroughly mixed with Jurkat-PD-1-NFAT-luc cells. The cells were incubated at 4°C for 1 hour. The cells were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. To remove unbound target antibodies, the cells were washed twice with pre-cooled DPBS. Goat anti-human Fab-FITC (800:1 dilution) was added at 100 μL / well, and the cells were incubated at 4°C for 1 hour. The cells were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. After washing twice with pre-cooled DPBS, the cells were resuspended in 200 μL of DPBS, and the binding affinity of the FGFR2 / PD-1 bispecific antibody to the cells was measured using a Beckman Coulter CytoFLEX flow cytometer. The obtained data were plotted and analyzed using GraphPad Prism6.

[0134] The experimental results are shown in Figure 6A. Both anti-FGFR2 / PD-1 bispecific antibodies a and b can specifically bind to PD-1 expressed on the cell surface. Anti-FGFR2 / PD-1 bispecific antibodies a and b, as well as the positive control anti-PD-1 monoclonal antibody, bind to Jurkat-PD1-NFAT-luc cells. 50 The nM values ​​were 0.4823, 0.6344, and 0.1046 nM, respectively. Compared to anti-PD-1 monoclonal antibodies, anti-FGFR2 / PD-1 bispecific antibodies a and b exhibited weaker activity in binding to the cell surface PD-1 protein.

[0135] Using complete culture medium, CHO-K1-PD-L1 cells were divided into 4 × 10⁶ cells. 5The solutions were diluted to cells / mL and added to a 96-well plate of type 3903 at 100 μL / well. After incubation overnight in a 37°C, 5% CO2 incubator, the medium was removed. Using analytical medium (RPMI 1640 + 1% FBS), the anti-FGFR2 / PD-1 bispecific antibody molecules a and b, as well as the anti-PD-1 monoclonal antibody, were diluted to 500 nM. A 10-step concentration gradient was obtained by 5-fold dilution (maximum concentration 500 nM, minimum concentration 2.56 × 10⁶). -4 Diluted to nM, 40 μL / well was added to a 96-well plate containing CHO-K1-PD-L1 cells. Jurkat-PD-1-NFAT-luc cells were analyzed using analytical medium (RPMI 1640 + 1% FBS) at 4 × 10⁶ levels. 5 The reagent was diluted to cells / mL and added to a 96-well plate containing CHO-K1-PD-L1 cells at 40 μL / well. After incubation in a 37°C, 5% CO2 incubator for 6 hours, the 96-well plate was removed, allowed to return to room temperature, and 80 μL / well of Bio-Glo assay reagent was added. The plate was incubated at room temperature for 5-10 minutes, and luminescence values ​​were read using a spectramax i3. All data were obtained from double wells. The obtained signal values ​​were averaged, fitted using a four-parameter method, and the curves were plotted.

[0136] The experimental results are shown in Figure 6B. IC of PD-1 and PD-L1 binding inhibition by anti-FGFR2 / PD-1 bispecific antibody molecules a and b. 50 These values ​​were 0.481 and 2.352 nM, respectively, indicating IC50 for PD1 and PD-L1 binding inhibition by anti-PD-1 monoclonal antibodies. 50 The concentration was 0.372 nM. This indicates that anti-FGFR2 / PD-1 bispecific antibody a and anti-PD-1 monoclonal antibody have equivalent ability to inhibit PD-1 and PD-L1 binding, while anti-FGFR2 / PD-1 bispecific antibody b has lower ability to inhibit PD-1 and PD-L1 binding than anti-PD-1 monoclonal antibody.

[0137] Example 7 ADCC activity of anti-FGFR2 / PD-1 bispecific antibody against SNU-16 cells and T cells In this experiment, we used human gastric cancer cells SNU-16, which highly express FGFR2 on the cell surface, and Jurkat-PD-1-NFAT-luc, which expresses PD-1, as target cells to measure the killing ability of NK cells against tumor cells and T cells.

[0138] 7.1 DCC activity of bispecific antibodies against SNU-16 tumor cells SNU-16 cells were isolated and incubated in RPMI 1640 medium containing 5% FBS for 2 × 10⁶ cells. 5 The solution was adjusted to cells / mL and added to the inner 60 wells of a 96-well plate at 50 μL / well, with the edge wells sealed with sterile water. Using RPMI 1640 medium containing 5% FBS, anti-FGFR2 / PD-1 bispecific antibodies a and b, as well as the positive control anti-FGFR2 monoclonal antibody, were diluted to 200 nM, 4-fold, and then added to the cell-containing wells at 50 μL / well. Incubation was performed in a 37°C, 5% CO2 incubator for 30 minutes. During this time, fresh human peripheral blood mononuclear cells (hPBMCs) were collected, and NK cells were isolated according to the instructions for the human NK Cell Isolation Kit (Miltenyi Biotec, #130-092-657). Using RPMI 1640 medium containing 5% FBS, the isolated NK cells were isolated to 4 × 10⁶ cells. 5 The solution was diluted to cells / mL, added to the wells at a rate of 100 μL / well, and incubated in a 37°C, 5% CO2 incubator for 24 hours. CyQUANT TMLDH was measured according to the instructions for the LDH Cytotoxicity Assay Kit (invitrogen, # C20300). The specific procedure is as follows: 20 μL of lysis buffer was added to the well used to detect the maximum LDH release, mixed by light tapping, and incubated for 45 minutes in a 37°C, 5% CO2 incubator. Then, 50 μL of sample was dispensed into each well of a new 96-well plate, and 50 μL / well of LDH measurement standard solution was added. The wells were mixed and incubated for 30 minutes at room temperature in the dark. 50 μL of stop solution was added, mixed by light tapping, and the OD values ​​at 490 nm and 680 nm were measured using a microplate reader. Absorbance = OD for each well 490 -OD 680 Cytotoxicity or mortality (%) = (Absorbance of experimental group - Absorbance of blank well) / (Total LDH absorbance - Absorbance of blank well) × 100.

[0139] The experimental results are shown in Figure 7A. Compared to the positive control anti-FGFR2 monoclonal antibody, K cells showed a weak killing effect against SNU-16 tumor cells under the action of anti-FGFR2 / PD-1 bispecific antibody a. Under the action of anti-FGFR2 / PD-1 bispecific antibody b, NK cells showed a significant killing effect against SNU-16 tumor cells. EC of anti-FGFR2 / PD-1 bispecific antibody b and the positive control anti-FGFR2 monoclonal antibody 50 The values ​​were 4.615 and 3.031 nM, respectively, indicating equivalent ADCC activity levels.

[0140] Therefore, introducing the point mutation S239D-I332E into the Fc region of the anti-FGFR2 / PD-1 bispecific antibody significantly improved the ADCC effect of anti-FGFR2 / PD-1 bispecific antibody b (Figure 7A).

[0141] 7.2 ADCC activity of bispecific antibodies against T cells ADCC activity was measured using Jurkat-PD-1-NFAT-luc cells, which express PD-1 on their cell surface, as target cells. The method was as described above. The difference was that the antibodies used were anti-FGFR2 / PD-1 bispecific antibodies a and b, an anti-MHC-I monoclonal antibody as a positive control, and an anti-PD-1 monoclonal antibody as a negative control. The initial antibody concentration was 50 nM, and after 4-fold serial dilution, it was added to wells containing cells. CyQUAN TM LDH levels were measured according to the instructions for the LDH Cytotoxicity Assay Kit (invitrogen, # C20300). Data analysis was performed using GraphPad Prism6.

[0142] The results are shown in Figure 7B. Under the action of the positive control anti-MHC-I monoclonal antibody, NK cells showed significant killing activity, but under the action of anti-FGFR2 / PD-1 bispecific antibodies a and b, as well as the negative control anti-PD-1 monoclonal antibody, they did not show significant killing activity against T cells.

[0143] Example 8: Inhibitory effect of anti-FGFR2 / PD-1 bispecific antibody on FGF1-induced Ba / F3-FGFR2IIIb cell proliferation After digesting 293FT cells with 0.25% pancreatin-EDTA, the 293FT cells were diluted with DMEM complete medium and added to a 6-well plate at a concentration of 700,000 cells / well. After the cells adhered to the cell wall, the packaged viral vector pLVX (with human FGFR2IIIb gene insertion) was reacted with the 293FT cells using Lipofectamine 3000 transfection reagent. After 48 hours, the 293FT cell medium was collected, centrifuged at 500g for 5 minutes, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter, and polybren co-infection agent was added at a ratio of 1000:1. 2 ml of the supernatant was added to a T25 petri dish (250,000 Ba / F3 cells had been added 24 hours prior). After 48 hours, the Ba / F3 cells were collected, cultured in complete medium supplemented with antibiotics, and passaged until cell proliferation stabilized to obtain a Ba / F3-FGFR2IIIb cell population. The Ba / F3-FGFR2 cell population was diluted to 2-3 cells / ml using culture medium, dispensed into 96-well plates, and single-clone sorting was performed to obtain the Ba / F3-FGFR2IIIb cell line. The FGFR2IIIb gene expression efficiency in the Ba / F3-FGFR2IIIb cell line was measured by flow cytometry. The highest FGFR2IIIb gene expression efficiency in the Ba / F3-FGFR2IIIb cell line was 9.26% (results not shown). Ba / F3-FGFR2IIIb cells cultured to the logarithmic growth phase were harvested by centrifugation. 1 × 10⁶ cells were collected in complete medium. 6The solutions were diluted to cells / ml and added to a 3903 clear-bottom 96-well plate at 100 μl / well, and incubated in a 37°C, 5% CO2 incubator. Using complete medium, anti-FGFR2 / PD-1 bispecific antibodies a and b, and anti-FGFR2 monoclonal antibody were diluted to 500 nM, serially diluted 5-fold, and added to a 96-well plate containing Ba / F3-FGFR2IIIb cells, and incubated in a 37°C, 5% CO2 incubator for 1 hour. Using complete medium, FGF1 was diluted to a concentration of 10 ng / ml and heparin was diluted to 5 μg / ml, and added to a 96-well plate containing Ba / F3-FGFR2IIIb cells and antibodies. After incubation for 2 hours in a 37°C, 5% CO2 incubator, 20 μl / well of MTS / PMS solution was added, and the mixture was incubated for 1-4 hours in a 37°C, 5% CO2 incubator. The mixture was then shaken to ensure uniform mixing, and the OD value at 490 nm was measured using a microplate reader. All data were obtained from double wells. The obtained signal values ​​were converted to inhibition rates, averaged, fitted using the four-parameter method, and the curves were plotted.

[0144] The experimental results are shown in Figure 8. EC of anti-FGFR2 / PD-1 bispecific antibodies a and b, and the positive control anti-FGFR2 monoclonal antibody. 50 The values ​​were 0.0870, 0.0515, and 0.1030 nM, respectively. This indicates that the anti-FGFR2 / PD-1 bispecific antibody b has a stronger ability to compete with FGF1 for the FGFR2IIIb receptor and can more effectively inhibit cell proliferation induced by the binding of FGF1 to FGFR2IIIb.

[0145] Example 9: Antitumor effect of anti-FGFR2 / PD-1 bispecific antibody in MC38 transplanted tumor model Mouse colon cancer MC38 cells cultured in vitro were harvested, and the cell suspension concentration was set to 2 × 10⁻⁶. 7The cell suspension was adjusted to cells / mL. The hair on the right rib cage of NOG mice was shaved, and 100 μl of the cell suspension was subcutaneously inoculated into the right rib cage of each NOG mouse under sterile conditions. After the tumor cells formed a solid tumor subcutaneously, the diameter of the transplanted tumor was measured using calipers. The average tumor volume was 50 mm². 3 ~100 mm 3 After maturation, the mice were randomly divided into three groups of nine: a blank control group receiving only PBS, a control dose group receiving 10 mg / kg of anti-PD-1 monoclonal antibody, and a dose group receiving 13.5 mg / kg of anti-FGFR2 / PD-1 bispecific antibody b. Intraperitoneal administration was performed twice a week for a total of five times. During the experiment, the diameter of the transplanted tumors was measured twice a week, and the body weight of the mice was measured simultaneously. The changes in tumor growth curves and mouse body weight curves over time were plotted for each group. The experimental results are shown in Figures 9A and 9B. In the MC38 cell transplanted tumor model, the inhibitory effect of anti-FGFR2 / PD-1 bispecific antibody b and anti-PD-1 monoclonal antibody on tumors was significantly higher compared to the control group. No significant difference was observed in the inhibitory effect of anti-FGFR2 / PD-1 bispecific antibody b and anti-PD-1 monoclonal antibody on tumors. Since the body weight of mice in each dose group did not decrease significantly even after several days of continuous administration, it was suggested that anti-FGFR2 / PD-1 bispecific antibody b and anti-PD-1 monoclonal antibody have relatively low toxic side effects in mice.

[0146] The results showed that in a transplanted tumor model of non-target tumor cells, the antitumor activity of anti-FGFR2 / PD-1 bispecific antibody b was not significantly different from that of anti-PD-1 monoclonal antibody. Both antibodies significantly suppressed tumor growth, indicating that the structure of the anti-FGFR2 / PD-1 bispecific antibody does not affect the efficacy of anti-PD-1 monoclonal antibody.

[0147] Example 10: Antitumor effect of anti-FGFR2 / PD-1 bispecific antibody in SNU-16 transplanted tumor model Human gastric cancer SNU-16 cells cultured in vitro were harvested, resuspended in serum-free medium, and then mixed with an equal volume of matrix gel to achieve a cell concentration of 8 × 10⁶. 7The concentration was adjusted to / ml. Under sterile conditions, 100 μl of the cell suspension was subcutaneously injected into the right rib region of NOG mice. Eight days after tumor inoculation, hPBMC cells were resuscitated, and the cell concentration was increased to 2.5 × 10⁶. 7 The concentration was adjusted to / ml. Except for three tumor-bearing mice that were not inoculated with hPBMC cells, the remaining tumor-bearing mice were each injected with 200 μl of hPBMC cell suspension via the tail vein. That is, 5 × 10 per mouse. 6 Individual hPBMC cells were injected. After the tumor cells formed a solid tumor subcutaneously, the diameter of the transplanted tumor was measured using calipers. The average tumor volume was approximately 200 mm³. 3 After growing to maturity, the mice were randomly divided into seven groups. Specifically, these included a blank control group administered only PBS, an hPBMC control group in which hPBMC cells were diluted with PBS, two monoclonal antibody control groups administered 5 mg / kg of anti-FGFR2 monoclonal antibody and 5 mg / kg of anti-PD-1 monoclonal antibody, respectively, a combination therapy control group administered 5 mg / kg of anti-FGFR2 monoclonal antibody and 5 mg / kg of anti-PD-1 monoclonal antibody in combination, and two dose groups administered 6.75 mg / kg of anti-FGFR2 / PD-1 bispecific antibody a and 6.75 mg / kg of anti-FGFR2 / PD-1 bispecific antibody b, respectively. The antibodies were administered twice a week for a total of five doses. During the experiment, the diameter of the transplanted tumors was measured twice a week, and the body weight of the mice was measured simultaneously. The changes in tumor growth curves and mouse body weight curves over time were plotted for each group.

[0148] The experimental results are shown in Figures 10A and 10B and Table 1. In a SNU-16 cell mixed hPBMC transplanted tumor model, there was no significant difference in the inhibitory effects of anti-FGFR2 / PD-1 bispecific antibodies a and b, and they were equivalent to the inhibitory effect of anti-FGFR2 monoclonal antibody. Compared to the combined administration of anti-FGFR2 monoclonal antibody and anti-PD-1 monoclonal antibody, the inhibitory effect was relatively superior. The inhibitory effect of anti-FGFR2 / PD-1 bispecific antibodies a and b was significantly superior to that of anti-PD-1 monoclonal antibody. Since the body weight of the mice did not significantly decrease even after several days of continuous administration, it was suggested that anti-FGFR2 / PD-1 bispecific antibodies a and b have fewer toxic side effects in mice.

[0149] The results showed that in a SNU-16 cell-grafted tumor model highly expressing FGFR2, the inhibitory activity of anti-FGFR2 / PD-1 bispecific antibodies a and b was superior to that of anti-PD-1 monoclonal antibodies, and both significantly inhibited tumor growth. This suggests that anti-FGFR2 / PD-1 bispecific antibodies specifically target FGFR2 tumor cells and exert superior antitumor effects. [Table 1]

[0150] Example 11: Stability test of anti-FGFR2 / PD-1 bispecific antibody b This test can be used to evaluate the stability of proteins with added additives, thereby revealing important information necessary for optimal formulation development.

[0151] Anti-FGFR2 / PD-1 bispecific antibody b was dialyzed overnight with Buffer 1 (20 mM acetate + 6% trehalose + 1% arginine hydrochloride + 0.2% Tween 80, pH 5.0) and Buffer 2 (20 mM histidine salt + 6% trehalose + 50 mM sodium chloride + 0.2% Tween 80, pH 5.5), and then concentrated to 5 mg / mL by ultrafiltration at 4000 rpm for 20 minutes. After aliquoting the samples, they were stored at 4°C, 25°C, and 37°C, respectively.

[0152] The experimental results are shown in Table 2. Under the same treatment conditions with different buffers, when anti-FGFR2 / PD-1 bispecific antibody b was added to buffer 1, significant aggregates formed on day 21, but no aggregates formed at all in buffer 2. In buffer 2, only a small amount of aggregates were detected on day 28, suggesting that anti-FGFR2 / PD-1 bispecific antibody b is more stable in a buffer containing acetate. [Table 2]

[0153] Consideration The above experiments demonstrate that the bispecific antibodies provided by the present invention simultaneously bind to FGFR2 and PD-1, activating the immune activity of T cells through binding to PD-1, and specifically killing tumor cells expressing the FGFR2 protein. The anti-FGFR2 / PD-1 bispecific antibodies can effectively inhibit the binding of PD-1 and PD-L1. Of these, bispecific antibody b showed a significant inhibitory effect on the proliferation of target cells SUN-16, and its cell proliferation inhibitory effect when competing with FGF1 for the FGFR2 receptor was relatively good. When the point mutation S239D-I332E was introduced into the Fc region of anti-FGFR2 / PD-1 bispecific antibody b, the ADCC activity of anti-FGFR2 / PD-1 bispecific antibody b was significantly enhanced (Figure 7A), but it did not show a significant killing effect on T cells. In transplanted tumor models, the anti-FGFR2 / PD-1 bispecific antibody showed excellent antitumor activity in both MC38 cells, which are non-target proteins, and SNU-16 cells, which highly express FGFR2.

[0154] During research on anti-FGFR2 / PD-1 bispecific antibodies, we also designed anti-FGFR3 / PD-1 bispecific antibodies. However, pharmacodynamic studies using multiple animal models revealed that the antitumor activity of the anti-FGFR3 / PD-1 bispecific antibody was significantly weaker than that of both the anti-FGFR2 / PD-1 bispecific antibody and the anti-PD-1 monoclonal antibody. This indicates that the combination of anti-FGFR3 monoclonal antibody and anti-PD-1 monoclonal antibody affects the activity of the anti-PD-1 monoclonal antibody. This further demonstrates the superiority of the combination of two targets in the anti-FGFR2 / PD-1 bispecific antibody of the present invention. While it does not affect the activation of T cell immune activity, it also exhibits excellent antibody activity against target cells, specifically binding to and killing tumor cells that express FGFR2.

[0155] All documents relating to the present invention are cited herein by reference, so that each document may be cited independently. Furthermore, after reading the foregoing, those skilled in the art may make various variations and modifications to the present invention, and it should be understood that equivalent forms of these variations are included within the scope of the claims of the present invention.

Claims

1. The first antigen-binding domain D1, The second antigen-binding domain D2, This includes, where D1 specifically binds to the target molecule FGFR2 protein. The aforementioned D2 specifically binds to the target molecule PD-1 protein. Here, D1 is an anti-FGFR2 antibody or its antigen-binding fragment, the anti-FGFR2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the following three heavy chain complementarity determining regions: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO:

3. A bispecific antibody characterized in that the light chain variable region includes the following three light chain complementarity determining regions: LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO:

6.

2. The D2 is an anti-PD-1 antibody or its antigen-binding fragment, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the following three heavy chain complementarity determining regions: HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9, and The bispecific antibody according to claim 1, characterized in that the light chain variable region includes the following three light chain complementarity determining regions: LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO:

12.

3. The anti-FGFR2 or anti-PD-1 antigen binding fragment is Fab, F(ab'), F(ab') 2 The bispecific antibody according to claim 1, characterized in that the anti-FGFR2 or anti-PD-1 antibody is an IgG antibody, selected from Fv or single-chain Fv (scFv).

4. The bispecific antibody according to claim 1, characterized in that D1 is an anti-FGFR2 antibody, and D2 is bound via a linker to a region of D1 selected from the group consisting of a heavy chain variable region, a heavy chain constant region, a light chain variable region, or a combination thereof.

5. The aforementioned bispecific antibody comprises a monomer and has a structure represented by formula I or formula II from the N-terminus to the C-terminus. 【Chemistry 2】 Here, VL A This indicates the light chain variable region of an anti-FGFR2 antibody or its antigen-binding fragment. VH A This indicates the heavy chain variable region of the anti-FGFR2 antibody or its antigen-binding fragment. VL B This indicates the light chain variable region of an anti-PD-1 antibody or its antigen-binding fragment. VH B This indicates the heavy chain variable region of the anti-PD-1 antibody or its antigen-binding fragment. CH exhibits a heavy chain steady region. CL represents the light chain constant region. L1 and L2 are, independently, couplings or linkers. "~" indicates a disulfide bond or covalent bond. The "-" indicates a peptide bond. The bispecific antibody according to claim 1, characterized in that the bispecific antibody has the activity to simultaneously bind to FGFR2 and PD-1.

6. The aforementioned anti-FGFR antibody, a) The heavy chain constant region has a mutation at amino acid position 239, preferably S293D, and / or b) The heavy chain constant region has a mutation at the 332nd amino acid, preferably I332E. The bispecific antibody according to claim 1, characterized in that it has a heavy chain constant region containing an amino acid mutation selected from the group consisting of the following.

7. The anti-FGFR2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14, and / or the anti-PD1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16, characterized in that the bispecific antibody according to claim 1.

8. The bispecific antibody according to claim 1, characterized in that the heavy chain constant region sequence of the anti-FGFR antibody is shown in SEQ ID NO: 23, or the heavy chain constant region sequence of the anti-FGFR antibody is shown in SEQ ID NO:

24.

9. The bispecific antibody comprises a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are as follows: a) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 29, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 28, b) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 30, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 28, or c) A polypeptide having both anti-FGFR activity and anti-PD1 activity, wherein the amino acid sequence of a) or b) is formed by the substitution, deletion, or addition of one or more amino acid residues, and is derived from a), b), or c). A bispecific antibody according to claim 1, characterized by being selected from the group consisting of the following.

10. A polynucleotide molecule characterized by encoding a bispecific antibody according to any one of claims 1 to 9.

11. An expression vector characterized by comprising the polynucleotide molecule described in claim 10.

12. A host cell comprising the expression vector described in claim 11.

13. a) A step of culturing the host cells described in claim 12 under expression conditions to express a bispecific antibody, b) A step of separating and purifying the bispecific antibody described in step a), A method for preparing a bispecific antibody according to any one of claims 1 to 9, characterized by including the following:

14. A pharmaceutical composition comprising an effective amount of a bispecific antibody according to any one of claims 1 to 9, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

15. The use of a bispecific antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 14 in the preparation of a therapeutic agent for cancer or tumor, wherein the cancer or tumor is FGFR2-positive cancer or tumor.

16. a) A bispecific antibody according to any one of claims 1 to 9, b) A complex portion selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides or enzymes, An immune complex characterized by containing [something].

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