Anti-FGFR2 / PD-1 bispecific antibody

Abstract

The present 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, wherein D1 is an anti-FGFR2 antibody or an antigen-binding fragment thereof, and D2 is an anti-PD-1 antibody or an antigen-binding fragment thereof. The anti-FGFR2 / PD-1 bispecific antibody of the present invention can inhibit the proliferation of FGFR2 target cells and kill the target cells by means of an ADCC effect, and can also block the binding of PD-1 to a ligand thereof, remove a T-cell suppressive state and exert an anti-tumor effect.

Description

An anti-FGFR2 / PD-1 bispecific antibody Technical Field

[0001] The present invention relates to the field of antibody drug technology, and specifically to an anti-FGFR2 / PD-1 bispecific antibody. Background Art

[0002] Fibroblast growth factor receptors (FGFRs) are highly conserved receptor tyrosine kinases. Four typical structures, FGFR1-4, and FGFR5, which lacks the intracellular tyrosine kinase domain, are commonly found. The FGFR extracellular domain contains three immunoglobulin-like domains. Carboxyl-selective isoformation of the third Ig domain can generate isoforms IIIb or IIIc, from FGFR1 to FGFR3. FGFR2-IIIb, expressed in epithelial cells, can be activated by FGFs and form a ternary complex with heparan sulfate proteoglycans, mediating signaling pathways such as RAS-RAF-MAPK, PI3K-AKT, signal transducers and activators of transcription, and phospholipase Cγ. These complexes participate in physiological processes such as angiogenesis, cell proliferation and migration, regulation of organ development, and wound healing. Abnormal FGFR2 signaling, including overexpression of FGFR2 and its ligands, mutations in the FGFR2 receptor, and conversion of its subtypes, can induce cancerous transformation in normal cells. This is exemplified by 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 target, such as Johnson & Johnson's erdafitinib, Incyte's pemigatinib, and BridgeBio Pharma's infigratinib, are small molecule inhibitors. There are fewer large molecule antibody drugs under development. For example, bemarituzumab, a monoclonal antibody targeting FGFR2 for the treatment of HER2-negative locally advanced or metastatic gastric cancer and gastroesophageal junction cancer, is currently in clinical development.

[0003] Programmed cell death protein 1 (PD-1), also known as CD279, belongs to the immunoglobulin superfamily. It is a type I membrane protein composed of 288 amino acids and is a member of the expanded CD28 / CTLA-4 family of T cell regulators. Its structure comprises an extracellular IgV domain, a transmembrane region, and an intracellular tail. The intracellular tail contains two phosphorylation sites, representing immunoreceptor tyrosine-based inhibitory and activation motifs, which bind to SHP-1 and SHP-2 phosphatases. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages. The PD-1 protein expressed on the surface of activated immune cells interacts with its ligands, PD-L1 and PD-L2, upregulating the E3 ubiquitin ligases CBL-b and c-CBL, triggering downregulation of immune cell receptors, inhibiting activation and cytokine release, or initiating programmed cell death. High expression of PD-L1 and PD-L2 has been found in a variety of solid tumors and hematologic malignancies, suggesting that tumor cells exploit the immunosuppressive response mediated by PD-1 and its ligands to achieve immune evasion. In 2018, nivolumab, the world's first PD-1 inhibitor, was approved for marketing in China as a second-line treatment for advanced non-small cell lung cancer with wild-type EGFR / ALK driver genes. Subsequently, sintilimab, the first domestically produced PD-1 antibody for the treatment of Hodgkin's lymphoma, was also approved for marketing. With further in-depth clinical research on PD-1, PD-1 drugs have achieved significant breakthroughs in oncology treatment, becoming the most popular tumor immunotherapy approach in recent years. Approved indications globally 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 target immune cells and viral molecules to tumor cells, enhancing their killing effect. They can also simultaneously bind to different antigens on tumor cells, enhancing their binding specificity and reducing off-target effects. Bispecific antibodies have broadened the application of antibody drugs and provided new research ideas for tumor immunity.

[0005] However, there is currently a lack of satisfactory bispecific antibodies targeting FGFR2 and PD-1 in the art. Therefore, there is an urgent need to develop a new bispecific antibody against FGFR2 and PD-1 in the art.

[0006] Summary of the Invention

[0007] The object of the present invention is to provide an anti-FGFR2 / PD-1 bispecific antibody.

[0008] The bispecific antibodies provided by the present invention can simultaneously specifically bind to FGFR2 and PD-1, inhibiting the proliferation of FGFR2 target cells and killing target cells through the ADCC effect, while protecting T cells from normal immune function and eliminating target cells. The present invention also aims to provide polynucleotide molecules encoding the bispecific antibodies; expression vectors containing the molecules; host cells containing the expression vectors; methods for preparing the bispecific antibodies; pharmaceutical compositions containing the bispecific antibodies; immunoconjugates containing the bispecific antibodies; uses of the bispecific antibodies or pharmaceutical compositions in the preparation of drugs for treating cancer or tumors; and methods for treating cancer or tumors with the fusion proteins, pharmaceutical compositions, or immunoconjugates.

[0009] In a first aspect of the present invention, a bispecific antibody is provided, comprising:

[0010] a first antigen binding domain D1; and

[0011] The second antigen-binding domain D2,

[0012] Wherein, the D1 specifically binds to the target molecule FGFR2 protein,

[0013] The D2 specifically binds to the target molecule PD-1 protein.

[0014] In another preferred embodiment, the D1 is an anti-FGFR2 antibody or an antigen-binding fragment thereof,

[0015] In another preferred embodiment, the D2 is an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0016] In another preferred embodiment, the antibodies include: animal-derived antibodies (such as mouse-derived antibodies), chimeric antibodies, and humanized antibodies.

[0017] In another preferred embodiment, the antibody fragment comprises a heavy chain variable region and a light chain variable region.

[0018] In another preferred embodiment, the antibody fragment comprises a single-chain variable region fragment (scFv) or a double-chain variable region fragment (dcFv).

[0019] In another preferred embodiment, the D1 and the D2 are connected via a linker.

[0020] In another preferred embodiment, D1 is an antibody, and the linker is (G4S)n, preferably, (G4S)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5 or 6), preferably, n=3 or 4; more preferably, the linker is as shown in SEQ ID NO.17 or SEQ ID NO.18.

[0021] In another preferred embodiment, D1 is an anti-FGFR2 antibody, and D2 is connected 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.

[0022] In another preferred embodiment, D1 is an anti-FGFR2 antibody, and D2 is connected to the end of the heavy chain constant region of D1 via a linker.

[0023] In another preferred embodiment, the bispecific antibody is a homologous or heterologous dimer.

[0024] In another preferred embodiment, the bispecific antibody comprises a monomer having a structure shown in Formula I or Formula II from N-terminus to C-terminus:

[0025] in,

[0026] VL A represents the light chain variable region of an anti-FGFR2 antibody or an antigen-binding fragment thereof;

[0027] VH A represents the heavy chain variable region of an anti-FGFR2 antibody or an antigen-binding fragment thereof;

[0028] VL B represents the light chain variable region of an anti-PD-1 antibody or an antigen-binding fragment thereof;

[0029] VH B represents the heavy chain variable region of an anti-PD-1 antibody or an antigen-binding fragment thereof;

[0030] CH represents the heavy chain constant region;

[0031] CL represents the light chain constant region;

[0032] L1 and L2 are each independently a bond or a linker;

[0033] “~” represents a disulfide bond or a covalent bond;

[0034] “-” represents a peptide bond;

[0035] The bispecific antibody has the activity of simultaneously binding to FGFR2 and PD-1.

[0036] 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.

[0037] In another preferred embodiment, the anti-FGFR2 or anti-PD-1 antibody is selected from a chimeric antibody, a murine antibody or a humanized antibody.

[0038] 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).

[0039] In another preferred embodiment, the constant region of the IgG antibody contains at least one amino acid mutation.

[0040] In another preferred embodiment, the D1 is an IgG antibody, and the D2 is a scFv; more preferably, the D2 is connected to the N-terminus or C-terminus of D1; and even more preferably, the D2 is connected to the heavy chain of D1.

[0041] In another preferred embodiment, the D2 comprises one, two, three or more anti-PD-1 scFvs.

[0042] In another preferred embodiment, the scFv comprises a VH-L1-VL structure or a VL-L1-VH structure from N-terminus to C-terminus.

[0043] In another preferred embodiment, the amino acid sequences of the linkers L1 and L2 are independently (G4S) n , n is selected from 1, 2, 3, 4, 5, 6.

[0044] In another preferred embodiment, the amino acid sequence of the linker L1 is shown as SEQ ID NO.17.

[0045] In another preferred embodiment, the amino acid sequence of the linker L2 is shown in SEQ ID NO.18.

[0046] In another preferred embodiment, the bispecific antibody is a homodimer comprising an anti-FGFR2 IgG antibody and two anti-PD-1 scFvs, wherein each scFv comprises a variable region VH and a variable region VL, VH and VL are connected by a linker L1 to form a VL-L1-VH structure, and each anti-PD-1 scFv is connected to the C-terminus of the anti-FGFR2 immunoglobulin antibody IgG via a linker L2.

[0047] In another preferred embodiment, the bispecific antibody comprises a heavy chain and a light chain, and the amino acid sequences of the heavy chain and the light chain are selected from the following group:

[0048] 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; and

[0049] 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,

[0050] d) A polypeptide derived from a) or b) having both anti-FGFR2 activity and anti-PD-1 activity, formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of a) or b).

[0051] In another preferred embodiment, the bispecific antibody comprises an active fragment and / or derivative of the bispecific antibody, wherein the active fragment and / or the derivative retains 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.

[0052] In another preferred embodiment, the derivative of the bispecific antibody is a polypeptide that has undergone one or more amino acid mutations (after amino acid deletion, insertion and / or substitution) of the bispecific antibody and retains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0053] In another preferred embodiment, the amino acid mutation is a conservative amino acid substitution.

[0054] In another preferred embodiment, the amino acid mutation is located in the framework region or the constant region.

[0055] In another preferred embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the following three heavy chain complementarity determining regions: HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and

[0056] The light chain variable region includes the following three light chain complementary determining regions: LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.6.

[0057] In another preferred embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the following three heavy chain complementarity determining regions: HCDR1 as shown in SEQ ID NO.7, HCDR2 as shown in SEQ ID NO.8, and HCDR3 as shown in SEQ ID NO.9; and

[0058] The light chain variable region includes the following three light chain complementary determining regions: LCDR1 as shown in SEQ ID NO.10, LCDR2 as shown in SEQ ID NO.11, and LCDR3 as shown in SEQ ID NO.12.

[0059] In another preferred example, the anti-FGFR2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, 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.

[0060] In another preferred example, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, 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.

[0061] In another preferred embodiment, the heavy chain constant region comprises an Fc fragment, and the Fc fragment is derived from IgG1 or IgG4.

[0062] In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG1.

[0063] In another preferred embodiment, the Fc fragment contains at least one amino acid mutation.

[0064] In another preferred embodiment, the mutation is used to enhance the ADCC effect of the bispecific antibody.

[0065] In another preferred embodiment, the Fc fragment derived from IgG1 has a mutation selected from the following group:

[0066] S239D, I332E, A330L, G236A, preferably S239D, I332E.

[0067] In another preferred embodiment, the heavy chain constant region includes CH1, CH2, and CH3.

[0068] In another preferred embodiment, the CH1, CH2, and CH3 amino acid sequences are independently shown as SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21.

[0069] In another preferred example, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.23.

[0070] In another preferred embodiment, the heavy chain constant region comprises an amino acid mutation selected from the group consisting of S239D and I332E.

[0071] In another preferred embodiment, the anti-FGFR2 antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof 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;

[0072] Preferably, the variable region comprises at least one amino acid mutation.

[0073] In another preferred embodiment, the heavy chain constant region of the anti-FGFR antibody comprises an amino acid mutation selected from the following group:

[0074] a) the heavy chain constant region has an amino acid mutation at position 239; preferably S293D; and / or

[0075] b) the heavy chain constant region has an amino acid mutation at position 332; preferably I332E.

[0076] In another preferred embodiment, the heavy chain constant region sequence of the anti-FGFR antibody is shown as SEQ ID NO.23; or the heavy chain constant region sequence of the anti-FGFR antibody is shown as SEQ ID NO.24.

[0077] 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.

[0078] In another preferred embodiment, the D1 is an IgG antibody, and the D2 is a scFv; preferably, the D2 is connected to the N-terminus or C-terminus of D1; more preferably, the D2 is connected to the heavy chain of D1.

[0079] In a second aspect of the present invention, a polynucleotide molecule is provided, wherein the polynucleotide molecule encodes the bispecific antibody provided in the first aspect of the present invention.

[0080] In the third aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide molecule according to the second aspect of the present invention.

[0081] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector according to the third aspect of the present invention.

[0082] In a fifth aspect of the present invention, a method for preparing the bispecific antibody provided in the first aspect of the present invention is provided, the preparation method comprising the following steps:

[0083] a) culturing the host cell according to the fourth aspect of the present invention under expression conditions to express the bispecific antibody;

[0084] b) isolating and purifying the bispecific antibody described in step a).

[0085] In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising an effective amount of the bispecific antibody provided in the first aspect of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0086] In the seventh aspect of the present invention, there is provided use of the bispecific antibody provided in the first aspect of the present invention or the pharmaceutical composition according to the sixth aspect of the present invention in the preparation of a drug for cancer or tumor;

[0087] Preferably, the cancer or tumor is an FGFR2-positive cancer or tumor.

[0088] In another preferred embodiment, the cancer or tumor is selected from the group consisting of lung cancer, bone cancer, stomach cancer, pancreatic cancer, 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 system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethra cancer, penis cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer. carcinoma, lymphoma, transitional cell carcinoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, soft tissue sarcoma, solid tumors in children, lymphocytic lymphoma, central nervous system tumors, primary central nervous system lymphoma, spinal tumors, brain stem glioma, pituitary adenoma, melanoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic leukemia, acute leukemia, or a combination thereof.

[0089] In an eighth aspect of the present invention, an immunoconjugate is provided, comprising:

[0090] a) the bispecific antibody according to any one of the first aspects of the present invention; and

[0091] b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0092] In another preferred embodiment, the conjugate moiety is selected from: fluorescent or luminescent markers, radioactive markers, contrast agents for magnetic resonance imaging or computerized X-ray tomography, or enzymes, radionuclides, biotoxins or cytokines that can produce detectable products.

[0093] In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate.

[0094] In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors or cancers.

[0095] In the ninth aspect of the present invention, a method for treating cancer or tumor is provided, comprising administering the bispecific antibody of the first aspect of the present invention, the pharmaceutical composition of the sixth aspect of the present invention, or the immunoconjugate of the eighth aspect of the present invention to a subject in need thereof.

[0096] In another preferred embodiment, the method further comprises administering the drug in combination with other anti-tumor drugs.

[0097] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1A shows a schematic diagram of the structure of anti-FGFR2 / PD-1 dual antibody a or b.

[0099] Figure 1B shows a schematic structural diagram of the anti-FGFR2 / PD-1 dual antibody c.

[0100] Figure 1C shows a schematic structural diagram of the anti-FGFR2 / PD-1 dual antibody d.

[0101] Figure 2A shows the UPLC-SEC detection profile of anti-FGFR2 / PD-1 dual antibody a.

[0102] Figure 2B shows the UPLC-SEC detection profile of anti-FGFR2 / PD-1 dual antibody b.

[0103] Figure 2C shows the UPLC-SEC detection profile of anti-FGFR2 / PD-1 dual antibody c.

[0104] Figure 2D shows the UPLC-SEC detection profile of anti-FGFR2 / PD-1 dual antibody d.

[0105] Figure 3A shows the mass spectrum of the intact molecular weight of the de-N-glycan of anti-FGFR2 / PD-1 dual antibody a.

[0106] Figure 3B shows the mass spectrum of the intact molecular weight of the anti-FGFR2 / PD-1 dual antibody b after de-N-glycan removal.

[0107] Figure 4A shows the DSC detection spectrum of anti-FGFR2 / PD-1 dual antibody a.

[0108] Figure 4B shows the DSC detection spectrum of anti-FGFR2 / PD-1 dual antibody b.

[0109] Figure 5A shows the ELISA detection of the binding of anti-FGFR2 / PD-1 dual antibodies a and b to FGFR2.

[0110] Figure 5B shows the binding of anti-FGFR2 / PD-1 dual antibodies c and d to FGFR2 detected by ELISA.

[0111] Figure 5C shows the ELISA detection of the binding of anti-FGFR2 / PD-1 dual antibodies a and b to PD-1.

[0112] Figure 5D shows the binding of anti-FGFR2 / PD-1 dual antibodies a and d to PD-1 detected by ELISA.

[0113] FIG6A shows the binding activity of anti-FGFR2 / PD-1 dual antibodies a and b to Jurkat-PD-1-NFAT-luc cells.

[0114] Figure 6B shows the activity of anti-FGFR2 / PD-1 dual antibodies a and b in blocking PD-1 / PD-L1 binding.

[0115] FIG7A shows the ADCC effects of anti-FGFR2 / PD-1 dual antibodies a and b on SNU-16 cells.

[0116] Figure 7B shows the ADCC effects of anti-FGFR2 / PD-1 dual antibodies a and b on T cells.

[0117] Figure 8 shows the inhibitory effects of anti-FGFR2 / PD-1 dual antibodies a and b on FGF1-induced proliferation of Ba / F3-FGFR2IIIb cells.

[0118] Figure 9A shows the anti-tumor effect of anti-FGFR2 / PD-1 dual antibody b in the MC38 transplant tumor model.

[0119] Figure 9B shows the toxic and side effects of anti-FGFR2 / PD-1 dual antibody b on mice in the MC38 transplant tumor model.

[0120] Figure 10A shows the anti-tumor effects of anti-FGFR2 / PD-1 dual antibodies a and b in the SNU-16 xenograft tumor model.

[0121] Figure 10B shows the toxic and side effects of anti-FGFR2 / PD-1 dual antibodies a and b on mice in the SNU-16 transplanted tumor model. DETAILED DESCRIPTION

[0122] After extensive and in-depth research, the inventors have developed a novel bispecific antibody that targets both the tumor cell surface molecule FGFR2 and the immune cell surface molecule PD-1. This bispecific antibody maintains the activity of both antibodies and can simultaneously bind to both FGFR2 and PD-1 antigens. The bispecific antibody of the present invention can be used to induce a T cell-mediated immune response while simultaneously killing FGFR2-expressing tumor cells through ADCC. Therefore, the bispecific antibody of the present invention can be developed into an anti-tumor drug with superior efficacy. This is the basis for the completion of the present invention.

[0123] the term

[0124] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0125] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0126] In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. One end of each light chain has a variable region (VL) and the other end has a constant region. The light chain constant region includes a domain CL; the constant region of the light chain pairs with the CH1 domain of the heavy chain constant region, and the variable region of the light chain pairs with the variable region of the heavy chain. Constant regions are not directly involved in antibody-antigen binding, but they exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 subtypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the heavy chain's CH1 domain and the light chain's CL domain. The two heavy chains of an antibody are covalently linked by an interpolypeptide disulfide bond formed between the hinge region.

[0127] The "immunoglobulin antibody IgG" described in the present invention is a molecule of approximately 150 kDa, which is composed of four peptide chains, including two identical gamma heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus having a tetrameric quaternary structure. The two heavy chains are interconnected by disulfide bonds and each is connected to a light chain. The resulting tetramer has two identical halves that form a fork-shaped or Y-like shape, with each end of the fork containing an identical antigen-binding site. IgG antibodies can be divided into multiple subclasses (e.g., IgG1, 2, 3, and 4) based on slight differences in the amino acid sequence in the constant region of the heavy chain.

[0128] As used herein, the term "bispecific antibody" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes. Based on their symmetry, bispecific antibodies can be classified as structurally symmetrical or asymmetrical. Based on the number of binding sites, bispecific antibodies can be classified as bivalent, trivalent, tetravalent, or multivalent.

[0129] In the present invention, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a class of substantially homogeneous populations, i.e., the individual antibodies contained in the population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which are typically mixtures of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they can be synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody.

[0130] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment, etc., that has 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). As used herein, the term "antigen-binding fragment" also encompasses other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies and bivalent nanobodies), small modular immunotherapies, and shark variable IgNAR domains.

[0131] As used herein, 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 is composed of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of the antibody. The Fc fragment (Fc) is a crystallizable fragment and is composed of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells. The term "F(ab')2" fragment antibody is obtained by digesting the entire IgG antibody with pepsin, removing most of the Fc region while retaining some of the hinge region intact, and has two antigen-binding F(ab') portions linked together by disulfide bonds.

[0132] In the present invention, the term "scFv" or "single-chain variable region fragment scFv" is a single-chain antibody (scFv), which refers to a fusion protein comprising the variable regions of the heavy chain VH and light chain VL of an immunoglobulin, wherein VH and VL are connected by a linker containing 15-25 amino acids, wherein the fusion protein retains the same antigenic specificity as the intact immunoglobulin.

[0133] As used herein, the term "Fv fragment" or "Fv antibody" refers to a minimal antibody fragment comprising the variable regions of the heavy and light chains, but without the constant regions, and possessing a complete antigen-binding site. Generally, an Fv fragment also includes a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.

[0134] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ in sequence, which contributes to the binding and specificity of each particular antibody for its specific antigen. However, this variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments within the heavy and light chain variable regions, known as the complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are known as the framework regions (FRs). The variable regions of native heavy and light chains each contain four FRs, which generally have a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop, which in some cases may form a partial β-pleated sheet structure. The CDRs in each chain are closely aligned by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).

[0135] As used herein, the term "framework region FR" refers to the amino acid sequence inserted between the CDRs, that is, it refers to those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light chain and heavy chain of immunoglobulins each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain can be represented as (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, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%. Knowing the amino acid sequence of the CDR, a person skilled in the art can easily determine the framework region FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H. As used herein, the term "human framework region" is 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.

[0136] As used herein, the term "linker" refers to one or more amino acid residues inserted into the immunoglobulin domain that provide sufficient mobility for the domains of the light chain and heavy chain to fold into an exchange dual variable region immunoglobulin. In the present invention, preferred linkers refer to linkers L1 and L2, wherein L1 connects the VH and VL of the single-chain antibody (scFv), and L2 connects the scFv between the CH1 and CH2 of the antibody heavy chain, or directly to the heavy chain of the antibody (such as the N-terminus or C-terminus of the antibody heavy chain) through L2. Examples of suitable linkers include glycine (Gly) or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker can vary with the type of secondary structural elements that need to be implemented in the linker.

[0137] In the present invention, the terms "anti", "binding", and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The equilibrium dissociation constant (KD) of an antibody or antigen is 2M or less, and the antibody binds to the antigen. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, surface plasmon resonance (SPR) is used to measure the binding affinity of the antibody to the antigen in a BIACORE instrument or an ELISA is used to measure the relative affinity of the antibody to the antigen.

[0138] In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody or a region of an antigen that is bound by an antibody.

[0139] As used herein, "an amino acid mutation in the heavy chain constant region" refers to an amino acid mutation at a corresponding position in naturally occurring human IgG1, for example, an amino acid mutation at position 239 or 332 in human IgG1.

[0140] As used herein, the bispecific antibodies of the present invention also include conservative variants thereof, which refer to polypeptides 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 similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0141] Table A

[0142] Bispecific antibodies

[0143] 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 portion and an anti-PD-1 antibody portion.

[0144] The bispecific antibodies of the present invention may be dimers, trimers, or multimers, preferably homodimers or heterodimers. The anti-FGFR2 or anti-PD-1 antibody portion of the bispecific antibodies of the present invention may include one or more antibodies or antigen-binding fragments thereof, preferably 1, 2, 3, 4, 5, or 6. The sequences of the present invention use the Kabat numbering system.

[0145] When constructing the bispecific antibodies of the present invention, issues related to the chemical and physical stability of the bispecific antibodies are also addressed, such as expressing physically stable molecules, increasing thermal and salt-dependent stability, reducing aggregation, increasing solubility at high concentrations, and maintaining affinity for the two antigens FGFR2 and PD-1, respectively.

[0146] Encoding nucleic acid and expression vector

[0147] Another aspect of the present invention provides a polynucleotide molecule encoding the bispecific antibody described above. The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. This is typically done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells using conventional methods.

[0148] The preparation method of the polynucleotide molecule of the present invention is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining a polynucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR method, or obtaining a polynucleotide molecule encoding the above-mentioned monoclonal antibody by artificial full sequence synthesis method.

[0149] Those skilled in the art will appreciate that the nucleotide sequence encoding the amino acid sequence of the bispecific antibody described above can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homolog. Polynucleotide homologs 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.

[0150] Another aspect of the present invention provides an expression vector containing the above-mentioned polynucleotide molecule.

[0151] These vectors can be used to transform appropriate host cells to enable them to express proteins. The expression vectors are conventional expression vectors in the art, and refer to expression vectors containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. The expression vectors can be viruses or plasmids, such as appropriate phages or phagemids. For more technical details, please refer to, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, edited by Sambrook et al. Many known techniques and protocols for nucleic acid manipulation can be found in, for example, Current Protocols in Molecular Biology, 2nd edition, edited by Ausubel et al. The expression vectors of the present invention are preferably pDR1, pcDNA3.1(+), pcDNA3.4, pcDNA3.1 / ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF, or pCHO 1.0, more preferably pcDNA3.4.

[0152] The present invention further provides a host cell, wherein the host cell contains the above expression vector.

[0153] The host cell of the present invention is any of the conventional host cells in the art, as long as the above-mentioned recombinant expression vector can be stably replicated and the nucleotides carried can be effectively expressed. The host cell includes prokaryotic expression cells and eukaryotic expression cells, and the host cell preferably includes: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21 (DE3) or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies). The aforementioned expression vector is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0154] Preparation method

[0155] The host cell culture methods and antibody isolation and purification methods described in the present invention are conventional methods in the art. For specific operating procedures, please refer to the corresponding cell culture technology manuals and antibody isolation and purification technology manuals. The method for preparing the anti-FGFR2 / PD-1 bispecific antibody disclosed in the present invention comprises: culturing the above-mentioned host cells under expression conditions to express the bispecific antibody that specifically binds to FGFR2 and PD-1; and isolating and purifying the anti-FGFR2 / PD-1 bispecific antibody. Using the above method, the recombinant protein can be purified into a substantially homogeneous substance.

[0156] The anti-FGFR2 / PD-1 bispecific antibody disclosed in the present invention can be isolated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer and pH changes can be used to elute the anti-FGFR2 / PD-1 bispecific antibody bound to the affinity column. The inventors of the present invention conducted testing experiments on the resulting anti-FGFR2 / PD-1 bispecific antibody, and the experimental results showed that the anti-FGFR2 / PD-1 bispecific antibody can bind well to target cells and antigens and has high affinity.

[0157] Pharmaceutical composition

[0158] Another aspect of the present invention provides a composition, preferably, the composition is a pharmaceutical composition.

[0159] In the present invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier to more stably exert therapeutic effects. These formulations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed herein while protecting the protein's multiple functional groups from degradation (including but not limited to aggregation, deamination, or oxidation).

[0160] Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5.0-8.0, preferably about 6.0-8.0, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection or intracavitary injection. Generally, for liquid preparations, it can usually remain stable for at least one year at 2-8°C; for lyophilized preparations, it can usually remain stable for at least six months at 30°C. The bispecific antibody preparation can be a suspension, aqueous injection, lyophilized preparation, etc. commonly used in the pharmaceutical field.

[0161] The pharmaceutical composition of the present invention contains 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 conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to 50 mg / kg body weight per day. In addition, the bispecific antibody of the present invention can also be used in conjunction with other therapeutic agents.

[0162] When using a pharmaceutical composition, a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to a mammal. This safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0163] application

[0164] Another aspect of the present invention provides use of the aforementioned bispecific antibody capable of specifically binding to FGFR2 and PD-1, or the aforementioned pharmaceutical composition, in the preparation of a drug for treating cancer or tumors.

[0165] The drugs for treating cancer or tumors referred to in the present invention refer to drugs that have the function of inhibiting and / or treating tumors, which may include delaying the development of tumor-related symptoms and / or reducing the severity of these symptoms, further including alleviating existing tumor-associated symptoms and preventing the occurrence of other symptoms, and also including reducing or preventing tumor metastasis, etc.

[0166] The tumors targeted by the drug of the present invention preferably include but are not limited to: lung cancer, bone cancer, stomach cancer, pancreatic cancer, 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 system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethra cancer, penis cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, soft tissue sarcoma, solid tumors of childhood, lymphocytic lymphoma, central nervous system tumors, primary central nervous system lymphoma, spinal tumors, brain stem glioma, pituitary adenoma, melanoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia, and combinations of the foregoing cancers.

[0167] When the bispecific antibodies and compositions of the present invention are administered to animals, including humans, the dosage varies depending on the patient's age and weight, the nature and severity of the disease, and the route of administration. Animal experimental results and various circumstances should be considered, and the total dosage should not exceed a certain range. Specifically, the intravenous dosage is 1-1800 mg / day.

[0168] The bispecific antibodies and compositions of the present invention can also be administered in combination with other anti-tumor drugs to achieve a more effective treatment of tumors. These anti-tumor drugs include but are not limited to: 1. Cytotoxic drugs: 1) Drugs that act on the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosoureas, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxaliplatin; antibiotics such as adriamycin / doxorubicin, actinomycin D, and dactinomycin; D), daunorubicin, epirubicin, mithramycin, etc.; 2) Drugs that affect nucleic acid metabolism: dihydrofolate reductase inhibitors such as methotrexate and pemetrexed; thymidine synthetase inhibitors such as fluorouracils (5-fluorouracil, capecitabine); purine nucleoside synthetase inhibitors such as 6-mercaptopurine; ribonucleotide reductase inhibitors such as hydroxycarbamide; DNA polymerase inhibitors such as cytosine arabinoside and gemcitabine; 3) Drugs that act on microtubules: docetaxel, vinblastine, vinorelbine, podophyllins, homoharringtonine, etc.; 2. Hormonal drugs: antiestrogens 1. Anti-tumor drugs: 1. Anti-tumor drugs: such as tamoxifen, droloxifene, exemestane, etc. 2. Aromatase inhibitors: such as aminoglutethimide, formestane, letrozole, anastrozole, etc. 3. Anti-androgen: flutamide 4. RH-LH agonists / antagonists: zoledrone, enantone, etc. 5. Biological response modifiers: such drugs mainly achieve anti-tumor effects by regulating the body's immune function, such as interferons (Interferon), interleukin-2 (Interleukin-2), thymosins, etc. 6. Monoclonal antibody drugs: trastuzumab, rituximab, cetuximab, bevacizumab, etc. The bispecific antibodies and compositions disclosed in the present invention can be used in combination with one or a combination of the above-mentioned anti-tumor drugs.

[0169] The main advantages of the present invention include

[0170] The positive and progressive effects of the anti-FGFR2 / PD-1 bispecific antibody of the present invention are:

[0171] (1) It can regulate T cell immune activity by binding to the T cell surface molecule PD-1 and specifically bind to tumor cells expressing FGFR2, inducing immune cells to target and kill FGFR2-positive tumor cells. In particular, it has a significant proliferation inhibitory effect on FGFR2-positive tumor cells, and the ADCC effect is also more significant;

[0172] (2) High specificity and good safety;

[0173] (3) high expression level;

[0174] (4)Stable structure.

[0175] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0176] The experimental materials and sources used in the following examples and the preparation methods of the experimental reagents are described in detail as follows.

[0177] Experimental Materials:

[0178] Competent cells: Brand: Sangon, product number: B528412.

[0179] 293F cells: Brand: GIBCO, Product No. R79007.

[0180] hPBMC: Brand: Auscell, catalog number: FPB004F-C-MLR.

[0181] 0.45μm filter: brand Millipore, product number SLHV013SL.

[0182] SNU-16 cells: Brand: Nanjing Kebai, product number: CBP60502.

[0183] Jurkat-PD1-NFAT-luc cells: Promega brand, product number J1252.

[0184] CHO-K1-PD-L1 cells: Promega brand, product number J1252.

[0185] Ba / F3 cells: brand: Seli, product number: SLB-CL0019A.

[0186] 293FT cells: brand GIBCO, product number R70007.

[0187] Hitrap Mabselect Sure affinity chromatography column: brand: Hitrap Mabselect Sure, product number: 11003493.

[0188] Waters MassPREP TM Micro Desalting Column: Brand: Waters, Model: 186004032.

[0189] Experimental reagents:

[0190] Endotoxin-free plasmid extraction kit: brand Tiangen, product number DP117.

[0191] DNA purification and recovery kit: brand Tiangen, product number DP214.

[0192] ClonExpressTM II One Step Cloning Kit: brand: ClonExpress, product number: C112.

[0193] HS DNA Polymerase: brand takara, product number R010B.

[0194] Coating solution: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, add double distilled water to make up to 1 L.

[0195] PBST: PBS + 0.05% Tween 20.

[0196] Tween 20: Brand Aladdin, item number T104863.

[0197] ELISA blocking solution: PBST + 1% BSA.

[0198] BSA: Brand: Bioscientific, item number: A60332.

[0199] Human-FGFR2-his protein: Brand: Kaixia, Product No.: FGR-HM1BD.

[0200] HRP-labeled goat anti-human Fc antibody: Brand: Biolon, Product No. BF03031.

[0201] TMB: Brand: BD Biosciences, Catalog No. 555214.

[0202] Stop solution: 2M sulfuric acid solution.

[0203] 0.25% Trypsin-EDTA: GIBCO brand, product number 25200-072.

[0204] 1640 complete medium: RPMI1640 medium + 10% FBS + 1% Pen Strep + 1% sodium pyruvate.

[0205] RPMI1640 culture medium: brand GIBCO, product number 22400089.

[0206] FBS: Brand: GIBCO, Product No. 10099-141.

[0207] Pen Strep: Brand GIBCO, item number 1514022.

[0208] Sodium pyruvate: brand GIBCO, product number 11360-070.

[0209] DMEM complete medium: DMEM high glucose medium + 10% FBS + 1% Pen Strep + 1% GLUMAX.

[0210] DMEM high glucose medium: brand GIBCO, product number 11965-092.

[0211] GlutaMAX: Brand: GIBCO, item number: 35050-061.

[0212] Luminescent Cell Viability Assay: Promega, product number G7572.

[0213] Goat anti-human Fab-FITC: Brand: Thermo Fisher Scientific, Cat. No. MA1-10379.

[0214] Glycosidase F: Brand: NEB, Product No. P0704L.

[0215] Experimental instruments:

[0216] Mastercycler nexus PCR instrument: purchased from Eppendorf.

[0217] Hitrap Mabselect Sure column: purchased from Cytiva.

[0218] HiLoad 26 / 600 Superdex 200 pg column: purchased from Cytiva.

[0219] SpectraMax i3x microplate reader: purchased from Molecular Devices.

[0220] SpectraMaxM5 microplate reader: purchased from Molecular Devices.

[0221] Coulter CytoFLEX flow cytometer: purchased from Beckman.

[0222] FD-0007 fully automatic capillary differential scanning calorimeter: purchased from Malvern.

[0223] LC / MS liquid chromatography-mass spectrometry instrument: purchased from Waters.

[0224] The sequences of the present invention are shown in Table B below:

[0225] Example 1. Construction of anti-FGFR2 / PD-1 dual antibody molecules

[0226] The present invention uses an anti-FGFR2 monoclonal antibody IgG and an anti-PD-1 monoclonal antibody scFv in tandem to construct an anti-FGFR2 / PD-1 bispecific antibody, the structure of which is shown in Figure 1A.

[0227] The anti-FGFR2 monoclonal antibody sequence in the anti-FGFR2 / PD-1 bispecific antibody was derived from the humanized monoclonal antibody disclosed in patent CN102905723A and served as a positive control for anti-FGFR2 monoclonal antibody. The anti-PD-1 monoclonal antibody sequence in the anti-FGFR2 / PD-1 bispecific antibody was derived from the humanized monoclonal antibody disclosed in patent CN201710054783.5 and served as a positive control for anti-PD-1 monoclonal antibody.

[0228] The scFv-VLVH of the anti-FGFR2 / PD-1 bispecific antibody is connected to 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) through the peptide linker L1 (SEQ ID NO.17) to obtain VL-L1-VH, namely PD-1-scFv-VLVH (SEQ ID NO.27).

[0229] The C-terminus of the anti-FGFR2 monoclonal antibody heavy chain (SEQ ID NO.25) and the N-terminus of the anti-PD-1 monoclonal antibody variable fragment PD-1-scFv-VLVH (SEQ ID NO.27) were connected through the peptide linker L2 (SEQ ID NO.18) to obtain the heavy chain of the anti-FGFR2 / PD-1 dual antibody a (SEQ ID NO.29), and the light chain of the anti-FGFR2 monoclonal antibody (SEQ ID NO.28) remained unchanged.

[0230] Mutations were performed on the corresponding sites on human IgG1 in the heavy chain constant region of an anti-FGFR2 monoclonal antibody. After screening, S239D and I332E were selected to obtain the anti-FGFR2 monoclonal antibody heavy chain HC-S239D-I332E.

[0231] The N-terminus of the anti-FGFR2 monoclonal antibody heavy chain HC-S239D-I332E (SEQ ID NO.26) and the anti-PD-1 monoclonal antibody variable fragment PD-1-scFv-VLVH (SEQ ID NO.27) were connected through the peptide linker L2 (SEQ ID NO.18) to obtain the heavy chain of the anti-FGFR2 / PD-1 dual antibody b (SEQ ID NO.30), and the light chain of the anti-FGFR2 monoclonal antibody (SEQ ID NO.28) remained unchanged.

[0232] At the same time, in the process of constructing anti-FGFR2 / PD-1 bispecific antibody molecules, different connection methods and configurations are designed to connect anti-FGFR2 monoclonal antibody IgG or its scFv and anti-PD-1 monoclonal antibody IgG or its scFv to form different forms of anti-FGFR2 / PD-1 bispecific antibody molecules.

[0233] 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) were connected by a peptide linker L1 (SEQ ID NO. 17) to obtain VH-L1-VL, i.e., FGFR2-scFv-VHVL. The C-terminus of the anti-PD-1 monoclonal antibody heavy chain and the N-terminus of the anti-FGFR2 monoclonal antibody variable fragment FGFR2-scFv-VHVL were connected by a peptide linker L2 (SEQ ID NO. 18) to obtain the heavy chain of the anti-FGFR2 / PD-1 dual antibody c. The light chain of the anti-PD-1 monoclonal antibody remained unchanged, and the structure is shown in Figure 1B.

[0234] 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 connected by a 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 anti-PD-1 monoclonal antibody variable fragment PD-1-scFv-VHVL were connected by a peptide linker L2 (SEQ ID NO.18) to obtain the heavy chain of the anti-FGFR2 / PD-1 dual antibody d. The light chain of the anti-FGFR2 monoclonal antibody (SEQ ID NO.28) remained unchanged. The structure is shown in Figure 1C.

[0235] To improve expression efficiency in CHO cells, the nucleic acid sequence of the anti-FGFR2-PD1 bispecific antibody was codon-optimized, primarily considering factors such as codon preference, GC content, mRNA secondary structure, and repetitive sequences. The nucleic acid sequence of the antibody was synthesized by Sangon Biotech Co., Ltd. The amino acid and nucleic acid sequences are listed in the sequence listing.

[0236] Example 2. Expression and purification of anti-FGFR2 / PD-1 dual antibody

[0237] DNA fragments encoding the heavy and light chains of the anti-FGFR2 / PD-1 bispecific antibodies a and b were subcloned into the pcDNA3.4 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells and / or CHO cells. After 5-7 days of cell culture, the culture medium was centrifuged at high speed, filtered through a 0.22μm filter, and loaded onto a Hitrap MabSelect Sure affinity chromatography column. The protein was eluted in a single step using 100mM citric acid, pH 3.5. The sample was recovered and dialyzed into PBS, pH 7.4. The purified protein was analyzed by UPLC-SEC.

[0238] The detection results of anti-FGFR2 / PD-1 bispecific antibodies a, b, c, and d proteins are shown in Figures 2A, 2B, 2C, and 2D. The antibody proteins were uniform in state, the monomer purity of anti-FGFR2 / PD-1 bispecific antibody a reached more than 93%, the monomer purity of anti-FGFR2 / PD-1 bispecific antibody b was slightly higher than that of bispecific antibody a, and the monomer purity reached more than 96%. The purity of anti-FGFR2 / PD-1 bispecific antibodies c and d was slightly lower than that of bispecific antibody a, and the monomer purity was above 86% and 88%.

[0239] Example 3. Detection of intact molecular weight of anti-FGFR2 / PD-1 dual antibody by removing N-glycans

[0240] 1 mg of each anti-FGFR2 / PD-1 bispecific antibody a and b was taken, and the sample was desalted into ultrapure water by ultrafiltration and concentrated to above 20 mg / ml. 60 μl of sample was taken, 59 μl of ultrapure water was added, and 1 μl of glycosidase F was added. After incubation in a 37°C water bath for 2 hours, the sample was purified by a Waters MassPREP TM The deglycosylated intact molecular weight of anti-FGFR2 / PD-1 bispecific antibody samples a and b was detected using LC / MS liquid chromatography-mass spectrometry using a Micro Desalting Column.

[0241] The experimental results are shown in Figures 3A and 3B. The theoretical deglycosylated molecular weight of the anti-FGFR2 / PD-1 bispecific antibody a is 196892.05 Da, and the molecular weight measured after mass spectrometry analysis is 196896.98 Da; the theoretical deglycosylated molecular weight of the anti-FGFR2 / PD-1 bispecific antibody b is 196979.98 Da, and the molecular weight measured after mass spectrometry analysis is 196986.28 Da. The theoretical molecular weights of the anti-FGFR2 / PD-1 bispecific antibodies a and b are basically consistent with the measured molecular weights.

[0242] Example 4. Thermal stability determination of anti-FGFR2 / PD-1 dual antibody

[0243] Differential scanning calorimetry (DSC) was used to characterize the temperature-dependent changes in the molar heat capacity of the anti-FGFR2 / PD-1 bispecific antibody to assess its thermal stability. Protein solutions of anti-FGFR2 / PD-1 bispecific antibodies a and b were diluted to 0.5 mg / mL with 1× PBS (pH 7.4) buffer. 500 μL of the solution was analyzed using an automated capillary differential scanning calorimeter. The resulting DSC spectra are shown in Figures 4A and 4B.

[0244] The experimental results showed that as the temperature increased, the Tm1 and Tm2 of FGFR2 / PD-1 dual antibody a were 62.74°C and 79.08°C, respectively, and the Tm1 and Tm2 of FGFR2 / PD-1 dual antibody b were 60.56°C and 81.01°C, respectively. Both anti-FGFR2-PD1 dual antibodies a and b had their first endothermic peak at about 60°C and their second endothermic peak at about 80°C, indicating that the thermal stability of anti-FGFR2 / PD-1 dual antibody a and b proteins was comparable.

[0245] Example 5. Determination of affinity of bispecific antibodies for antigens by enzyme-linked immunosorbent assay (ELISA)

[0246] 5.1 Detection of the affinity of anti-FGFR2 / PD-1 dual antibodies for FGFR2

[0247] Dilute the recombinant Human-FGFR2-His protein to 200 ng / ml in the coating solution, add 100 μl / well to the ELISA plate, and incubate at room temperature for 2 hours or overnight at 4°C. Remove the coating solution (use absorbent paper to remove any remaining droplets), add 200 μl / well of blocking solution to the ELISA plate, and incubate at room temperature for 1-2 hours. Remove the blocking solution (use absorbent paper to remove any remaining droplets), and then dilute the anti-FGFR2 / PD-1 dual antibodies a and b, as well as the anti-FGFR2 monoclonal antibody to 10 μg / ml in the blocking solution. Fourfold dilutions were made to form 12 concentration gradients (highest concentration 10 μg / ml, lowest concentration 0.002 ng / ml), and 100 μl / well were added to the blocked ELISA plate in sequence and incubated at room temperature for 1 hour. Wash the plate 3 times with PBST (remove residual droplets with absorbent paper), dilute HRP-labeled goat anti-human Fc antibody at 1:3000 with blocking solution, add 100 μl / well to the enzyme-labeled plate, and place at room temperature for 30 minutes. Wash the plate 3 times with PBST (remove residual droplets with absorbent paper), add TMB colorimetric solution at 100 μl / well, place at room temperature in the dark for 5 minutes, add 70 μl / well stop solution to stop the substrate color reaction, read the OD value at 450 nm with a microplate reader, analyze the data with GraphPad, plot and calculate EC 50 .

[0248] The experimental results are shown in Figure 5A. The EC of anti-FGFR2 / PD-1 bispecific antibody molecules a, b and the positive control anti-FGFR2 monoclonal antibody bound to Human-FGFR2-His 50 (Unit: nM) were 0.0568, 0.0512 and 0.0392 respectively, and the affinities of the three were comparable.

[0249] The same method was used to determine the affinity of the anti-FGFR2 / PD-1 bispecific antibody molecules c, d and the positive control anti-FGFR2 monoclonal antibody to Human-FGFR2-His. The experimental results are shown in Figure 5B. 50 (Unit: nM) were 0.6244, 0.0615 and 0.0297 respectively. The affinities of anti-FGFR2 / PD-1 dual antibody d and the positive control anti-FGFR2 monoclonal antibody were comparable. Compared with the positive control anti-FGFR2 monoclonal antibody, the affinity of anti-FGFR2 / PD-1 dual antibody c was significantly decreased.

[0250] The structures of anti-FGFR2 / PD-1 bispecific antibodies a, b, and d are similar, all of which are in the form of a series connection of anti-FGFR2 monoclonal antibody IgG and anti-PD-1 monoclonal antibody scFv, while the anti-FGFR2 / PD-1 bispecific antibody c is in the form of a series connection of anti-PD-1 monoclonal antibody IgG and anti-FGFR2 monoclonal antibody scFv. Among them, the affinities of bispecific antibodies a and b are better than those of bispecific antibody c, indicating that the bispecific antibody molecules formed by the series connection of anti-FGFR2 monoclonal antibody IgG and anti-PD-1 monoclonal antibody scFv have a higher affinity for binding to FGFR2 antigen.

[0251] 5.2 Detection of the affinity of anti-FGFR2 / PD-1 dual antibodies for PD-1

[0252] Dilute recombinant Human-PD-1-His protein to 200 ng / ml in coating solution and add 100 μl / well to the ELISA plate. Incubate at room temperature for 2 hours or overnight at 4°C. Remove the coating solution (use absorbent paper to remove any remaining droplets) and add 200 μl / well of blocking solution to the ELISA plate. Incubate at room temperature for 1-2 hours. Remove the blocking solution (use absorbent paper to remove any remaining droplets). Dilute anti-FGFR2 / PD-1 dual antibodies a and b and anti-PD-1 monoclonal antibody to 10 μg / ml in blocking solution. Make four-fold dilutions to form 12 concentration gradients (highest concentration 10 μg / ml, lowest concentration 0.002 ng / ml). Add 100 μl / well to the blocked ELISA plate and incubate at room temperature for 1 hour. Wash the plate 3 times with PBST (remove residual droplets with absorbent paper), dilute HRP-labeled goat anti-human FC antibody at 1:3000 with blocking solution, add 100 μl / well to the ELISA plate, and place at room temperature for 30 minutes. Wash the plate 3 times with PBST (remove residual droplets with absorbent paper), add TMB at 100 μl / well, place at room temperature in the dark for 5 minutes, add 70 μl / well of stop solution to stop the substrate color reaction, read the OD value at 450 nm with a microplate reader, analyze the data with GraphPad, plot and calculate EC 50 .

[0253] The experimental results are shown in Figure 5C. The EC of anti-FGFR2 / PD-1 bispecific antibody molecules a and b, as well as the positive control anti-PD-1 monoclonal antibody combined with Human-PD-1-His 50 (Unit: nM) were 0.1216, 0.1411 and 0.0586, respectively. The affinity of bispecific antibodies a and b was lower than that of monoclonal antibodies, indicating that the variable regions of the anti-PD-1 monoclonal antibody were connected in a VL-VH manner and then linked to the heavy chain of the anti-FGFR2 monoclonal antibody to form a bispecific antibody with slightly reduced binding ability to the PD-1 antigen.

[0254] The same method was used to determine the affinities of anti-FGFR2 / PD-1 bispecific antibody molecules a, d and the positive control anti-PD-1 monoclonal antibody to Human-PD-1-His. The experimental results are shown in Figure 5D . The EC50 (unit: nM) were 0.1599, 0.3564, and 0.0856, respectively. The affinity of anti-FGFR2 / PD-1 bispecific antibody a to Human-PD-1-His was slightly higher than that of anti-FGFR2 / PD-1 bispecific antibody d, indicating that the variable regions of the anti-PD-1 monoclonal antibody were linked in a VL-VH manner, and the bispecific antibody molecule formed by tandem with the anti-FGFR2 monoclonal antibody scFv had a higher affinity for binding to the PD-1 antigen.

[0255] Example 6. Blocking activity of anti-FGFR2 / PD-1 dual antibodies on PD-1 / PD-L1 binding

[0256] Jurkat-PD-1-NFAT-luc cells expressing PD-1 on their cell surface were used as target cells. The binding affinity of anti-FGFR2 / PD-1 dual antibody to the cells was determined by flow cytometry. The cell density was adjusted to 1.5×10 6 Cells / mL, 100 μL / well added to a 96-well plate. Anti-FGFR2 / PD-1 dual antibodies a and b and the positive control anti-PD-1 monoclonal antibody were diluted to 200 nM with DPBS containing 1% BSA, diluted 4-fold into 12 concentration gradients, and 100 μL / well added to a 96-well plate and mixed evenly with Jurkat-PD-1-NFAT-luc cells. Incubate at 4°C for 1 hour. Centrifuge at 400g for 5 minutes and discard the supernatant. Wash twice with pre-chilled DPBS to remove unbound test antibodies. Add 100 μL of goat anti-human Fab-FITC (800:1 dilution) to each well and incubate at 4°C for 1 hour. Centrifuge at 400g for 5 minutes and discard the supernatant. The cells were washed twice with pre-cooled DPBS and finally resuspended in 200 μL of DPBS. The binding affinity of the anti-FGFR2 / PD-1 dual antibody to the cells was determined by Beckman Coulter CytoFLEX flow cytometer, and the data were analyzed by GraphPad Prism6.

[0257] The experimental results are shown in Figure 6A. Both anti-FGFR2 / PD-1 dual antibodies a and b can specifically bind to PD-1 expressed on the cell surface. EC binding of anti-FGFR2 / PD-1 dual antibodies a, b and positive control anti-PD-1 monoclonal antibody to Jurkat-PD1-NFAT-luc cells 50 They were 0.4823, 0.6344 and 0.1046 nM respectively, indicating that the activity of anti-FGFR2 / PD-1 dual antibodies a and b in binding to cell surface PD-1 protein was relatively weaker than that of anti-PD-1 monoclonal antibody.

[0258] CHO-K1-PD-L1 cells were diluted to 4 × 10 5 Cells / mL, 100 μL / well were added to a 3903 model 96-well plate, placed in a 37°C incubator with 5% CO2 for overnight incubation, and the culture medium was removed for later use. Anti-FGFR2 / PD-1 bispecific antibodies a and b and anti-PD-1 monoclonal antibody were diluted to 500 nM in assay medium (RPMI 1640 + 1% FBS), and then diluted 5-fold to 10 concentrations (the highest concentration was 500 nM, the lowest concentration was 2.56×10 -4 nM), 40 μL / well was added to a 96-well plate containing CHO-K1-PD-L1 cells; Jurkat-PD-1-NFAT-luc cells were diluted to 4×10 5 Cells / mL were added to a 96-well plate containing CHO-K1-PD-L1 cells at 40 μL / well. After incubation at 37°C, 5% CO2 for 6 hours, the 96-well plate was removed and equilibrated to room temperature. 80 μL of Bio-Glo detection reagent was added to each well and incubated at room temperature for 5-10 minutes. Luminescence values ​​were read using a SpectraMax i3. All data were collected in duplicate. The averaged signal values ​​were then fitted using the 4-parameter method to plot the curve.

[0259] The experimental results are shown in Figure 6B. The anti-FGFR2 / PD-1 dual antibody molecules a and b blocked the IC 50 The IC values ​​of anti-PD-1 monoclonal antibodies blocking PD1 and PD-L1 binding were 0.481 and 2.352 nM, respectively. 50 It is 0.372nM, indicating that the ability of the anti-FGFR2 / PD-1 bispecific antibody molecule a to block the binding of PD-1 and PD-L1 is comparable to that of the anti-PD-1 monoclonal antibody, and the ability of the anti-FGFR2 / PD-1 bispecific antibody molecule b to block the binding of PD-1 and PD-L1 is lower than that of the anti-PD-1 monoclonal antibody.

[0260] Example 7. ADCC Effects of Anti-FGFR2 / PD-1 Dual Antibodies on SNU-16 Cells and T Cells

[0261] In this experiment, human gastric cancer cells SNU-16 with high expression of cell surface FGFR2 and Jurkat-PD-1-NFAT-luc expressing PD-1 were used as target cells to detect the killing ability of NK cells against tumor cells and T cells.

[0262] 7.1 ADCC Effects of Bispecific Antibodies on SNU-16 Tumor Cells

[0263] SNU-16 cells were taken and adjusted to 2×10 5 cells / mL, 50 μL / well was added to the inner 60 wells of the 96-well plate, and the edge wells were sealed with sterile water. Anti-FGFR2 / PD-1 dual antibodies a, b and positive control anti-FGFR2 monoclonal antibody were diluted to 200nM with RPMI 1640 medium containing 5% FBS, and added to the wells containing cells after 4-fold dilution, 50 μL per well. Incubate in a 37°C, 5% CO2 incubator for 30 minutes. During this period, fresh human peripheral blood mononuclear cells (hPBMC) were taken and NK cells were sorted according to the instructions of the human NK Cell Isolation Kit (Miltenyi Biotec, #130-092-657). The sorted NK cells were adjusted to 4×10 5 cells / mL, 100 μL / well was added to the above wells and incubated in a 37°C, 5% CO2 incubator for 24 hours. TM LDH was detected according to the instructions of LDH Cytotoxicity Assay Kit (invitrogen, #C20300). Specifically: Add 20 μL of lysis solution to the well used to detect the maximum release of LDH, tap to mix, and incubate in a 37°C, 5% CO2 incubator for 45 minutes. Afterwards, take 50 μL of sample to a new 96-well plate and add 50 μL of LDH detection working solution to each well. Mix well and incubate at room temperature in the dark for 30 minutes. Add 50 μL of reaction termination solution, tap to mix, and use a microplate reader to detect the OD value at 490nm and 680nm. The absorbance of each well = OD 490 -OD 680 Cytotoxicity or mortality (%) = (absorbance of experimental group - absorbance of blank well) / (absorbance of total LDH well - absorbance of blank well) × 100.

[0264] The experimental results are shown in Figure 7A. Compared with the positive control anti-FGFR2 monoclonal antibody, the killing effect of NK cells on SNU-16 tumor cells was weaker under the action of anti-FGFR2 / PD-1 dual antibody a; however, under the action of anti-FGFR2 / PD-1 dual antibody b, NK cells had a significant killing effect on SNU-16 tumor cells. 50 They are 4.615 and 3.031 nM respectively, with comparable ADCC effect levels.

[0265] Therefore, after the S239D-I332E point mutation was performed on the Fc region of the anti-FGFR2 / PD-1 dual antibody, the ADCC effect of the anti-FGFR2 / PD-1 dual antibody b was significantly enhanced ( Figure 7A ).

[0266] 7.2 ADCC Effects of Bispecific Antibodies on T Cells

[0267] Jurkat-PD-1-NFAT-luc cells expressing PD-1 on their cell surface were used as target cells to detect ADCC effects. The method was as described above. The difference was that the antibodies used were anti-FGFR2 / PD-1 dual antibodies a and b, as well as positive control anti-MHC-I monoclonal antibody and negative control anti-PD-1 monoclonal antibody. The initial concentration of the antibodies was 50 nM, and they were diluted 4-fold in stages and added to the wells containing cells. According to the CyQUANT TM LDH was detected according to the instructions of the LDH Cytotoxicity Assay Kit (Invitrogen, #C20300). Data were analyzed using GraphPad Prism 6.

[0268] The results are shown in Figure 7B . NK cells had a significant killing effect under the action of the positive control anti-MHC-I monoclonal antibody, but had no significant killing effect on T cells under the action of anti-FGFR2 / PD-1 dual antibodies a and b and the negative control anti-PD-1 monoclonal antibody.

[0269] Example 8. Inhibitory effect of anti-FGFR2 / PD-1 dual antibody on FGF1-induced proliferation of Ba / F3-FGFR2IIIb cells

[0270] 293FT cells were digested with 0.25% trypsin-EDTA, diluted with complete DMEM medium, and plated into 6-well plates at a density of 700,000 cells per well. After the cells adhered, the packaged viral vector pLVX (containing the human FGFR2IIIb gene) was transfected with the 293FT cells using Lipofectamine 3000 transfection reagent. After 48 hours, the 293FT cell culture medium was collected and centrifuged at 500g for 5 minutes. The supernatant was removed and filtered through a 0.45μm filter. Polybrene was added at a 1000:1 ratio to promote infection, and 2ml of the supernatant was added to a T25 culture dish (to which 250,000 Ba / F3 cells had been added 24 hours earlier). After 48 hours, the Ba / F3 cells were harvested and passaged in complete medium containing the resistance factor until stable cell growth was achieved, resulting in a Ba / F3-FGFR2IIIb cell population. The Ba / F3-FGFR2 cell population was diluted with culture medium to 2-3 cells / ml and added to a 96-well plate for monoclonal isolation to obtain the Ba / F3-FGFR2IIIb cell line. The FGFR2IIIb gene expression efficiency of the Ba / F3-FGFR2IIIb cell line was tested by flow cytometry, and the highest FGFR2IIIb gene expression efficiency in the Ba / F3-FGFR2IIIb cell line was 9.26% (results not shown).

[0271] Ba / F3-FGFR2IIIb cells cultured to the logarithmic phase were collected by centrifugation and diluted to 1×10 cells with complete culture medium. 6 Cells / ml, 100 μl / well was added to a 3903 white clear-bottom 96-well plate and placed in a 37°C, 5% CO2 incubator until ready to use. Anti-FGFR2 / PD-1 dual antibodies a and b and anti-FGFR2 monoclonal antibody were diluted to 500 nM in complete culture medium and serially diluted 5-fold. The plates were added to a 96-well plate containing Ba / F3-FGFR2IIIb cells and incubated in a 37°C, 5% CO2 incubator for 1 hour. FGF1 was diluted to a concentration of 10 ng / ml with complete culture medium, and heparin was diluted to a concentration of 5 μg / ml. The plates were added to a 96-well plate containing Ba / F3-FGFR2IIIb cells and antibodies, and the plates were incubated in a 37°C, 5% CO2 incubator for 2 h. Then, 20 μl of MTS / PMS solution was added to each well. The plates were incubated in a 37°C, 5% CO2 incubator for 1-4 h, and then shaken to mix. The OD value at 490 nm was detected by a microplate reader. All data were obtained in duplicate. The obtained signal values ​​were converted into inhibition rates, and the average was taken to draw the curve using the 4-parameter method.

[0272] The experimental results are shown in Figure 8. The EC values ​​of anti-FGFR2 / PD-1 dual antibodies a, b and positive control anti-FGFR2 monoclonal antibody are 50The values ​​were 0.0870, 0.0515 and 0.1030 nM respectively, indicating that the anti-FGFR2 / PD-1 dual antibody b has a stronger ability to compete with FGF1 for the FGFR2IIIb receptor, and can better block the cell proliferation stimulated by the binding of FGF1 to FGFR2IIIb.

[0273] Example 9. Anti-tumor effect of anti-FGFR2 / PD-1 dual antibody in MC38 transplanted tumor model

[0274] The mouse colon cancer MC38 cells cultured in vitro were collected and the cell suspension concentration was adjusted to 2×10 7 cells / mL. The right flank of NOG mice was shaved and 100 μl of cell suspension was inoculated subcutaneously under sterile conditions. After the tumor cells formed solid tumors under the mouse skin, the diameter of the transplanted tumor was measured with a vernier caliper. When the average tumor volume grew to 50 mm 3 -100mm 3 The animals were then randomly divided into three groups, each with nine animals: a blank control group, injected with PBS alone; a control group receiving a 10 mg / kg dose of the anti-PD-1 monoclonal antibody; and a group receiving a 13.5 mg / kg dose of the anti-FGFR2 / PD-1 bispecific antibody b, administered intraperitoneally twice weekly for a total of five doses. Throughout the experiment, the diameter of the transplanted tumors was measured twice weekly, and the mice were weighed. Tumor growth curves and mouse weight curves over time were plotted for each group.

[0275] The experimental results are shown in Figures 9A and 9B. In the MC38 cell transplant tumor model, the inhibitory effects of anti-FGFR2 / PD-1 dual antibody b and anti-PD-1 monoclonal antibody on tumors were significantly higher than those in the control group. There was no significant difference in the inhibitory effects of anti-FGFR2 / PD-1 dual antibody b and anti-PD-1 monoclonal antibody on tumors; the body weight of mice in each dose group did not decrease significantly after continuous administration for several days, indicating that anti-FGFR2 / PD-1 dual antibody b and anti-PD-1 monoclonal antibody had relatively small toxic side effects on mice.

[0276] The results showed that in the non-target tumor cell transplant tumor model, the anti-tumor activity of anti-FGFR2 / PD-1 dual antibody b was not significantly different from that of anti-PD-1 monoclonal antibody, and both were able to significantly inhibit tumor growth, indicating that the structure of anti-FGFR2 / PD-1 dual antibody does not affect the effect of anti-PD-1 monoclonal antibody.

[0277] Example 10. Anti-tumor effect of anti-FGFR2 / PD-1 dual antibody in SNU-16 transplanted tumor model

[0278] Human gastric cancer SNU-16 cells cultured in vitro were collected, resuspended in serum-free medium, and mixed with an equal volume of matrigel to adjust the cell concentration to 8×10 7Under sterile conditions, 100 μl of cell suspension was inoculated subcutaneously on the right flank of NOG mice. On the 8th day after tumor inoculation, hPBMC cells were revived and the cell concentration was adjusted to 2.5×10 7 Except for the three tumor-bearing mice that were not inoculated with hPBMC cells, 200 μl of hPBMC cell suspension was injected into the tail vein of each tumor-bearing mouse, that is, 5×10 6 When the tumor cells formed solid tumors under the mouse skin, the diameter of the transplanted tumor was measured with a vernier caliper. When the average tumor volume grew to 200 mm 3 Animals were randomly divided into seven groups, including a blank control group (PBS alone) and an hPBMC control group (hPBMC diluted with PBS). Two monoclonal antibody control groups were administered with 5 mg / kg of anti-FGFR2 monoclonal antibody and 5 mg / kg of anti-PD-1 monoclonal antibody, respectively; a combination control group was administered with 5 mg / kg of anti-FGFR2 monoclonal antibody and 5 mg / kg of anti-PD-1 monoclonal antibody; and two dose groups were administered with 6.75 mg / kg of anti-FGFR2 / PD-1 dual antibody a and 6.75 mg / kg of anti-FGFR2 / PD-1 dual antibody b. Dosing was performed twice weekly for a total of five doses. Throughout the experiment, the diameter of the transplanted tumors was measured twice weekly, and the mice were weighed. Tumor growth curves and mouse weight curves were plotted over time for each group.

[0279] The experimental results are shown in Figures 10A and 10B and Table 1. In the SNU-16 cell mixed hPBMC xenograft tumor model, the tumor inhibitory effects of anti-FGFR2 / PD-1 dual antibodies a and b were not significantly different and were comparable to those of the anti-FGFR2 monoclonal antibody; the inhibitory effects were relatively better than those of the combination of anti-FGFR2 monoclonal antibody and anti-PD-1 monoclonal antibody; the tumor inhibitory effects of anti-FGFR2 / PD-1 dual antibodies a and b were significantly superior to those of the anti-PD-1 monoclonal antibody; the body weight of the mice did not decrease significantly after continuous administration for several days, indicating that the toxic side effects of anti-FGFR2 / PD-1 dual antibodies a and b on mice were relatively small.

[0280] The results showed that in the SNU-16 cell transplant tumor model with high FGFR2 expression, the anti-FGFR2 / PD-1 dual antibodies a and b had better tumor inhibitory activity than the anti-PD-1 monoclonal antibody, and both were able to significantly inhibit tumor growth, indicating that the anti-FGFR2 / PD-1 dual antibodies can specifically target FGFR2 tumor cells and exert a good anti-tumor effect.

[0281] Table 1 Anti-tumor effects of anti-FGFR2 / PD-1 dual antibodies in the SNU-16 xenograft tumor model

[0282] Example 11. Stability Study of Anti-FGFR2 / PD-1 Dual Antibody b

[0283] This experiment can be used to evaluate the stability of proteins in the presence of excipients, revealing important information needed to develop an optimal formulation.

[0284] Anti-FGFR2 / PD-1 dual antibody b was dialyzed overnight against 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 concentrated to 5 mg / mL by ultrafiltration at 4000 rpm for 20 min. Samples were aliquoted and stored at 4°C, 25°C, and 37°C.

[0285] The experimental results are shown in Table 2. Under the same treatment conditions in different buffers, anti-FGFR2 / PD-1 dual antibody b appeared obvious aggregates on the 21st day after being placed in buffer 1, but no aggregates appeared in buffer 2. A small amount of aggregates could be detected in buffer 2 until the 28th day, indicating that anti-FGFR2 / PD-1 dual antibody b is more stable in a buffer containing acetate.

[0286] Table 2 Preliminary screening results of anti-FGFR2 / PD-1 dual antibody preparations

[0287] discuss

[0288] The above experiments show that the bispecific antibodies provided by the present invention can simultaneously bind to FGFR2 and PD-1, release the immune function of T cells by binding to PD-1, and specifically kill tumor cells expressing FGFR2 protein. The anti-FGFR2 / PD-1 bispecific antibody can effectively block the binding of PD-1 and PD-L1. Among them, bispecific antibody b has a significant proliferation inhibitory effect on the target cell SUN-16, and its effect of competing with FGF1 for FGFR2 receptors to inhibit cell proliferation is also relatively good; after the S239D-I332E point mutation of the Fc region of the anti-FGFR2 / PD-1 bispecific antibody, the ADCC effect of the anti-FGFR2 / PD-1 bispecific antibody b was significantly enhanced (Figure 7A), but it had no significant killing effect on T cells; in the transplant tumor model, whether it is MC38 cells with non-target proteins or SNU-16 cells with high FGFR2 expression, the anti-FGFR2 / PD-1 bispecific antibody showed good anti-tumor activity.

[0289] During the research on anti-FGFR2 / PD-1 bispecific antibodies, anti-FGFR3 / PD-1 bispecific antibodies were also designed. However, in multiple animal model efficacy research experiments, it was found that the anti-tumor activity of the anti-FGFR3 / PD-1 bispecific antibody was significantly weaker than that of the anti-FGFR2 / PD-1 bispecific antibody and the anti-PD-1 monoclonal antibody, indicating that the combination of anti-FGFR3 monoclonal antibody and anti-PD-1 monoclonal antibody will affect the activity of the anti-PD-1 monoclonal antibody. This also once again verifies the superior efficacy of the two-target combination in the anti-FGFR2 / PD-1 bispecific antibody of the present invention. While not affecting the release of T cell immune activity, the antibody effect on target cells is also good, and can specifically bind to and kill tumor cells expressing FGFR2.

[0290] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A bispecific antibody, characterized in that: The bispecific antibody comprises: A first antigen binding domain D1; and The second antigen binding domain D2, Wherein, the D1 specifically binds to the target molecule FGFR2 protein, The D2 specifically binds to the target molecule PD-1 protein; Wherein, D1 is an anti-FGFR2 antibody or an antigen-binding fragment thereof, wherein the anti-FGFR2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following three heavy chain complementarity determining regions: HCDR1 as shown in SEQ ID NO.1, HCDR2 as shown in SEQ ID NO.2, and HCDR3 as shown in SEQ ID NO.3; and The light chain variable region includes the following three light chain complementary determining regions: LCDR1 as shown in SEQ ID NO.4, LCDR2 as shown in SEQ ID NO.5, and LCDR3 as shown in SEQ ID NO.

6.

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

12.

3. The bispecific antibody according to claim 1, characterized in that 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.

4. The bispecific antibody according to claim 1, characterized in that D1 is an anti-FGFR2 antibody, and D2 is connected 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 through a linker.

5. The bispecific antibody according to claim 1, characterized in that The bispecific antibody comprises a monomer having a structure shown in Formula I or Formula II from the N-terminus to the C-terminus: in, V L A represents a light chain variable region of an anti-FGFR2 antibody or an antigen-binding fragment thereof; VH A represents the heavy chain variable region of an anti-FGFR2 antibody or an antigen-binding fragment thereof; V L B represents a light chain variable region of an anti-PD-1 antibody or an antigen-binding fragment thereof; VH B represents a heavy chain variable region of an anti-PD-1 antibody or an antigen-binding fragment thereof; CH represents heavy chain constant region; CL represents the light chain constant region; L1 and L2 are each independently a bond or a linker; "~" represents a disulfide bond or a covalent bond; "-" represents a peptide bond; The bispecific antibody has the activity of simultaneously binding to FGFR2 and PD-1.

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

7. The bispecific antibody according to claim 1, characterized in that 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.

8. The bispecific antibody according to claim 1, wherein The heavy chain constant region sequence of the anti-FGFR antibody is shown as SEQ ID NO.23; or the heavy chain constant region sequence of the anti-FGFR antibody is shown as SEQ ID NO.

24.

9. The bispecific antibody according to claim 1, characterized in that The bispecific antibody comprises a heavy chain and a light chain, and 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 derived from a), b) or c) having both anti-FGFR activity and anti-PD1 activity, formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence in a) or b).

10. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the bispecific antibody according to any one of claims 1 to 9.

11. An expression vector, characterized in that: The expression vector contains the polynucleotide molecule according to claim 10.

12. A host cell, characterized in that The host cell contains the expression vector according to claim 11.

13. A method for preparing a bispecific antibody according to any one of claims 1 to 9, characterized in that: The preparation method comprises the following steps: a) culturing the host cell according to claim 12 under expression conditions, thereby expressing the bispecific antibody; b) isolating and purifying the bispecific antibody described in step a).

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the bispecific antibody according to any one of claims 1 to 9 and one or more pharmaceutically acceptable carriers, diluents or excipients.

15. Use of the bispecific antibody according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 14 in the preparation of a medicament for treating cancer or tumor; preferably, the cancer or tumor is a FGFR2-positive cancer or tumor.

16. An immunoconjugate, characterized in that: The immunoconjugate comprises: a) the bispecific antibody according to any one of claims 1 to 9; and b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide or an enzyme.

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