Anti-FGFR2 / PD-1 bispecific antibody

Abstract

The invention relates to an anti-FGFR2 (Fibroblast Growth Factor Receptor 2) / PD-1 (Protein The anti-FGFR2 / PD-1 bispecific antibody comprises a first antigen binding domain D1 and a second antigen binding domain D2, 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 can inhibit the proliferation of FGFR2 target cells and kill the target cells through an ADCC effect, and also can block the combination of PD-1 and ligands thereof, relieve the inhibition state of T cells and play an anti-tumor role.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular, to an anti-FGFR2 / PD-1 bispecific antibody. Background Art

[0002] Fibroblast growth factor receptors (FGFRs) are highly conserved receptor tyrosine kinases. There are four typical structures, FGFR1-4, and FGFR5, which lacks the intracellular tyrosine kinase domain. The extracellular domain of FGFR contains three immunoglobulin-like domains, among which the carboxyl selective isomerization of the third Ig domain can produce the IIIb or IIIc subtypes of FGFR1 to FGFR3. FGFR2-IIIb expressed in epithelial cells can be activated by FGFs and form a ternary complex with heparin sulfate proteoglycans, mediating signal transduction pathways such as RAS-RAF-MAPK, PI3K-AKT, signal transducers, transcription activators, and phospholipase Cγ, and participating 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, mutation of FGFR2 receptors, and conversion of its subtypes, can induce canceration of normal cells, such as FGFR2 single nucleotide polymorphisms in breast cancer cells, overexpression of FGFR2-IIIb and its ligands in pancreatic cancer cells, etc. Currently, the drugs approved for this target, such as erdafitinib from Johnson & Johnson, pemigatinib from Incyte, and infigratinib from BridgeBio Pharma, are all small molecule inhibitors. There are few large molecule antibody drugs under development, such as bemarituzumab monoclonal antibody targeting FGFR2 for the treatment of HER2-negative locally advanced or metastatic gastric cancer and gastroesophageal junction cancer, which is still in the clinical development stage.

[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 includes an extracellular IgV domain, a transmembrane region, and an intracellular tail. The intracellular tail contains two phosphorylation sites of the inhibitory and activation motifs of the immune receptor tyrosine, which can 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 E3-ubiquitin ligases CBL-b and c-CBL, triggering downregulation of immune cell receptors, inhibiting their activation and cytokine release, or completely initiating programmed death of immune cells. High expression of PD-L1 and PD-L2 has been found in a variety of solid tumors and hematological malignancies, indicating that tumor cells use the immunosuppressive response mediated by PD-1 and its ligands to enable tumor cells to escape immune function. In 2018, the world's first PD-1 inhibitor Nivolumab was approved for marketing in China for the second-line treatment of advanced non-small cell lung cancer with wild-type EGFR / ALK driver genes. Subsequently, the first domestically produced PD-1 antibody drug Sintilimab for the treatment of Hodgkin's lymphoma was also approved for marketing. With more in-depth clinical research on PD-1, PD-1 drugs have made major breakthroughs in the field of tumor treatment and have become the most popular tumor immunotherapy method in recent years. Approved indications worldwide include melanoma, lymphoma, lung cancer, gastric cancer, liver cancer, colorectal cancer, etc.

[0004] Bispecific antibodies (BsAb), also known as bifunctional antibodies, are specific drugs that simultaneously target two different antigens or different epitopes of the same antigen. BsAb can act on tumor cells with immune cells and virus molecules, enhancing the killing effect on target cells. It can also bind to different antigens of tumor cells at the same time, enhancing its binding specificity and reducing off-target effects. Bispecific antibodies have broadened the application field of antibody drugs and provided new research ideas for tumor immunity.

[0005] However, there is currently a lack of satisfactory bispecific antibodies against 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. Summary of the invention

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

[0007] The bispecific antibody provided by the present invention can specifically bind to FGFR2 and PD-1 at the same time, inhibit the proliferation of FGFR2 target cells and kill target cells through the ADCC effect, while protecting T cells from normal immune function and eliminating target cells. The purpose of the present invention is also to provide a polynucleotide molecule encoding the bispecific antibody; to provide an expression vector comprising the molecule; to provide a host cell comprising the expression vector; to provide a method for preparing the bispecific antibody; to provide a pharmaceutical composition comprising the bispecific antibody; to provide an immunoconjugate comprising the bispecific antibody; to provide the use of the bispecific antibody or the pharmaceutical composition in the preparation of a drug for treating cancer or tumors; to provide a method for treating cancer or tumors with the fusion protein, the pharmaceutical composition or the immunoconjugate.

[0008] In a first aspect of the present invention, a bispecific antibody is provided, comprising:

[0009] A first antigen binding domain D1; and

[0010] The second antigen binding domain D2,

[0011] Wherein, the D1 specifically binds to the target molecule FGFR2 protein,

[0012] The D2 specifically binds to the target molecule PD-1 protein.

[0013] In another preferred embodiment, the D1 is an anti-FGFR2 antibody or an antigen-binding fragment thereof,

[0014] In another preferred embodiment, the D2 is an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0015] In another preferred embodiment, the antibodies include: animal-derived antibodies (such as mouse-derived antibodies), chimeric antibodies, and humanized antibodies.

[0016] In another preferred embodiment, the antibody fragment comprises a heavy chain variable region and a light chain variable region.

[0017] In another preferred embodiment, the antibody fragment comprises a single-chain variable region fragment (scFv) and a double-chain variable region fragment (dcFv).

[0018] In another preferred embodiment, the D1 and the D2 are connected via a connector.

[0019] 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.

[0020] In another preferred example, 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.

[0021] 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.

[0022] In another preferred embodiment, the bispecific antibody is a homologous or heterologous dimer.

[0023] In another preferred embodiment, the bispecific antibody comprises a monomer having a structure shown in Formula I or Formula II from the N-terminus to the C-terminus:

[0024]

[0025]

[0026] in,

[0027] V L A represents a light chain variable region of an anti-FGFR2 antibody or an antigen-binding fragment thereof;

[0028] VH A represents the heavy chain variable region of an anti-FGFR2 antibody or an antigen-binding fragment thereof;

[0029] V L B represents a light chain variable region of an anti-PD-1 antibody or an antigen-binding fragment thereof;

[0030] VH B represents a heavy chain variable region of an anti-PD-1 antibody or an antigen-binding fragment thereof;

[0031] CH represents heavy chain constant region;

[0032] CL represents the light chain constant region;

[0033] L1 and L2 are each independently a bond or a linker;

[0034] “~” represents a disulfide bond or a covalent bond;

[0035] “-” represents a peptide bond;

[0036] The bispecific antibody has the activity of simultaneously binding to FGFR2 and PD-1.

[0037] 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.

[0038] 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.

[0039] 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).

[0040] In another preferred embodiment, the constant region of the IgG antibody contains at least one amino acid mutation.

[0041] In another preferred embodiment, D1 is an IgG antibody, and D2 is a scFv; more preferably, D2 is connected to the N-terminus or C-terminus of D1; and even more preferably, D2 is connected to the heavy chain of D1.

[0042] In another preferred embodiment, the D2 comprises one, two, three or more anti-PD-1 scFvs.

[0043] In another preferred embodiment, the scFv comprises a VH-L1-VL structure or a VL-L1-VH structure from the N-terminus to the C-terminus.

[0044] 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.

[0045] In another preferred example, the amino acid sequence of the linker L1 is shown as SEQ ID NO.17.

[0046] In another preferred example, the amino acid sequence of the linker L2 is shown as SEQ ID NO.18.

[0047] 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 through a linker L2.

[0048] 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:

[0049] 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

[0050] 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,

[0051] d) A polypeptide derived from a) or b) which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence in a) or b) and has both anti-FGFR2 activity and anti-PD-1 activity.

[0052] 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.

[0053] In another preferred embodiment, the derivative of the bispecific antibody is a polypeptide of the bispecific antibody that has undergone one or several amino acid mutations (after amino acid deletion, insertion and / or substitution) and retains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0054] In another preferred embodiment, the amino acid mutation is a conservative amino acid substitution.

[0055] In another preferred embodiment, the amino acid mutation is located in the framework region or the constant region.

[0056] 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 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, HCDR3 as shown in SEQ ID NO.3; and

[0057] 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.

[0058] 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 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, HCDR3 as shown in SEQ ID NO.9; and

[0059] 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.

[0060] 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.

[0061] In another preferred example, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region 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.

[0062] In another preferred embodiment, the heavy chain constant region comprises an Fc fragment, and the Fc fragment is derived from IgG1 or IgG4.

[0063] In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG1.

[0064] In another preferred embodiment, the Fc fragment contains at least one amino acid mutation.

[0065] In another preferred embodiment, the mutation is used to improve the ADCC effect of the bispecific antibody.

[0066] In another preferred embodiment, the Fc fragment derived from IgG1 has a mutation selected from the following group:

[0067] S239D, I332E, A330L, G236A, preferably S239D, I332E.

[0068] In another preferred example, the heavy chain constant region includes CH1, CH2, and CH3.

[0069] In another preferred example, 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.

[0070] In another preferred example, the heavy chain constant region amino acid sequence is shown as SEQ ID NO.23.

[0071] In another preferred example, the heavy chain constant region comprises an amino acid mutation selected from the following group: S239D, I332E.

[0072] 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, 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;

[0073] Preferably, the variable region comprises at least one amino acid mutation.

[0074] In another preferred embodiment, the heavy chain constant region of the anti-FGFR antibody comprises an amino acid mutation selected from the following group:

[0075] a) the heavy chain constant region has an amino acid mutation at position 239; preferably S293D; and / or

[0076] b) the heavy chain constant region has an amino acid mutation at position 332; preferably I332E.

[0077] In another preferred example, 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.

[0078] 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.

[0079] In another preferred example, 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.

[0080] In the 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.

[0081] In the third aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide molecule as described in the second aspect of the present invention.

[0082] 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.

[0083] 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:

[0084] a) culturing the host cell according to the fourth aspect of the present invention under expression conditions, thereby expressing the bispecific antibody;

[0085] b) isolating and purifying the bispecific antibody described in step a).

[0086] In the 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.

[0087] 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 provided in the sixth aspect of the present invention in the preparation of a drug for cancer or tumor;

[0088] Preferably, the cancer or tumor is a FGFR2 positive cancer or tumor.

[0089] In another preferred embodiment, the cancer or tumor is selected from: lung cancer, bone cancer, gastric 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, urethral 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 pelvic 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, acute leukemia, or a combination thereof.

[0090] In an eighth aspect of the present invention, an immunoconjugate is provided, comprising:

[0091] a) the bispecific antibody according to any one of the first aspects of the present invention; and

[0092] b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide or an enzyme, and the like.

[0093] In another preferred embodiment, the conjugate portion 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.

[0094] In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate.

[0095] In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors or cancers.

[0096] In the ninth aspect of the present invention, a method for treating cancer or tumor is provided, the method comprising administering the bispecific antibody described in the first aspect of the present invention, the pharmaceutical composition described in the sixth aspect of the present invention, or the immunoconjugate described in the eighth aspect of the present invention to a subject in need thereof.

[0097] In another preferred embodiment, the method further comprises administering the drug in combination with other anti-tumor drugs.

[0098] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1A A schematic diagram of the structure of anti-FGFR2 / PD-1 dual antibody a or b is shown.

[0100] Figure 1B A schematic diagram of the structure of the anti-FGFR2 / PD-1 dual antibody c is shown.

[0101] Figure 1C A schematic diagram of the structure of the anti-FGFR2 / PD-1 dual antibody d is shown.

[0102] Figure 2A The UPLC-SEC detection spectrum of anti-FGFR2 / PD-1 dual antibody a is shown.

[0103] Figure 2B The UPLC-SEC detection spectrum of anti-FGFR2 / PD-1 dual antibody b is shown.

[0104] Figure 2C The UPLC-SEC detection spectrum of anti-FGFR2 / PD-1 dual antibody c is shown.

[0105] Figure 2D The UPLC-SEC detection spectrum of anti-FGFR2 / PD-1 dual antibody d is shown.

[0106] Figure 3A The mass spectrum of the N-deglycosylated intact molecular weight of anti-FGFR2 / PD-1 dual antibody a is shown.

[0107] Figure 3B The mass spectrum of the N-deglycosylated intact molecular weight of the anti-FGFR2 / PD-1 dual antibody b is shown.

[0108] Figure 4A The DSC detection spectrum of anti-FGFR2 / PD-1 dual antibody a is shown.

[0109] Figure 4B The DSC detection spectrum of anti-FGFR2 / PD-1 dual antibody b is shown.

[0110] Figure 5A ELISA detection of the binding of anti-FGFR2 / PD-1 dual antibodies a and b to FGFR2 is shown.

[0111] Figure 5B ELISA detection of the binding of anti-FGFR2 / PD-1 dual antibodies c, d to FGFR2 is shown.

[0112] Figure 5C ELISA detection of the binding of anti-FGFR2 / PD-1 dual antibodies a and b to PD-1 is shown.

[0113] Figure 5D ELISA detection of the binding of anti-FGFR2 / PD-1 dual antibodies a and d to PD-1 is shown.

[0114] Fig. 6A The binding activity of anti-FGFR2 / PD-1 dual antibodies a and b to Jurkat-PD-1-NFAT-luc cells is shown.

[0115] Figure 6B The activity of anti-FGFR2 / PD-1 dual antibodies a and b in blocking PD-1 / PD-L1 binding was shown.

[0116] Fig. 7A The ADCC effect of anti-FGFR2 / PD-1 dual antibodies a and b on SNU-16 cells is shown.

[0117] Figure 7B The ADCC effect of anti-FGFR2 / PD-1 dual antibodies a and b on T cells is shown.

[0118] Figure 8 The results showed that anti-FGFR2 / PD-1 dual antibodies a and b had inhibitory effects on FGF1-induced proliferation of Ba / F3-FGFR2IIIb cells.

[0119] Fig.9A The anti-tumor effect of anti-FGFR2 / PD-1 dual antibody b in the MC38 transplanted tumor model was shown.

[0120] Fig. 9B The toxic and side effects of anti-FGFR2 / PD-1 dual antibody b on mice in the MC38 transplant tumor model were shown.

[0121] Fig. 10A The anti-tumor effects of anti-FGFR2 / PD-1 dual antibodies a and b in the SNU-16 transplanted tumor model were shown.

[0122] Fig. 10B The toxic and side effects of anti-FGFR2 / PD-1 dual antibodies a and b on mice in the SNU-16 transplanted tumor model are shown. DETAILED DESCRIPTION

[0123] After extensive and in-depth research, the inventors have obtained a new bispecific antibody targeting the tumor cell surface molecule FGFR2 and the immune cell surface molecule PD-1, which can maintain the activity of the antibodies at both ends and can bind to FGFR2 and PD-1 antigens at the same time. The bispecific antibody of the present invention can be used to achieve T cell-mediated immune response and kill tumor cells expressing FGFR2 through ADCC effect. Therefore, the bispecific antibody of the present invention can be developed as an anti-tumor drug with superior efficacy. The present invention was completed on this basis.

[0124] the term

[0125] 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.

[0126] 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.

[0127] 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 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, and 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, and the light chain constant region includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.

[0128] The "immunoglobulin antibody IgG" described in the present invention is a molecule of about 150 kDa, which is composed of four peptide chains, containing two identical γ heavy chains of about 50 kDa, and two identical light chains of about 25 kDa, thereby having a tetrameric quaternary structure. The two heavy chains are interconnected by a disulfide bond and each is connected to a light chain. The resulting tetramer has two identical halves, which form a fork-shaped or Y-like shape, and each end of the fork contains an identical antigen binding site. IgG antibodies can be divided into multiple subclasses (e.g., IgG1, 2, 3, 4) based on slight differences in the amino acid sequence in the constant region of the heavy chain.

[0129] In the present invention, the term "bispecific antibody (or bispecific antibody)" refers to an antibody molecule that can specifically bind to two antigens (targets) or two epitopes at the same time. According to symmetry, bispecific antibodies can be divided into structurally symmetrical and asymmetrical molecules. According to the number of binding sites, bispecific antibodies can be divided into divalent, trivalent, tetravalent and multivalent molecules.

[0130] 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 naturally occurring mutations that may exist. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture 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 advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture and will not be contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population, and this should not be interpreted as requiring any special method to produce the antibody.

[0131] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment or a single Fv fragment, etc., which 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 expression "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, mini-antibodies, nanobodies (e.g., monovalent nanobodies and bivalent nanobodies, etc.), small modular immunopharmaceuticals, and shark variable IgNAR domains, etc.

[0132] In the present invention, the terms "Fab" and "Fc" refer to the fact that papain can cleave antibodies into two identical Fab segments and one Fc segment. The Fab segment 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 segment is the crystallizable fragment (Fc), which is composed of the CH2 and CH3 domains of the antibody. The Fc segment 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') parts linked together by disulfide bonds.

[0133] In the present invention, the term "scFv" or "single chain variable region fragment scFv" is a single chain antibody (single chain antibody fragment, 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.

[0134] In the present invention, the term "Fv fragment" or "Fv antibody" refers to a minimum antibody fragment containing the variable region of the heavy chain and the variable region of the light chain of the antibody, but without the constant region, and having all the antigen binding sites. Generally, the Fv fragment also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.

[0135] In the present invention, the term "variable" means that some parts of the variable region in the antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conservative part of the variable region is called the framework region (FR). The variable region of the natural heavy chain and light chain each contains four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region 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, Volume I, pp. 647-669 (1991)).

[0136] As used herein, the term "framework region FR" refers to the amino acid sequence inserted between CDRs, that is, refers to those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. The light chain and heavy chain of an immunoglobulin each have four FRs, which are 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 therefore 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 therefore be expressed 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 regions 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 substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

[0137] As used herein, the term "linker" refers to one or more amino acid residues inserted into the immunoglobulin domain to provide sufficient mobility for the domains of the light chain and the 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 a single-chain antibody (scFv), and L2 connects the scFv between the CH1 and CH2 of the antibody heavy chain, or directly connects to the heavy chain of the antibody through L2 (such as the N-terminus or C-terminus of the antibody heavy chain). Suitable examples of linkers include glycine (Gly) or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker may vary with the type of secondary structural elements that need to be implemented in the linker.

[0138] 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 the antigen to which it is directed. -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The 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, the binding affinity of the antibody to the antigen is measured in a BIACORE instrument using surface plasmon resonance (SPR) or the relative affinity of the antibody to the antigen is measured using ELISA.

[0139] 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.

[0140] 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.

[0141] As used herein, the bispecific antibodies of the present invention also include conservative variants thereof, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0142] Table A

[0143]

[0144]

[0145] Bispecific Antibodies

[0146] 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.

[0147] The bispecific antibody of the present invention may be a dimer, trimer or multimer, preferably a homologous or heterologous dimer. The anti-FGFR2 or anti-PD-1 antibody portion of the bispecific antibody of the present invention may include one or more antibodies or antigen-binding fragments thereof, preferably 1, 2, 3, 4, 5 or 6. The sequence of the present invention adopts the Kabat system numbering rules.

[0148] 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 two antigens, FGFR2 and PD-1, respectively.

[0149] Encoding nucleic acid and expression vector

[0150] Another aspect of the present invention provides a polynucleotide molecule encoding the bispecific antibody described above. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

[0151] 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.

[0152] Those skilled in the art will appreciate that the nucleotide sequence encoding the amino acid sequence of the bispecific antibody described above may be appropriately replaced, deleted, altered, inserted or added to provide a polynucleotide homologue. The polynucleotide homologues of the present invention may be prepared by replacing, deleting or adding one or more bases of the gene encoding the bispecific antibody within the range of maintaining the antibody activity.

[0153] Another aspect of the present invention provides an expression vector, which contains the above-mentioned polynucleotide molecule.

[0154] These vectors can be used to transform into appropriate host cells to enable them to express proteins. The expression vector is a conventional expression vector in the art, which refers to an expression vector 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 vector can be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Molecular Cloning: A Laboratory Manual, Second 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, Second Edition, edited by Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.4, pcDNA3.1 / ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO1.0, and more preferably pcDNA3.4.

[0155] The present invention further provides a host cell, wherein the host cell contains the above expression vector.

[0156] The host cell described in the present invention is any conventional host cell in the art, as long as the above-mentioned recombinant expression vector can be stably replicated by itself and the nucleotide 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, and 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.

[0157] Preparation method

[0158] The host cell culture method and the antibody separation and purification method described in the present invention are conventional methods in the art. For specific operation methods, please refer to the corresponding cell culture technology manual and antibody separation and purification technology manual. The preparation method of the anti-FGFR2 / PD-1 bispecific antibody disclosed in the present invention includes: culturing the above-mentioned host cells under expression conditions to express bispecific antibodies that can specifically bind to FGFR2 and PD-1; and separating and purifying the anti-FGFR2 / PD-1 bispecific antibody. Using the above method, the recombinant protein can be purified into a substantially uniform substance.

[0159] The anti-FGFR2 / PD-1 bispecific antibody disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high salt buffer, pH change, etc. can be used to elute the anti-FGFR2 / PD-1 bispecific antibody bound to the affinity column. The inventors of the present invention conducted a detection experiment on the obtained 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 a high affinity.

[0160] Pharmaceutical composition

[0161] Another aspect of the present invention provides a composition, preferably, the composition is a pharmaceutical composition.

[0162] In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to more stably exert the therapeutic effect. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation).

[0163] 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 injection), 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, water injection, lyophilized preparation, etc. commonly used in the pharmaceutical field.

[0164] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned bispecific antibody of the present invention (or its conjugate) 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 preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 micrograms / kg body weight to 50 mg / kg body weight per day. In addition, the bispecific antibody of the present invention can also be used with other therapeutic agents.

[0165] When using the pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal. The safe and effective amount is usually 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 10 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0166] application

[0167] Another aspect of the present invention provides the use of the above-mentioned bispecific antibody capable of specifically binding to FGFR2 and PD-1, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating cancer or tumor.

[0168] 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.

[0169] The tumors targeted by the drug of the present invention preferably include but are not limited to: lung cancer, bone cancer, gastric 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, urethral cancer, penis cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvic 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.

[0170] 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, disease characteristics and severity, and route of administration. The results of animal experiments and various conditions can be referred to, and the total dosage cannot exceed a certain range. Specifically, the dosage for intravenous injection is 1-1800 mg / day.

[0171] The bispecific antibodies and compositions of the present invention can also be administered in combination with other anti-tumor drugs to achieve the purpose of more effective tumor treatment. 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, etc.; thymidine synthetase inhibitors such as fluorouracils (5-fluorouracil, capecitabine), etc.; purine nucleoside synthetase inhibitors such as 6-mercaptopurine, etc.; ribonucleotide reductase inhibitors such as hydroxycarbamide, etc.; DNA polymerase inhibitors such as cytosinearabinoside and gemcitabine, etc.; 3) Drugs that act on microtubules: docetaxel, vincristine, vinorelbine, podophyllotoxin, homoharringtonine, etc.; 2. Hormonal drugs: anti-estrogens 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 agonist / antagonist: Noradrenaline, Enanton, etc. 5. Biological response modifier drugs: 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 thereof disclosed in the present invention can be used in combination with one or a combination of the above-mentioned anti-tumor drugs.

[0172] The main advantages of the present invention include

[0173] The positive improvement effects of the anti-FGFR2 / PD-1 bispecific antibody of the present invention are:

[0174] (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;

[0175] (2) High specificity and good safety;

[0176] (3) High expression level;

[0177] (4)Stable structure.

[0178] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually carried out according to 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.

[0179] The experimental materials and sources used in the following examples and the preparation methods of the experimental reagents are described in detail as follows. Experimental Materials:

[0180] Competent cells: Brand: Sangon, catalog number: B528412.

[0181] 293F cells: Brand: GIBCO, Product No.: R79007.

[0182] hPBMC: Brand: Auscell, catalog number: FPB004F-C-MLR.

[0183] 0.45μm filter: brand Millipore, product number SLHV013SL.

[0184] SNU-16 cells: Brand: Nanjing Kebai, catalog number: CBP60502.

[0185] Jurkat-PD1-NFAT-luc cells: brand: Promega, product number: J1252.

[0186] CHO-K1-PD-L1 cells: brand: Promega, product number: J1252.

[0187] Ba / F3 cells: Brand: Seli, Product No.: SLB-CL0019A.

[0188] 293FT cells: brand: GIBCO, product number: R70007.

[0189] Hitrap Mabselect Sure affinity chromatography column: brand: Hitrap Mabselect Sure, product number: 11003493.

[0190] Waters MassPREP TM Micro Desalting Column: Brand: Waters, Model: 186004032. Experimental reagents:

[0191] Endotoxin-free plasmid extraction kit: brand Tiangen, product number DP117.

[0192] DNA purification and recovery kit: brand Tiangen, product number DP214.

[0193] ClonExpressTM II One Step Cloning Kit: brand: ClonExpress, product number: C112.

[0194] HS DNA Polymerase: brand takara, product number R010B.

[0195] Coating solution: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, add double distilled water to make up to 1 L.

[0196] PBST: PBS + 0.05% Tween 20.

[0197] Tween 20: Brand Aladdin, item number T104863.

[0198] ELISA blocking solution: PBST + 1% BSA.

[0199] BSA: Brand: BSA, item number: A60332.

[0200] Human-FGFR2-his protein: Brand: Kaixia, product number: FGR-HM1BD.

[0201] HRP-labeled goat anti-human Fc antibody: brand: Biolon, product number: BF03031.

[0202] TMB: Brand: BD Biosciences, catalog number 555214.

[0203] Stop solution: 2M sulfuric acid solution.

[0204] 0.25% Pancreatin-EDTA: Brand: GIBCO, Product No. 25200-072.

[0205] 1640 complete culture medium: RPMI1640 culture medium + 10% FBS + 1% Pen Strep + 1% sodium pyruvate. 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. DMEM high glucose medium: brand GIBCO, item number 11965-092.

[0210] GlutaMAX: Brand: GIBCO, item number: 35050-061.

[0211] Luminescent Cell Viability Assay: Promega, catalog number G7572. Goat anti-human Fab-FITC: Thermo Fisher, catalog number MA1-10379.

[0212] Glycosidase F: Brand: NEB, Product No. P0704L.

[0213] Experimental instruments:

[0214] Mastercycler nexus PCR instrument: purchased from Eppendorf.

[0215] Hitrap Mabselect Sure column: purchased from Cytiva.

[0216] HiLoad 26 / 600 Superdex 200 pg column: purchased from Cytiva.

[0217] SpectraMax i3x microplate reader: purchased from Molecular Devices.

[0218] SpectraMaxM5 microplate reader: purchased from Molecular Devices.

[0219] Coulter CytoFLEX flow cytometer: purchased from Beckman.

[0220] FD-0007 fully automatic capillary differential scanning calorimeter: purchased from Malvern.

[0221] LC / MS liquid chromatography-mass spectrometry instrument: purchased from Waters.

[0222] The sequence of the present invention is shown in Table B below:

[0223]

[0224]

[0225]

[0226]

[0227] Example 1. Construction of anti-FGFR2 / PD-1 dual antibody molecule

[0228] The present invention uses a tandem approach of anti-FGFR2 monoclonal antibody IgG and anti-PD-1 monoclonal antibody scFv to construct an anti-FGFR2 / PD-1 bispecific antibody, the structure of which is as follows: Figure 1A shown.

[0229] Among them, the anti-FGFR2 monoclonal antibody sequence in the anti-FGFR2 / PD-1 bispecific antibody is derived from the humanized monoclonal antibody disclosed in the CN102905723A patent, which serves as the anti-FGFR2 monoclonal antibody positive control. The anti-PD-1 monoclonal antibody sequence in the anti-FGFR2 / PD-1 bispecific antibody is derived from the humanized monoclonal antibody disclosed in the CN201710054783.5 patent, which serves as the anti-PD-1 monoclonal antibody positive control.

[0230] 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 a peptide linker L1 (SEQ ID NO.17) to obtain VL-L1-VH, i.e. PD-1-scFv-VLVH (SEQ ID NO.27).

[0231] 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 by 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.

[0232] The corresponding sites on human IgG1 were selected to mutate the heavy chain constant region of the anti-FGFR2 monoclonal antibody. After screening, S239D and I332E were selected to obtain the anti-FGFR2 monoclonal antibody heavy chain HC-S239D-I332E.

[0233] 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.

[0234] 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.

[0235] 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 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 peptide linker L2 (SEQ ID NO.18) to obtain the heavy chain of the anti-FGFR2 / PD-1 dual antibody c, and the light chain of the anti-PD-1 monoclonal antibody remained unchanged, with the structure as shown Figure 1B shown.

[0236] 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 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 peptide linker L2 (SEQ ID NO.18) to obtain the heavy chain of the anti-FGFR2 / PD-1 dual antibody d, and the light chain of the anti-FGFR2 monoclonal antibody (SEQ ID NO.28) remained unchanged, with the structure as shown Figure 1C shown.

[0237] In order to improve the expression efficiency of antibody molecules in CHO cells, the nucleic acid sequence of the anti-FGFR2-PD1 bispecific antibody molecule was codon optimized, mainly considering factors such as codon preference, GC content, secondary structure of mRNA, and repeat sequence. The nucleic acid sequence of the antibody was synthesized by Sangon Biotech Co., Ltd. The amino acid and nucleic acid sequences are shown in the sequence table.

[0238] Example 2. Expression and purification of anti-FGFR2 / PD-1 dual antibody

[0239] The DNA fragments of the heavy chain and light chain of the anti-FGFR2 / PD-1 bispecific antibody a and b were subcloned into the vector pcDNA3.4, 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 and filtered through a 0.22μm filter membrane and then loaded onto a Hitrap Mabselect Sure affinity chromatography column. The protein was eluted in one step with 100mM citric acid and pH 3.5 elution buffer, and the target sample was recovered and dialyzed into PBS at pH 7.4. The purified protein was detected by UPLC-SEC.

[0240] The test results of anti-FGFR2 / PD-1 dual antibody a, b, c and d proteins are as follows Figure 2A , 2B , 2C and 2D, where the antibody protein status is uniform, the monomer purity of anti-FGFR2 / PD-1 bispecific antibody a is more than 93%, the monomer purity of anti-FGFR2 / PD-1 bispecific antibody b is slightly higher than that of bispecific antibody a, and the monomer purity is more than 96%, and the purity of anti-FGFR2 / PD-1 bispecific antibodies c and d is slightly lower than that of bispecific antibody a, and the monomer purity is above 86% and 88%.

[0241] Example 3. Detection of intact molecular weight of anti-FGFR2 / PD-1 dual antibody after N-glycosylation

[0242] 1 mg of each anti-FGFR2 / PD-1 bispecific antibody a and b were taken, and the samples were desalted into ultrapure water by ultrafiltration, and the samples were concentrated to more than 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 samples were purified by using a Waters MassPREP TM The LC / MS liquid chromatography-mass spectrometry of the Micro Desalting Column was used to detect the desugared intact molecular weight of the anti-FGFR2 / PD-1 bispecific antibody a and b samples.

[0243] The experimental results are as follows Figure 3A and 3BAs shown, the theoretical desugared molecular weight of the anti-FGFR2 / PD-1 bispecific antibody a is 196892.05 Da, and the measured molecular weight after mass spectrometry analysis is 196896.98 Da; the theoretical desugared molecular weight of the anti-FGFR2 / PD-1 bispecific antibody b is 196979.98 Da, and the measured molecular weight 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.

[0244] Example 4. Thermal stability determination of anti-FGFR2 / PD-1 dual antibody

[0245] Differential Scanning Calorimeter (DSC) was used to identify the change in the molar heat capacity of the anti-FGFR2 / PD-1 dual antibody with temperature to evaluate its thermal stability. The protein solutions of anti-FGFR2 / PD-1 dual antibodies a and b were diluted to 0.5 mg / mL with 1× PBS (pH 7.4) buffer, and 500 μL was taken for detection using an automatic capillary differential scanning calorimeter to obtain the DSC spectrum as shown below: Figure 4A and 4B shown.

[0246] The experimental results showed that with the increase of temperature, the Tm1 and Tm2 of FGFR2 / PD-1 dual antibody a were 62.74℃ and 79.08℃, respectively, and the Tm1 and Tm2 of FGFR2 / PD-1 dual antibody b were 60.56℃ and 81.01℃, respectively. Both anti-FGFR2-PD1 dual antibodies a and b had the first endothermic peak at about 60℃ and the second endothermic peak at about 80℃, indicating that the thermal stability of anti-FGFR2 / PD-1 dual antibody a and b proteins is comparable.

[0247] Example 5. Determination of affinity of the double antibody for the antigen by enzyme-linked immunosorbent assay (ELISA)

[0248] 5.1 Detection of the affinity of anti-FGFR2 / PD-1 dual antibodies to FGFR2

[0249] Dilute the recombinant Human-FGFR2-His protein to 200ng / ml with the coating solution, add 100μl / well to the ELISA plate, and place at room temperature for 2 hours or overnight at 4 degrees Celsius. Remove the coating solution (use absorbent paper to remove residual droplets), add 200μl / well of blocking solution to the ELISA plate, and place at room temperature for 1-2 hours. Remove the blocking solution (use absorbent paper to remove residual droplets), and then dilute the anti-FGFR2 / PD-1 dual antibodies a and b, as well as the anti-FGFR2 monoclonal antibody to 10μg / ml with the blocking solution, and dilute four times to form 12 concentration gradients (highest concentration 10μg / ml, lowest concentration 0.002ng / ml), and add 100μl / well to the blocked ELISA plate in turn, and place 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 with blocking solution at 1:3000, 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 colorimetric solution at 100 μl / well, place at room temperature away from light for 5 minutes, add stop solution at 70 μl / well to stop the substrate colorimetric reaction, read the OD value at 450 nm with an ELISA reader, analyze the data with GraphPad, plot and calculate EC 50 .

[0250] The experimental results are as follows Figure 5A As shown, the EC of anti-FGFR2 / PD-1 bispecific antibody molecules a, b and positive control anti-FGFR2 monoclonal antibody binding to Human-FGFR2-His 50 (Unit: nM) were 0.0568, 0.0512 and 0.0392 respectively, and the affinities of the three were comparable.

[0251] 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 As shown, EC 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.

[0252] 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 molecules, 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.

[0253] 5.2 Detection of the affinity of anti-FGFR2 / PD-1 dual antibodies to PD-1

[0254] Dilute the recombinant Human-PD-1-His protein to 200ng / ml with the coating solution, add 100μl / well to the ELISA plate, and place at room temperature for 2 hours or overnight at 4 degrees Celsius. Remove the coating solution (use absorbent paper to remove residual droplets), add 200μl / well of blocking solution to the ELISA plate, and place at room temperature for 1-2 hours. Remove the blocking solution (use absorbent paper to remove residual droplets), and then dilute the anti-FGFR2 / PD-1 dual antibodies a and b, and the anti-PD-1 monoclonal antibody to 10μg / ml with the blocking solution, and dilute four times to form 12 concentration gradients (highest concentration 10μg / ml, lowest concentration 0.002ng / ml), and add 100μl / well to the blocked ELISA plate in turn, and place 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 with blocking solution at 1:3000, 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 away from light for 5 minutes, add stop solution at 70 μl / well to stop the substrate color reaction, read the OD value at 450nm with an ELISA reader, analyze the data with GraphPad, plot and calculate EC 50 .

[0255] The experimental results are as follows Figure 5C As shown, the EC of anti-FGFR2 / PD-1 bispecific antibody molecules a and b, and the positive control anti-PD-1 monoclonal antibody binding to 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 anti-PD-1 monoclonal antibodies were connected in a VL-VH manner and then linked to the heavy chain of anti-FGFR2 monoclonal antibodies to form a bispecific antibody with slightly reduced binding ability to PD-1 antigen.

[0256] The same method was used to determine the affinity of anti-FGFR2 / PD-1 bispecific antibody molecules a, d and positive control anti-PD-1 monoclonal antibody to Human-PD-1-His. The experimental results are shown in Figure 5D As shown, the EC50 (unit: nM) are 0.1599, 0.3564 and 0.0856, respectively. The affinity of anti-FGFR2 / PD-1 bispecific antibody a to Human-PD-1-His is slightly higher than that of anti-FGFR2 / PD-1 bispecific antibody d, indicating that the variable regions of the anti-PD-1 monoclonal antibody are linked in a VL-VH manner and then connected in series with the anti-FGFR2 monoclonal antibody scFv to form a bispecific antibody molecule with a higher affinity for binding to the PD-1 antigen.

[0257] Example 6. Blocking activity of anti-FGFR2 / PD-1 dual antibodies on PD-1 / PD-L1 binding

[0258] Jurkat-PD-1-NFAT-luc expressing PD-1 on the cell surface was used as the target cell. The binding affinity of anti-FGFR2 / PD-1 dual antibody to the cell was determined by flow cytometry. The cell density was adjusted to 1.5×10 with DPBS containing 1% BSA. 6 cells / mL, 100μL / well added to 96-well plate. Anti-FGFR2 / PD-1 dual antibodies a, b and positive control anti-PD-1 monoclonal antibody were diluted to 200nM with DPBS containing 1% BSA, diluted 4 times into 12 concentration gradients, 100μL / well added to 96-well plate, and mixed evenly with Jurkat-PD-1-NFAT-luc cells. Incubate at 4℃ for 1h. Centrifuge at 400g for 5min and discard the supernatant. Wash twice with pre-cooled DPBS to remove unbound antibodies to be tested. Add 100μL of goat anti-human Fab-FITC (800:1 dilution) to each well and incubate at 4℃ for 1h. Centrifuge at 400g for 5min 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.

[0259] The experimental results are as follows Fig. 6A As shown, 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 50They are 0.4823, 0.6344 and 0.1046 nM respectively, indicating that compared with the anti-PD-1 monoclonal antibody, the activity of anti-FGFR2 / PD-1 dual antibodies a and b in binding to cell surface PD-1 protein is relatively weaker.

[0260] CHO-K1-PD-L1 cells were diluted to 4 × 10 5 cells / mL, 100 μL / well was added to a 3903 model 96-well plate, placed in an incubator at 37°C and 5% CO2 for overnight incubation, and the 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 with assay medium (RPMI 1640 + 1% FBS), and then 5-fold gradient dilutions were made into 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, 40μL / well was added to a 96-well plate containing CHO-K1-PD-L1 cells, placed in an incubator at 37°C, 5% CO2 for 6 hours, then the 96-well plate was taken out and equilibrated to room temperature, 80μL of Bio-Glo detection reagent was added to each well, incubated at room temperature for 5-10 minutes, and the luminescence value was read with spectramax i3. All data were obtained in duplicate wells, and the average of the obtained signal values ​​was fitted and the curve was drawn using the 4-parameter method.

[0261] The experimental results are as follows Figure 6B As shown, the IC of anti-FGFR2 / PD-1 dual antibody molecules a and b blocking the binding of PD-1 and PD-L1 50 The IC values ​​of anti-PD-1 monoclonal antibodies blocking the binding of PD1 and PD-L1 are 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.

[0262] Example 7. ADCC effect of anti-FGFR2 / PD-1 dual antibody on SNU-16 cells and T cells

[0263] In this experiment, human gastric cancer cells SNU-16 with high expression of FGFR2 on the cell surface 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.

[0264] 7.1 ADCC effect of dual antibodies on SNU-16 tumor cells

[0265] SNU-16 cells were taken and adjusted to 2×10 cells / mL using RPMI 1640 medium containing 5% FBS. 5 cells / mL, 50μL / 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, 50μL 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 human NK Cell IsolationKit (Miltenyi Biotec, #130-092-657). The sorted NK cells were adjusted to 4×10 with RPMI 1640 medium containing 5% FBS. 5 cells / mL, 100 μL / well was added to the above wells and incubated in a 37°C, 5% CO2 incubator for 24 h. TM LDH Cytotoxicity Assay Kit (invitrogen, #C20300) instructions for LDH detection. Specifically as follows: Add 20 μL of lysis solution to the well used to detect the maximum amount of LDH release, tap to mix, and incubate in a 37°C, 5% CO2 incubator for 45 minutes. Afterwards, take 50 μL of each 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 stop solution, tap to mix, and use an enzyme reader to detect the OD values ​​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.

[0266] The experimental results are as follows Fig. 7AAs shown in the figure, 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; 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, and the ADCC effect levels are comparable.

[0267] Therefore, after the S239D-I332E point mutation was performed in 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 ( Fig. 7A ).

[0268] 7.2 ADCC effect of bispecific antibodies on T cells

[0269] Jurkat-PD-1-NFAT-luc expressing PD-1 on the cell surface was used as the target cell to detect the ADCC effect. The method was as described above. The difference was that the antibodies used were anti-FGFR2 / PD-1 dual antibodies a and b, positive control anti-MHC-I monoclonal antibody, and negative control anti-PD-1 monoclonal antibody. The initial concentration of the antibody was 50 nM, and it was diluted 4 times in stages and added to the wells containing cells. According to CyQUANT TM LDH was detected according to the instructions of LDH Cytotoxicity Assay Kit (invitrogen, #C20300) and data were analyzed using GraphPad Prism6.

[0270] The results are as follows Figure 7B As shown, 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.

[0271] Example 8. Inhibitory effect of anti-FGFR2 / PD-1 dual antibody on FGF1-induced proliferation of Ba / F3-FGFR2IIIb cells

[0272] After 293FT cells were digested with 0.25% trypsin-EDTA, they were diluted with DMEM complete medium and added to a 6-well plate, with 700,000 cells per well. After the cells adhered to the wall, the packaged viral vector pLVX (human FGFR2IIIb gene was inserted into the vector) was reacted with 293FT cells using Lipofectamine 3000 transfection reagent. After 48 hours, the 293FT cell culture medium was collected, centrifuged at 500g for 5 minutes and the supernatant was taken. The supernatant was filtered with a 0.45μm filter, and polybrene co-infection agent was added at 1000:1. 2ml of the supernatant was added to a T25 culture dish (250,000 Ba / F3 cells were added 24 hours ago). After 48 hours, Ba / F3 cells were collected and cultured with complete medium containing resistance until the cell growth was stable to obtain a Ba / F3-FGFR2IIIb cell population. The Ba / F3-FGFR2 cell population was diluted to 2-3 cells / ml with culture medium and added to a 96-well plate for monoclonal sorting to obtain the Ba / F3-FGFR2IIIb cell line. The FGFR2IIIb gene expression efficiency of the Ba / F3-FGFR2IIIb cell line was detected by flow cytometry, and the highest FGFR2IIIb gene expression efficiency of the Ba / F3-FGFR2IIIb cell line was 9.26% (results not shown).

[0273] Ba / F3-FGFR2IIIb cells cultured to the logarithmic phase were collected by centrifugation and diluted to 1×10 cells with complete medium. 6 cells / ml, 100 μl / well was added to a 3903 white transparent bottom 96-well plate, and placed in an incubator at 37°C and 5% CO2 for use. Anti-FGFR2 / PD-1 dual antibodies a, b and anti-FGFR2 monoclonal antibody were diluted to 500 nM with complete culture medium, and diluted 5-fold in stages, added to a 96-well plate containing Ba / F3-FGFR2IIIb cells, and incubated in an incubator at 37°C and 5% CO2 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, and added to a 96-well plate containing Ba / F3-FGFR2IIIb cells and antibodies. The plates were incubated in an incubator at 37°C and 5% CO2 for 2 h. Then, 20 μl of MTS / PMS solution was added to each well. The plates were incubated in an incubator at 37°C and 5% CO2 for 1-4 h, and then shaken to mix. The OD value at 490 nm was detected by an enzyme reader. All data were obtained in duplicate wells. The obtained signal values ​​were converted into inhibition rates, and the average was taken to draw curves using the 4-parameter method.

[0274] The experimental results are as follows Figure 8 As shown, the EC values ​​of anti-FGFR2 / PD-1 dual antibodies a, b and positive control anti-FGFR2 monoclonal antibody 50The values ​​are 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.

[0275] Example 9. Anti-tumor effect of anti-FGFR2 / PD-1 dual antibody in MC38 transplanted tumor model

[0276] The mouse colon cancer MC38 cells cultured in vitro were collected and the concentration of the cell suspension was adjusted to 2×10 7 The right flank of NOG mice was shaved, and 100 μl of cell suspension was inoculated subcutaneously under sterile conditions. When the tumor cells formed solid tumors under the skin of the mice, the diameter of the transplanted tumor was measured with a vernier caliper. When the average tumor volume grew to 50 mm 3 -100 mm 3 The animals were then randomly divided into 3 groups, with 9 animals in each group, including a blank control group, which was injected with PBS only as a control; a 10 mg / kg control dose group of anti-PD-1 monoclonal antibody; and a 13.5 mg / kg dose group of anti-FGFR2 / PD-1 bispecific antibody b, which was intraperitoneally injected twice a week for a total of 5 times. During the entire experiment, the diameter of the transplanted tumor was measured twice a week, and the weight of the mice was weighed at the same time, and the growth curve of the tumor over time and the weight curve of the mice in each group were drawn.

[0277] The experimental results are as follows Fig.9A and 9B As shown, 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, and 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 dosage 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 have less toxic side effects on mice.

[0278] 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.

[0279] Example 10. Anti-tumor effect of anti-FGFR2 / PD-1 dual antibody in SNU-16 transplanted tumor model

[0280] Human gastric cancer SNU-16 cells cultured in vitro were collected, resuspended in serum-free medium, and mixed with an equal volume of matrix gel to adjust the cell concentration to 8×10 7 / ml, and inoculated 100 μl of cell suspension subcutaneously on the right flank of NOG mice under sterile conditions. On the 8th day after tumor inoculation, hPBMC cells were revived and the cell concentration was adjusted to 2.5×10 7 / ml, except for the 3 tumor-bearing mice that were not inoculated with hPBMC cells, each of the remaining tumor-bearing mice was injected with 200 μl of hPBMC cell suspension through the tail vein, that is, each mouse was injected with 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 The animals were randomly divided into 7 groups, including a blank control group-PBS was injected only as a control; hPBMC control group-PBS diluted hPBMC cells as a control; anti-FGFR2 monoclonal antibody 5mg / kg and anti-PD-1 monoclonal antibody 5mg / kg were administered separately, two monoclonal antibody control groups; anti-FGFR2 monoclonal antibody 5mg / kg and anti-PD-1 monoclonal antibody 5mg / kg were combined, one combined drug control group; anti-FGFR2 / PD-1 dual antibody a 6.75mg / kg and anti-FGFR2 / PD-1 dual antibody b 6.75mg / kg were administered separately, two dose groups. The drug was administered twice a week for a total of 5 times. During the entire experiment, the diameter of the transplanted tumor was measured twice a week, and the mouse body weight was weighed at the same time, and the tumor growth curve and mouse body weight curve of each group were drawn over time.

[0281] The experimental results are as follows Fig. 10A , 10B As shown in Table 1, in the SNU-16 cell mixed hPBMC transplant tumor model, the inhibitory effects of anti-FGFR2 / PD-1 dual antibodies a and b on tumors had no significant difference and were equivalent to those of anti-FGFR2 monoclonal antibodies; the inhibitory effect was relatively better than that of the combination of anti-FGFR2 monoclonal antibodies and anti-PD-1 monoclonal antibodies; the inhibitory effects of anti-FGFR2 / PD-1 dual antibodies a and b on tumors were significantly better than those of anti-PD-1 monoclonal antibodies; the weight of mice did not decrease significantly after continuous administration for several days, indicating that anti-FGFR2 / PD-1 dual antibodies a and b had less toxic side effects on mice.

[0282] The results showed that in the SNU-16 cell transplant tumor model with high expression of FGFR2, the inhibitory activity of anti-FGFR2 / PD-1 dual antibodies a and b on tumors was better than that of anti-PD-1 monoclonal antibody, and both were able to significantly inhibit tumor growth, indicating that anti-FGFR2 / PD-1 dual antibodies can specifically target FGFR2 tumor cells and exert a good anti-tumor effect.

[0283] Table 1 Anti-tumor effects of anti-FGFR2 / PD-1 dual antibodies in SNU-16 transplanted tumor model

[0284]

[0285] Example 11. Stability study of anti-FGFR2 / PD-1 dual antibody b

[0286] 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.

[0287] Anti-FGFR2 / PD-1 dual antibody b was dialyzed overnight in buffer 1 (20mM acetate + 6% trehalose + 1% arginine hydrochloride + 0.2% Tween 80, pH 5.0) and buffer 2 (20mM histidine salt + 6% trehalose + 50mM sodium chloride + 0.2% Tween 80, pH 5.5), and ultrafiltration was concentrated to 5mg / mL at 4000rpm for 20min. The samples were stored at 4℃, 25℃, and 37℃ respectively.

[0288] The experimental results are shown in Table 2. Under the same treatment conditions in different buffers, anti-FGFR2 / PD-1 dual antibody b showed obvious aggregates on the 21st day when 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.

[0289] Table 2 Preliminary screening results of anti-FGFR2 / PD-1 dual antibody preparations

[0290]

[0291] discuss

[0292] From the above experiments, it can be seen that the bispecific antibody provided by the present invention can bind to FGFR2 and PD-1 at the same time, release the immune activity function of T cells by binding to PD-1, and specifically kill tumor cells expressing FGFR2 protein. Anti-FGFR2 / PD-1 dual antibody can effectively block the binding of PD-1 and PD-L1, among which dual antibody b has a significant proliferation inhibitory effect on target cell SUN-16, and its effect of competing with FGF1 for FGFR2 receptor to inhibit cell proliferation is also relatively good; after the Fc region of the anti-FGFR2 / PD-1 dual antibody is subjected to S239D-I332E point mutation, the ADCC effect of anti-FGFR2 / PD-1 dual antibody b is significantly enhanced ( Fig. 7A ), but had no obvious killing effect on T cells; in the transplanted tumor model, both the non-target protein MC38 cells and the FGFR2-overexpressing SNU-16 cells, the anti-FGFR2 / PD-1 dual antibody showed good anti-tumor activity.

[0293] In the process of studying 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 anti-FGFR3 / PD-1 bispecific antibodies was significantly weaker than that of anti-FGFR2 / PD-1 bispecific antibodies and anti-PD-1 monoclonal antibodies, indicating that the combination of anti-FGFR3 monoclonal antibodies and anti-PD-1 monoclonal antibodies will affect the activity of anti-PD-1 monoclonal antibodies. This also once again verifies the superiority of the combination of two targets in the anti-FGFR2 / PD-1 bispecific antibodies of the present invention. While not affecting the release of T cell immune activity, the antibody effect on target cells is also better, and can specifically bind to and kill tumor cells expressing FGFR2.

[0294] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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 or 2, 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.

4. The bispecific antibody according to any one of claims 1 to 3, 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.

5. The bispecific antibody according to any one of claims 1 to 4, 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).

6. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the bispecific antibody according to any one of claims 1 to 4.

7. An expression vector, characterized in that: The expression vector contains the polynucleotide molecule according to claim 6.

8. A host cell, characterized in that The host cell contains the expression vector according to claim 7.

9. A method for preparing a bispecific antibody according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: a) culturing the host cell according to claim 8 under expression conditions, thereby expressing the bispecific antibody; b) isolating and purifying the bispecific antibody described in step a).

10. 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 4 and one or more pharmaceutically acceptable carriers, diluents or excipients.

11. Use of the bispecific antibody according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating cancer or tumor; preferably, the cancer or tumor is a FGFR2-positive cancer or tumor.

12. An immunoconjugate, characterized in that: The immunoconjugate comprises: a) the bispecific antibody according to any one of claims 1 to 4; 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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