Use of a bispecific antibody against CLDN 18.2 and CD47 for treating diseases

Abstract

The present invention discloses the use of a bispecific antibody against CLDN 18.2 and CD47 for treating diseases. The present invention discloses the use of the bispecific antibody or its antigen-binding fragment in the preparation of a drug for diagnosing, preventing and / or treating pancreatic cancer and / or colorectal cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to CD47 and a second antigen-binding domain that specifically binds to CLDN 18.2. The present invention also discloses the use of the bispecific antibody or its antigen-binding fragment in combination with a platinum drug in the preparation of a drug for diagnosing, preventing and / or treating gastric cancer, colorectal cancer and pancreatic cancer, as well as a pharmaceutical composition and a kit comprising the bispecific antibody or its antigen-binding fragment. The bispecific antibody of the present invention has excellent effects in the application of treating tumors, can effectively inhibit gastric cancer, colorectal cancer and pancreatic cancer, and has no unexpected toxicity. It can effectively inhibit tumor growth when used alone or in combination with oxaliplatin.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a bispecific antibody against CLDN 18.2 and CD47 for treating diseases. Background Art

[0002] Bispecific antibodies refer to an antibody molecule that can bind to two different antigens or two different antigenic epitopes of the same antigen. They can enhance the tumor cell killing function by interacting with target cells and functional cells, and have broad application prospects in the targeted treatment of tumors.

[0003] CLDN 18.2

[0004] In 1998, researchers at Kyoto University in Japan first discovered and named Claudins. Claudins are integrin membrane proteins present in the tight junctions of epithelial cells and endothelial cells, forming paracellular barriers and pores, and participating in controlling the flow of molecules in the intercellular space between epithelial cells. They have four transmembrane domains, of which both the N-terminus and the C-terminus are present in the cytoplasm. Studies have found that Claudins proteins are closely related to the maintenance of osmotic pressure, barrier function, and cell polarity of epithelial cells, and are involved in the immune defense process against pathogens. There are changes in expression patterns during the occurrence and development of tumors. Research on the targeted treatment of tumors using the Claudins lineage as specific marker proteins has received widespread attention.

[0005] CLDN 18.2 (Claudins 18.2) protein belongs to the Claudins family and is one of the subtypes of Claudins 18. It is often abnormally highly expressed in the occurrence and development of various primary malignant tumors. It was initially found to be continuously and stably expressed in various gastric cancer tissues, but subsequent studies have shown that it can also be abnormally activated and overexpressed in a variety of primary malignant tumors such as breast cancer, colorectal cancer, liver cancer, and non-small cell lung cancer, especially digestive system malignancies such as gastric cancer, pancreatic cancer, and esophageal cancer.

[0006] Under normal circumstances, the CLDN 18.2 protein is buried in the cell membrane of epithelial cells, but the tight junctions of cells in malignant tumors are destroyed, exposing CLDN 18.2 on the surface of tumor cells and making it an attackable target. Therefore, CLDN 18.2 has become an anti-tumor target with great potential. New drug projects targeting CLDN 18.2 include monoclonal antibodies, bispecific antibodies, CAR-T and other types, and the competition is fierce.

[0007] Zolbetuximab (IMAB362) is a drug targeting CLDN 18.2. It is a chimeric IgG1 monoclonal antibody that specifically binds to CLDN18.2 on the surface of tumor cells, thereby inducing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, and inhibiting cell proliferation. Preclinical studies have successfully demonstrated its powerful ability to eliminate cancer cells and control the disease. Subsequently, its good clinical efficacy and safety were also evaluated through multiple Phase I / II clinical trials.

[0008] CD47

[0009] CD47 belongs to the immunoglobulin superfamily, with a molecular weight of about 50kDa. It is a transmembrane glycoprotein with 5 transmembrane domains and is widely expressed on the surface of almost all normal cells. Currently, there are three known natural ligands of CD47: Integrin, Thrombospondin-1 (TSP-1) and Signal-regulatory protein α (SIRPα). CD47 and its ligands are mainly involved in cell adhesion, cell migration, cell phagocytosis and maintaining immune homeostasis.

[0010] Macrophages are an important part of the human body's innate immunity, phagocytizing senescent and dead cells in the human body. In tumor tissues, macrophages can clear tumor cells by phagocytosis, but studies have found that their phagocytosis is inhibited by the CD47-SIRPα immune checkpoint pathway. When CD47 and SIRPα bind, it causes phosphorylation of tyrosine in ITIM (Immunoreceptor tyrosine-based inhibitory motif). The phosphorylated ITIM then recruits and activates the tyrosine esterase SHP1 / 2 protein. The activated tyrosine esterase further dephosphorylates a series of proteins in the cell and disrupts related biological functions, inhibiting the phagocytic function of tumor cells, and ultimately preventing tumor cells from being cleared and escaping immune surveillance. Therefore, blocking SIRPα or CD47 can activate the killing effect of macrophages on tumor cells, making the development of CD47 antibodies a way to treat tumors.

[0011] In summary, the two targets, CLDN 18.2 and CD47, are specifically present on the surface of tumor cells, while CD47 is highly related to the immune microenvironment. Attacking both targets simultaneously has a foreseeable synergistic effect in anti-tumor targeted therapy. Anti-CLDN18.2 and CD47 bispecific antibodies can selectively target cancer cells expressing CLDN 18.2 and CD47, kill tumor cells through Fc-terminal functions such as ADCC and ADCP, and block the CD47 / SIRPa interaction on target cells, activate the innate immune system against the cells, and further kill target cells. Moreover, when bispecific antibodies with one arm binding to CLDN 18.2 and the other arm binding to CD47 selectively target cells expressing two antigens, in terms of affinity, the binding of bispecific antibodies to two antigens on cells expressing both antigens simultaneously can lead to increased affinity compared to either arm. It is also expected that bispecific antibodies may have weaker activity against cells expressing only CD47 (but not CLDN 18.2). This may help avoid targeting normal cells that express certain CD47 but not CLDN 18.2, and may increase the safety and / or tolerability of anti-CLDN 18.2 and CD47 bispecific antibodies.

[0012] Patent WO2021003082 discloses a bispecific antibody against CLDN 18.2 and CD47, which can bind to cancer cells expressing CLDN 18.2 and CD47, induce ADCC, and block the binding of CD47 to SIRPα, inducing macrophage-mediated phagocytosis, and has very little binding to human red blood cells and low toxicity, which is consistent with the above speculation. However, it is unknown how effective this type of bispecific antibody is in the anti-tumor effect in vivo, and whether the results of in vitro experiments can be maintained in the complex environment of the body. Summary of the invention

[0013] In order to solve the problem that there is no report on effective in vivo treatment of bispecific antibodies against CLDN 18.2 and CD47 in the prior art, the present invention provides a bispecific antibody against CLDN 18.2 and CD47 and its use. The inventors found that the bispecific antibody against CLDN 18.2 and CD47 has excellent tumor treatment effect and no unexpected toxicity.

[0014] To solve the above technical problems, one of the technical solutions provided by the present invention is: use of a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 in the preparation of a medicament for diagnosing, preventing and / or treating pancreatic cancer and / or colorectal cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to CD47 and a second antigen-binding domain that specifically binds to CLDN 18.2.

[0015] The present invention provides a sequence list of the bispecific antibody against CLDN 18.2 and CD47 (including CDR regions, light chain and heavy chain variable regions and full sequences).

[0016] The amino acid sequence of LCDR1 of the first antigen binding domain is shown in SEQ ID NO:1, the amino acid sequence of LCDR2 is shown in SEQ ID NO:2, the amino acid sequence of LCDR3 is shown in SEQ ID NO:3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:6.

[0017] As described in one of the technical solutions, the second antigen binding domain comprises an immunoglobulin single variable domain VHH.

[0018] In a preferred technical solution of the present invention, the amino acid sequence of HCDR1 of the second antigen binding domain is shown as SEQ ID NO:7, the amino acid sequence of HCDR2 is shown as SEQ ID NO:8, and the amino acid sequence of HCDR3 is shown as SEQ ID NO:9.

[0019] As used in one of the technical solutions, the framework regions of the light chain variable regions and / or heavy chain variable regions of the first antigen binding domain and the second antigen binding domain are human framework regions.

[0020] In a preferred technical solution of the present invention, the amino acid sequence of the light chain variable region of the first antigen binding domain is shown in SEQ ID NO:10.

[0021] In a preferred technical solution of the present invention, the amino acid sequence of the heavy chain variable region of the first antigen binding domain is shown in SEQ ID NO:12.

[0022] In a preferred technical solution of the present invention, the amino acid sequence of the heavy chain variable region of the second antigen binding domain is shown in SEQ ID NO:14.

[0023] As described in one of the technical solutions, the light chain constant region of the first antigen binding domain is a human κ light chain constant region, and / or the heavy chain constant region of the first antigen binding domain is a human IgG1 heavy chain constant region.

[0024] In a preferred technical solution of the present invention, the amino acid sequence of the light chain constant region of the first antigen binding domain is shown in SEQ ID NO:11.

[0025] In a preferred technical solution of the present invention, the amino acid sequence of the heavy chain constant region of the first antigen binding domain is shown in SEQ ID NO:13.

[0026] In the application as described in one of the technical solutions, the heavy chain of the first antigen binding domain and the heavy chain of the second antigen binding domain are connected by a linker.

[0027] In a preferred technical solution of the present invention, the heavy chain of the second antigen-binding domain is connected to the C-terminus of the heavy chain of the first antigen-binding domain via a linker.

[0028] In a more preferred embodiment of the present invention, the amino acid sequence of the heavy chain of the first antigen binding domain is as shown in SEQ ID NO:16, and / or the amino acid sequence of the linker is as shown in SEQ ID NO:18.

[0029] In the application as described in one of the technical solutions, the bispecific antibody comprises a long chain and a short chain, the short chain comprises the amino acid sequence shown in SEQ ID NO:15, and the long chain comprises the amino acid sequence shown in SEQ ID NO:17.

[0030] The present invention provides a bispecific antibody against CLDN 18.2 and CD47 as described in the present invention, such as an injection, for use in a product for treating and / or preventing tumors. In addition, the present invention also provides a bispecific antibody against CLDN 18.2 and CD47, such as an injection, for use in a product for treating and / or preventing tumors in combination with a chemotherapeutic drug.

[0031] In order to solve the above technical problems, the second technical solution provided by the present invention is: use of a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 in combination with a platinum drug in the preparation of a drug for diagnosing, preventing and / or treating tumors, wherein the bispecific antibody or the antigen-binding fragment thereof is as defined in the application described in one of the technical sub-solutions, and the tumor is a CLDN 18.2 and / or CD47 positive tumor;

[0032] In a preferred technical solution of the present invention, the tumor is a digestive system tumor;

[0033] In a more preferred technical solution of the present invention, the digestive system tumor is pancreatic cancer or gastrointestinal tumor;

[0034] In a further preferred technical solution of the present invention, the gastrointestinal tumor is gastric cancer or colorectal cancer.

[0035] As described in the second technical solution, the platinum drug is selected from cisplatin, carboplatin and oxaliplatin.

[0036] In a specific technical solution of the present invention, the platinum drug is oxaliplatin.

[0037] In the above application, the chemotherapy drug is a basic chemotherapy regimen recommended in the tumor clinical treatment guidelines, such as a chemotherapy regimen based on platinum drugs (cisplatin, carboplatin, oxaliplatin), etc. Platinum drugs have a direct cytotoxic effect on tumor cells by inhibiting the replication and transcription of tumor cell DNA, and have a broad-spectrum anti-cancer activity. They are the most commonly used chemotherapy drugs. Chemical drugs directly kill tumor cells, can release substances such as intracellular tumor-associated antigens, and thus enhance tumor immunogenicity, and have a synergistic effect with immunomodulatory drugs.

[0038] Those skilled in the art can know from platinum drugs that the present invention can specifically use cisplatin, carboplatin, oxaliplatin, etc., but in actual clinical applications, cisplatin is relatively toxic and carboplatin is inconvenient to use, so oxaliplatin is a relatively superior technical solution, but the scope of protection of the present invention should not be limited thereto.

[0039] In order to solve the above technical problems, the third technical solution provided by the present invention is: a pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 as defined in the application described in one of the technical solutions, and a platinum drug.

[0040] In a specific technical solution of the present invention, the platinum drug is selected from cisplatin, carboplatin and oxaliplatin, preferably oxaliplatin.

[0041] In order to solve the above technical problems, the fourth technical solution provided by the present invention is: a set of medicine boxes, which includes medicine box A and medicine box B, wherein:

[0042] The drug kit A comprises a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 as defined in the application described in one of the technical solutions.

[0043] The drug kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

[0044] In a preferred technical solution of the present invention, the chemotherapeutic agent is preferably a platinum drug.

[0045] In a more preferred technical solution of the present invention, the platinum drug is selected from cisplatin, carboplatin and oxaliplatin, preferably oxaliplatin.

[0046] The antibody of the present invention is composed of two identical light chains and two identical heavy chains covalently cross-linked, containing 577 amino acids, and its heavy chain VH and light chain VL together constitute a domain that can specifically bind to CD47, and the C-terminus is connected to a heavy chain antibody VHH domain targeting CLDN18.2. The anti-CLDN18.2 end of the antibody of the present invention has cross-reactions with humans, mice, and crab-eating macaques, and the anti-CD47 end has cross-reactions with humans and crab-eating macaques. The antibody of the present invention can target the CLDN 18.2 molecules on the surface of tumor cells, block the immunosuppression mediated by the CD47 / SIRPα interaction, activate the activity of NK cells and macrophages of the body through the Fc end, kill CLDN 18.2-positive tumor cells through Fc-end functions such as ADCC and ADCP, and exert an anti-tumor effect.

[0047] On this basis, the anti-tumor efficacy of the antibody of the present invention in a mouse solid tumor model was explored.

[0048] The present invention provides the effect of the antibody of the present invention in treating cancer in tumor-bearing mouse models of three cell lines, BxPC3, HT29 and NUGC4, which overexpress CLDN 18.2. The therapeutic effect of the antibody of the present invention in combination with the commonly used platinum chemical drug oxaliplatin in the clinic was explored in a tumor-bearing mouse model of the NUGC4 human gastric cancer cell line overexpressing CLDN 18.2. The therapeutic effect of the antibody of the present invention and the CLDN18.2 target monoclonal antibody drug IMAB362 and the CD47 target monoclonal antibody drug Hu5F9-G4 were compared in the HT29 human colon cancer model overexpressing CLDN18.2. (IMAB362, a human-mouse chimeric antibody against CLDN18.2 developed by Astellas, and the IMAB362 used in this application was obtained in-house according to the antibody sequence reported in patent US20180127489; Hu5F9-G4, a humanized monoclonal antibody against CD47 developed by Forty Seven, and the Hu5F9-G4 used in this application was obtained in-house according to the antibody sequence reported in patent US20200283520A1.)

[0049] The antibody of the present invention has a dose-dependent anti-tumor effect in the tumor-bearing mouse models of three cell lines, BxPC3, HT29 and NUGC4, which overexpress CLDN 18.2. The high-dose tumor inhibition rate exceeds 80%, and the highest can exceed 100%, achieving the effect of completely inhibiting tumor growth. Compared with monoclonal antibodies, it has significant anti-tumor advantages and has good synergy when combined with chemotherapy drugs, indicating that the antibody of the present invention has good therapeutic potential for CLDN 18.2-positive pancreatic cancer, colorectal cancer, gastric cancer and other cancers in clinical practice.

[0050] The reagents and raw materials used in the present invention are commercially available.

[0051] The positive and progressive effects of the present invention are:

[0052] The present invention uses an anti-CLDN 18.2 and CD47 bispecific antibody in the application of treating tumors with excellent effect, can effectively inhibit gastric cancer, colorectal cancer, and pancreatic cancer, and has no unexpected toxicity. It can effectively inhibit tumor growth when used alone or in combination with oxaliplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1A The affinity of the bispecific antibody A of the present invention to CLDN18.2 was determined using the BLI method.

[0054] Figure 1B The affinity between the dual antibody A of the present invention and CD47 was determined using the BLI method.

[0055] Figure 1C The ELISA method was used to determine the binding activity of the dual antibody A of the present invention to CD47.

[0056] Figure 2 The efficacy of dual antibody A on BxPC3-hCLDN18.2 tumor-bearing mice transplanted tumors - tumor volume.

[0057] Figure 3 This is a tumor display diagram showing the efficacy of dual antibody A on BxPC3-hCLDN18.2 tumor-bearing mouse transplanted tumors.

[0058] Figure 4 The efficacy of dual antibody A on BxPC3-hCLDN18.2 tumor-bearing mice transplanted tumors - tumor weight.

[0059] Figure 5 This is the effect of dual antibody A on the body weight of BxPC3-hCLDN18.2 tumor-bearing mice.

[0060] Figure 6 The efficacy of dual antibody A on transplanted tumors in HT29-hCLDN18.2 tumor-bearing mice - tumor volume.

[0061] Figure 7 This is the tumor display diagram of the efficacy of dual antibody A on HT29-hCLDN18.2 tumor-bearing mouse transplanted tumors.

[0062] Figure 8 The efficacy of dual antibody A on transplanted tumors in HT29-hCLDN18.2 tumor-bearing mice - tumor weight.

[0063] Fig. 9 This is the effect of dual antibody A on the body weight of HT29-hCLDN18.2 tumor-bearing mice.

[0064] Fig.10The efficacy of dual antibody A on NUGC-4-hCLDN18.2 tumor-bearing mouse transplanted tumors - tumor volume.

[0065] Fig.11 This is the efficacy of dual antibody A on NUGC-4-hCLDN18.2 tumor-bearing mouse transplanted tumors - tumor display diagram (X: the tumor of this mouse was completely eliminated).

[0066] Fig.12 The efficacy of dual antibody A on NUGC-4-hCLDN18.2 tumor-bearing mice transplanted tumors - tumor weight.

[0067] Fig.13 This is the effect of dual antibody A on the body weight of NUGC-4-hCLDN18.2 tumor-bearing mice.

[0068] Fig.14 The efficacy of the administered antibody on HT29-hCLDN18.2 mouse transplanted tumors - tumor volume.

[0069] Fig.15 The tumor display shows the efficacy of the administered antibody on HT29-hCLDN18.2 tumor-bearing mouse transplanted tumors.

[0070] Fig.16 The efficacy of the administered antibody on the transplanted tumors in HT29-hCLDN18.2 tumor-bearing mice - tumor weight.

[0071] Fig.17 The figure shows the effect of the antibody of the present invention on the body weight of HT29-hCLDN18.2 tumor-bearing mice.

[0072] Fig.18 The efficacy of dual anti-A combined with oxaliplatin on NUGC-4-hCLDN18.2 mouse transplanted tumors - tumor volume.

[0073] Fig.19 This is the tumor display diagram of the efficacy of dual anti-A combined with oxaliplatin on NUGC-4-hCLDN18.2 tumor-bearing mouse transplanted tumors.

[0074] Fig. 20 The efficacy of dual anti-A combined with oxaliplatin on NUGC-4-hCLDN18.2 tumor-bearing mouse transplanted tumors - tumor weight.

[0075] Fig.21 This is the effect of dual anti-A combined with oxaliplatin on the body weight of NUGC-4-hCLDN18.2 tumor-bearing mice.

[0076] Fig.22a The ability of bispecific antibody A to block the binding of human CD47 to the receptor SIRPα on NUGC-4 cells.

[0077] Figure 22bThe ability of dual antibody A to block the binding of human CD47 to the receptor SIRPα on hCLDN18.2-NUGC-4 cells.

[0078] Fig.23a and Figure 23b The inhibitory effect of dual antibody A on tumor growth in mice. DETAILED DESCRIPTION

[0079] Explanation of terms

[0080] In some embodiments, the pharmaceutical composition of the present invention comprises suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used in the present invention, "pharmaceutically acceptable carriers" or "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents and absorption delay agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may include standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. The pharmaceutical preparation or pharmaceutical composition comprising the present invention may be prepared by mixing the antibody or antigen-binding fragment thereof of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized preparation or an aqueous solution. The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activity that will not adversely affect each other. For example, it is desirable to also provide other anti-tumor active ingredients, such as other antibodies, anti-tumor active agents, small molecule drugs or immunomodulators, etc. The active ingredient is suitably combined in an amount effective for the purpose of use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies of the present invention or their Fabs, and the matrix is ​​a formed article, such as a film or microcapsule form.

[0081] In the present invention, the amino acid sequences of the above-listed CDRs are all shown according to the Kabat definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., USDepartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and NorthCDR definition based on affinity propagation clustering using a large number of crystal structures. It will be appreciated by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0082] The three-letter codes and one-letter codes for amino acids used in the present invention are known to those skilled in the art, or are described in the literature (J. Biol. Chem, 243, p3558 (1968)).

[0083] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0084] In the present embodiment, each group of mice was euthanized at the end of the experiment. Using Excel statistical software: the mean value was calculated as AVE; the SD value was calculated as STDEV; the SEM value was calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism 6.0 software was used for drawing, and the P value of the difference between groups was analyzed by Student's t test.

[0085] In the embodiment of the present invention, the tumor volume (TV) is calculated as follows: TV = 0.5 × major diameter × minor diameter 2 .

[0086] Tumor inhibition rate TGItv (%): used to evaluate the anti-tumor activity of drugs, tumor inhibition rate (%) = [1-(TVt-TVinitial) / (CVt- CVinitial)] × 100%, wherein TVt represents the tumor volume of the treatment group at each measurement; TVinitial represents the tumor volume of the treatment group when grouped medication; CVt represents the tumor volume of the control group at each measurement; CVinitial represents the tumor volume of the control group when grouped medication.

[0087] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the examples and drawings. In the following examples, "bi-antibody A" is the bispecific antibody against CLDN 18.2 and CD47 of the present invention. The applicant's prior patent application 202111214360.8 and PCT / CN2022 / 082008 are cited and referenced in their entirety in this application. It is understood that the specific embodiments described herein are only used to explain the present invention, not to limit the present invention.

[0088] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0089] Preparation Example 1 Preparation of the Antibody of the Present Invention

[0090] 1. Construction of Antibody Expression Plasmid

[0091] The heavy and light chain genes of the antibody were ligated to the cloning T vector and confirmed by sequencing. The light and heavy chain genes were subcloned into Mu-H and After the Mu-P expression vector was confirmed by sequencing and restriction digestion, the recombinant plasmid was finally obtained.

[0092] 2. Antibody Expression

[0093] The recombinant plasmid was co-transfected into CHO-K1 host cells by electroporation for expression, and affinity purified by conventional methods in the art.

[0094] Sequence and structure of the antibody double antibody A of the present invention

[0095] The CLDN18.2-binding arm adopts an anti-CLDN18.2 single-domain antibody NA3S-H1 that specifically recognizes human CLDN18.2 but not CLDN18.1 (the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 7, 8 and 9, respectively; the amino acid sequence of VHH is shown in SEQ ID NO: 14), and the CD47-binding arm adopts the antigen-binding domain of the anti-CD47 humanized antibody A7H3L3 (the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 4, 5 and 6, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 1, 2 and 3, respectively; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10). The heavy chain variable region of antibody A7H3L3 was fused to the human IgG1 heavy chain constant region (SEQ ID NO: 13) to form the heavy chain of antibody A7H3L3 (SEQ ID NO: 16), and the light chain variable region was fused to the human κ light chain constant region (SEQ ID NO: 11) to form the light chain of antibody A7H3L3 (SEQ ID NO: 15).

[0096] The anti-CLDN18.2 single-domain antibody NA3S-H1 has been published in WO2020238730A1, and its in vitro ADCC and CDC cell killing effects and tumor inhibition tests in the human CLDN18.2-HEK29T-SCID tumor transplant model have shown excellent efficacy. The humanized antibody A7H3L3 binds weakly to CD47 on red blood cells and is an ideal candidate for bispecific antibodies. At the same time, the design of the bispecific antibody will make the anti-CD47 antibody A7H3L3 binding arm better bind to tumor cells expressing dual targets because of the binding to CLDN18.2, and better block SIRP α inhibitory signals.

[0097] The structure of the anti-CD47-CLDN18.2 bispecific antibody bispecific antibody A of the present invention is as follows:

[0098] Long chain structure (from N-terminus to C-terminus):

[0099] VH CD47 -CH1-Hinge-CH2-CH3-Linker-VHH CLDN18.2

[0100] Short chain structure (from N-terminus to C-terminus):

[0101] V L CD47 -CL(i.e. SEQ ID NO:15)

[0102] The sequence of the linker is (SEQ ID NO: 18): GGGGSGGGGS

[0103] The full-length long chain sequence of the complete bispecific antibody DiasA is shown in (SEQ ID NO: 17).

[0104] Example 1

[0105] In vitro pharmacodynamics of the dual antibody A of the present invention

[0106] (1) Antigen affinity, binding activity, and specificity

[0107] The antigen affinity study was performed using BLI (biomembrane interference) technology, and the results showed that the affinity of the bispecific antibody A of the present invention to human CLDN18.2 was high, and the affinity constant KD was 1.87×10 -9 M, see Appendix Figure 1A ; The affinity to human CD47 is low, and the affinity constant KD = 2.18 × 10 -8 M, see Appendix Figure 1B .

[0108] The antigen binding activity was studied by ELISA. The results showed that the bispecific antibody A of the present invention could specifically bind to CLDN18.2 (EC50 = 121 ng / mL) and CD47 (EC50 = 46.2 ng / mL), and presented a typical dose-effect curve. Figure 1C .

[0109] The competitive binding test showed that the bispecific antibody A of the present invention can effectively block the binding of CD47 and the ligand SIRPα at the ELISA level, and the binding activity result is IC50=2.2 μg / mL.

[0110] (2) Red blood cell agglutination test

[0111] The results of the red blood cell agglutination experiment showed that neither the bispecific antibody A of the present invention nor the negative control caused human red blood cell agglutination, which was significantly better than the positive control drug Hu5F9-G4 for the CD47 target (the control drug was homemade according to the antibody sequence reported in patent US20200283520A1), indicating that it has good safety for human red blood cells.

[0112] Example 2

[0113] Evaluation of the anti-tumor effect of the antibody of the present invention in a CB-17SCID mouse model with subcutaneous transplantation of BxPC-3-hCLDN18.2 pancreatic cancer cells

[0114] BxPC3 cells expressing hCLDN18.2 (purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.) were subcutaneously inoculated into 39 CB-17SCID mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Shanghai Branch), with 1×10 7 cells. On the 10th day after cell inoculation, 32 mice were randomly divided into 4 groups according to the tumor volume and weight of the animals, with 8 mice in each group, including PBS control group, low-dose group of dual anti-A (1 mg / kg), medium-dose group of dual anti-A (3 mg / kg) and high-dose group of dual anti-A (10 mg / kg). The day of grouping was defined as D1, and drug administration began on D1, alternating between tail vein injection and intraperitoneal injection, and the drug was administered twice a week. During the experiment, the general condition of the animals was observed every day, and the tumor volume and body weight were measured twice a week.

[0115] In this experiment, drugs were administered until D50. At the end of the experiment on D54, the mice were euthanized and the tumors were removed and weighed.

[0116] At the end of the experiment, the tumor volume, tumor weight and corresponding tumor inhibition rate of each group are shown in Table 1. The tumor growth curve was drawn according to the tumor volume during the experiment, as shown in Figure 2 As shown. Compared with the PBS control group, each group of dual anti-A can inhibit the growth of BxPC3-hCLDN18.2 tumors to varying degrees. The tumor inhibition rate TGITV was calculated based on the tumor volume. The TGITV of the three dose groups of 1, 3, and 10 mg / kg were 47.12%, 85.90%, and 86.48%, respectively. The D54 tumor volume of each group of mice was statistically compared. Compared with the PBS control group, the reduction in tumor volume in the 3 mg / kg and 10 mg / kg dose groups was extremely significant (P < 0.01).

[0117] The tumor display image and tumor weight of mice in this experiment are shown in Figure 3 and Figure 4As shown. The tumor inhibition rate TGItw was calculated according to the tumor weight, and the statistical analysis of TGItw and P value of each group is shown in Table 1. Compared with the PBS control group, the antibody of the present invention can reduce the weight of BxPC3-hCLDN18.2 tumor in a dose-dependent manner. The TGItw of the three dose groups of 1, 3, and 10 mg / kg were 44.16%, 80.00%, and 74.46%, respectively. The tumor weight inhibition rate of the 10 mg / kg dose group was lower than that of the 3 mg / kg dose group, which may be due to the longer administration time, so that the drug efficacy has reached saturation. The D54 tumor weight of each group of mice was compared with the PBS control group. Compared with the PBS control group, the difference in the reduction of tumor weight between the 3 mg / kg dose group and the 10 mg / kg dose group was extremely significant (P < 0.01).

[0118] Table 1 Efficacy of dual antibody A on BxPC3-hCLDN18.2 tumor-bearing mice transplanted tumors

[0119]

[0120] During the drug administration, the body weight of mice in each group showed a normal growth trend. Figure 5 The mice in each group were generally observed to have no obvious abnormalities, indicating that the animals tolerated the current dosage well. The weight data of mice in each group are shown in Table 2.

[0121] Table 2 Body weight data of BxPC3-hCLDN18.2 tumor-bearing mice (g)

[0122]

[0123] Example 3

[0124] Evaluation of the anti-tumor effect of the antibody of the present invention in a CB-17SCID mouse model with subcutaneous transplantation of HT29-hCLDN18.2 colorectal cancer cells

[0125] HT29 cells expressing hCLDN18.2 (purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.) were subcutaneously inoculated into 39 CB-17SCID mice, with each mouse inoculated with 5×10 6 cells. On the 6th day after cell inoculation, 32 mice were randomly divided into 4 groups according to the tumor volume and weight of the animals, with 8 mice in each group, including PBS control group, low-dose group of dual anti-A (1 mg / kg), medium-dose group of dual anti-A (3 mg / kg) and high-dose group of dual anti-A (10 mg / kg). The day of grouping was defined as D1, and drug administration began on D1, alternating between tail vein injection and intraperitoneal injection, and the drug was administered twice a week. During the experiment, the general condition of the animals was observed every day, and the tumor volume and body weight were measured twice a week.

[0126] In this experiment, drugs were administered until D32. At the end of the experiment on D36, the mice were euthanized and the tumors were removed and weighed.

[0127] At the end of the experiment, the tumor volume, tumor weight and corresponding tumor inhibition rate of each group are shown in Table 3. The tumor growth curve was drawn according to the tumor volume during the experiment, as shown in Figure 6 As shown. Compared with the PBS control group, each group of dual anti-A can inhibit the growth of HT29-hCLDN18.2 tumors to varying degrees. The tumor inhibition rate TGITV was calculated based on the tumor volume. The TGITV of the three dose groups of 1, 3, and 10 mg / kg were 39.30%, 48.54%, and 82.27%, respectively. The D36 tumor volume of each group of mice was statistically compared. Compared with the PBS control group, the reduction in tumor volume in the 1 mg / kg dose group, the 3 mg / kg dose group, and the 10 mg / kg dose group was extremely significant (P < 0.01).

[0128] The tumor display image and tumor weight of mice in this experiment are shown in Figure 7 and Figure 8 As shown. The tumor inhibition rate TGItw was calculated according to the tumor weight, and the statistical analysis of TGItw and P value of each group is shown in Table 3. Compared with the PBS control group, the antibody of the present invention can reduce the weight of HT29-hCLDN18.2 tumor in a dose-dependent manner, and the TGItw of the three dose groups of 1, 3, and 10 mg / kg were 23.29%, 31.53%, and 73.46%, respectively. The D36 tumor weight of each group of mice was statistically compared. Compared with the PBS control group, the reduction in tumor weight in the 3 mg / kg dose group was significant (P < 0.05), and the reduction in tumor weight in the 10 mg / kg dose group was extremely significant (P < 0.01).

[0129] Table 3 Efficacy of dual antibody A on HT29-hCLDN18.2 tumor-bearing mice transplanted tumors

[0130]

[0131] During the drug administration, the body weight of mice in each drug administration group showed a normal growth trend. After D29, the body condition of mice in the control group deteriorated and their body weight decreased due to the influence of tumor volume. Fig. 9 The mice in each group were generally observed to have no obvious abnormalities, indicating that the animals tolerated the current dosage well. The weight data of mice in each group are shown in Table 4.

[0132] Table 4 Body weight data of HT29-hCLDN18.2 tumor-bearing mice (g)

[0133] Grouping D1 D4 D8 D11 D15 D18 D22 D25 D29 D32 D36 G1-PBS 18.75 18.50 18.54 18.71 19.09 18.72 18.83 18.56 19.03 18.22 17.65 G2-Dual Antibody A-1mg / kg 19.62 19.19 19.92 19.95 19.60 19.73 20.21 19.74 19.96 19.38 19.54 G3-Dual Antibody A-3mg / kg 18.88 18.63 19.22 18.90 19.08 19.08 19.44 19.13 19.53 19.20 19.50 G4-Dual Antibody A-10mg / kg 18.84 18.78 19.30 19.10 19.21 18.99 19.58 18.97 19.49 19.01 19.46

[0134] Example 4

[0135] Evaluation of the anti-tumor effect of the antibody of the present invention in a CB-17SCID mouse model with subcutaneous transplantation of NUGC-4-hCLDN18.2 gastric cancer cells

[0136] NUGC-4 cells expressing hCLDN18.2 (the gastric cancer cell line NUGC-4 overexpressing exogenous human CLDN18.2 was constructed by lentiviral transfection; the original NUGC-4 cell line expressed endogenous CD47 and was purchased from the BNCC strain bank, number BNCC341962) were subcutaneously inoculated into 44 CB-17SCID mice, with each mouse inoculated with 3×10 6 cells. On the 6th day after cell inoculation, 28 mice were randomly divided into 4 groups according to the tumor volume of the animals, including PBS control group, low-dose group of dual anti-A (3 mg / kg), medium-dose group of dual anti-A (10 mg / kg) and high-dose group of dual anti-A (20 mg / kg). The day of grouping was defined as D1, and drug administration began on D1, with tail vein injection twice a week. During the experiment, the general condition of the animals was observed every day, and the tumor volume and body weight were measured twice a week.

[0137] In this experiment, drugs were administered until D27. At the end of the experiment on D29, the mice were euthanized and the tumors were removed and weighed.

[0138] At the end of the experiment, the tumor volume, tumor weight and corresponding tumor inhibition rate of each group are shown in Table 5. The tumor growth curve was drawn according to the tumor volume during the experiment, as shown in Fig.10 As shown. Compared with the PBS control group, each group of dual anti-A can inhibit the growth of NUGC-4-hCLDN18.2 tumors to varying degrees. The tumor inhibition rate TGITV was calculated based on the tumor volume. The TGITV of the three dose groups of 3, 10, and 20 mg / kg were 32.28%, 90.07%, and 106.32%, respectively. The D29 tumor volume of each group of mice was statistically compared. Compared with the PBS control group, the reduction in tumor volume in the 10 mg / kg dose group and the 20 mg / kg dose group was extremely significant (P < 0.01). In this experiment, by the end of the D29 experiment, one mouse in the medium dose 10 mg / kg group of dual anti-A had complete tumor remission (tumor disappeared), and three mice had partial tumor remission (tumor volume was less than the group volume); the tumors of animals in the high dose 20 mg / kg group of dual anti-A were completely relieved.

[0139] The tumor display image and tumor weight of mice in this experiment are shown in Fig.11 and Fig.12As shown. The tumor inhibition rate TGItw was calculated according to the tumor weight, and the statistical analysis of TGItw and P value of each group is shown in Table 5. Compared with the PBS control group, the dual anti-A antibody can reduce the weight of NUGC-4-hCLDN18.2 tumors in a dose-dependent manner. The TGItw of the three dose groups of 3, 10, and 20 mg / kg were 29.23%, 86.15%, and 100.00%, respectively. The D29 tumor weight of each group of mice was statistically compared. Compared with the PBS control group, the reduction in tumor weight in the 10 mg / kg dose group and the 20 mg / kg dose group was extremely significant (P < 0.01).

[0140] Table 5 Efficacy of dual antibody A on NUGC-4-hCLDN18.2 tumor-bearing mice transplanted tumors

[0141]

[0142] On D8, the weight of the PBS control group decreased abnormally because the water bottle of one cage of mice in this group leaked, causing the mice to lose water and weight. After the water bottle was replaced, the weight of the mice quickly returned to normal. During the drug administration process, the weight of mice in each group showed a normal growth trend, such as Fig.13 As shown in Table 6, no obvious abnormalities were observed in general observation, indicating that the animals tolerated the drug well at the current dosage. The weight data of mice in each test group are shown in Table 6.

[0143] Table 6 Body weight data of NUGC-4-hCLDN18.2 tumor-bearing mice (g)

[0144]

[0145]

[0146] Example 5

[0147] Evaluation of the anti-tumor effect of the antibody of the present invention compared with the single-target antibody in the CB-17SCID mouse model with subcutaneous transplantation of HT29-hCLDN18.2 colorectal cancer cells

[0148] The hCLDN18.2-expressing HT29-hCLDN18.2 cells were subcutaneously inoculated into 34 CB-17 SCID mice, with each mouse inoculated with 5×10 6cells. On the 7th day after cell inoculation, 28 animals were randomly divided into 4 groups according to the tumor volume and weight of the animals, including PBS control group, CLDN18.2 monoclonal antibody (IMAB362) group (10 mg / kg), CD47 monoclonal antibody (Hu5F9-G4) group (10 mg / kg) and dual anti-A group (10 mg / kg). The day of grouping was defined as D1, and medication began on the day of grouping. CLDN18.2 monoclonal antibody (IMAB362), CD47 monoclonal antibody (Hu5F9- G4) and dual anti-A antibody were administered alternately by tail vein injection and intraperitoneal injection twice a week. During the experiment, the general state of the animals was observed every day, and the tumor volume and body weight were measured twice a week.

[0149] In this experiment, drugs were administered until D26. At the end of the experiment on D29, the mice were euthanized and the tumors were removed and weighed.

[0150] The tumor growth curve was drawn according to the tumor volume during the experiment. Fig.14 As shown. TGITV was calculated according to the terminal tumor volume, as shown in Table 7. Compared with the PBS control group, each drug-treated group can inhibit the growth of HT29-hCLDN18.2 tumors to varying degrees. Among them, the TGITV of the CLDN18.2 monoclonal antibody (IMAB362) group, the CD47 monoclonal antibody (Hu5F9-G4) group, and the dual anti-A group were 26.60%, 21.07%, and 84.99%, respectively. Compared with the PBS control group, there was no significant difference in the reduction of tumor volume in the CLDN18.2 monoclonal antibody (IMAB362) group and the CD47 monoclonal antibody (Hu5F9-G4) group (P>0.05), and the reduction of tumor volume in the dual anti-A group was statistically significant. And the tumor volume of the dual anti-A group was significantly reduced compared with the CLDN18.2 monoclonal antibody (IMAB362) group and the CD47 monoclonal antibody (Hu5F9-G4) group, which was statistically significant.

[0151] Table 7 Efficacy of the administered antibody on HT29-hCLDN18.2 tumor-bearing mice transplanted tumors (TGItv)

[0152]

[0153]

[0154] The tumor display image and tumor weight of mice in this experiment are shown in Fig.15 and Fig.16, TGItw was calculated according to the tumor weight, as shown in Table 8. The TGItw of the CLDN18.2 monoclonal antibody (IMAB362) group, the CD47 monoclonal antibody (Hu5F9-G4) group, and the dual antibody A group were 20.59%, 16.30%, and 78.67%, respectively. Compared with the PBS control group, there was no significant difference in the reduction of tumor weight in the CLDN18.2 monoclonal antibody (IMAB362) group and the CD47 monoclonal antibody (Hu5F9-G4) group (P>0.05), and the reduction of tumor weight in the dual antibody A group was statistically significant. And the tumor weight of the dual antibody A group was significantly reduced compared with the CLDN18.2 monoclonal antibody (IMAB362) group and the CD47 monoclonal antibody (Hu5F9-G4) group, which was statistically significant.

[0155] Table 8 The efficacy of the administered antibody on HT29-hCLDN18.2 tumor-bearing mice transplanted tumors (TGItw)

[0156]

[0157] During the drug administration process, the body weight of mice in each group showed a normal and steady growth trend. Some mice had a slight decrease in body weight in the later stage due to the large tumor volume. Fig.17 As shown in Table 9, no obvious abnormalities were observed in general observation, indicating that all mice tolerated the current dosage well. The weight data of mice in each group are shown in Table 9.

[0158] Table 9 Body weight data of different groups of HT29-hCLDN18.2 tumor-bearing mice (g)

[0159]

[0160] Example 6

[0161] Evaluation of the anti-tumor effect of the antibody of the present invention combined with oxaliplatin in a CB-17SCID mouse model with subcutaneous transplantation of HT29-hCLDN18.2 cells

[0162] The hCLDN18.2-expressing NUGC-4-hCLDN18.2 cells were subcutaneously inoculated into 64 CB-17SCID mice, with 3×10 6cells. On the 7th day after cell inoculation, 48 animals were randomly divided into 6 groups according to the tumor volume of the animals, including PBS control group, oxaliplatin monotherapy group (4 mg / kg), dual anti-A low-dose group (3 mg / kg), dual anti-A high-dose group (6 mg / kg), dual anti-A low-dose combination group (4 mg / kg + 3 mg / kg) and dual anti-A high-dose combination group (4 mg / kg + 6 mg / kg). The day of grouping was defined as D1, and medication began on the day of grouping. Oxaliplatin was injected into the tail vein once a week; dual anti-A antibodies were injected alternately by intraperitoneal injection and tail vein injection twice a week.

[0163] In this study, drug administration was continued until D24 and the study ended on D27. The tumors were weighed and removed on the same day.

[0164] The tumor growth curve was drawn according to the tumor volume during the experiment. Fig.18 The TGItv was calculated based on the terminal tumor volume, as shown in Table 10.

[0165] Compared with the PBS control group, each drug-treated group could inhibit the growth of NUGC-4-hCLDN18.2 tumors to varying degrees. Among them, the TGITV of the oxaliplatin monotherapy group, the low-dose dual-antibody A group, and the high-dose dual-antibody A group were 26.74%, 42.28%, and 60.31%, respectively, and the TGITV of the low-dose dual-antibody A combination group and the high-dose dual-antibody A combination group were 73.41% and 79.79%, respectively. The TGITV of the low-dose dual-antibody A combination group was significantly higher than that of the oxaliplatin monotherapy group and the low-dose dual-antibody A group at the same dose, and the differences in tumor volume between the groups were statistically significant; the TGITV of the high-dose dual-antibody A combination group was significantly higher than that of the oxaliplatin monotherapy group and the high-dose dual-antibody A group at the same dose, and the differences in tumor volume between the groups were statistically significant.

[0166] Table 10 Efficacy of dual anti-A combined with oxaliplatin on NUGC-4-hCLDN18.2 mouse transplanted tumors (TGItv)

[0167]

[0168] The tumor display image and tumor weight of mice in this experiment are shown in Fig.19 and Fig. 20TGItw was calculated according to the tumor weight, as shown in Table 11. The TGItw of the oxaliplatin monotherapy group, the low-dose dual-antibody A group, and the high-dose dual-antibody A group were 23.19%, 34.78%, and 55.07%, respectively, and the TGItw of the low-dose dual-antibody A combination group and the high-dose dual-antibody A combination group were 62.32% and 72.46%, respectively. The TGItw of the low-dose dual-antibody A combination group was significantly higher than that of the oxaliplatin monotherapy group and the low-dose dual-antibody A group at the same dose, and the differences in tumor weight between the groups were statistically significant; the tumor inhibition rate TGItw of the high-dose dual-antibody A combination group was significantly higher than that of the oxaliplatin monotherapy group and the high-dose dual-antibody A group at the same dose, and the difference in tumor weight was statistically significant compared with the oxaliplatin monotherapy group; compared with the high-dose dual-antibody A group, the difference in tumor weight was not statistically significant.

[0169] Table 11 Efficacy of dual anti-A combined with oxaliplatin on NUGC-4-hCLDN18.2 mouse transplanted tumors (TGItw)

[0170]

[0171] During the drug administration, the body weight of mice in each group remained stable. Fig.21 As shown in Table 12, no obvious abnormalities were observed in general observation, indicating that the animals tolerated the drug well at the current dosage. The weight data of mice in each test group are shown in Table 12.

[0172] Table 12 Body weight data of different groups of NUGC-4-hCLDN18.2 tumor-bearing mice (g)

[0173] BW D1 D4 D7 D10 D14 D17 D21 D24 D27 G1-PBS 17.20 17.38 17.57 18.02 17.90 18.71 18.40 19.11 18.46 G2-Oxa-4mg / kg,QW 17.50 17.25 17.64 17.70 17.36 17.60 17.58 17.67 16.94 G3-Dual Antibody A-3mg / kg, BIW 17.68 18.34 18.22 18.35 18.42 18.64 17.84 18.49 18.59 G4-Dual Antibody A-6mg / kg, BIW 18.21 18.54 18.50 18.52 18.26 18.61 18.31 18.80 18.44 G5-Oxa-4mg / kg+Dual Antibody A-3mg / kg,QW+BIW 17.58 17.67 17.72 17.48 17.68 17.87 17.35 17.52 17.65 G6-Oxa-4mg / kg+Dual Antibody A-6mg / kg,QW+BIW 18.06 18.24 18.41 18.04 18.25 18.57 17.87 17.70 17.88

[0174] Example 7 Ability of anti-CD47-CLDN18.2 bispecific antibody to block the binding of CD47 to SIRPα on tumor cells expressing single and dual targets

[0175] The ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of CD47 to SIRPα on NUGC-4 and the above-mentioned hCLDN18.2-NUGC-4 cells was determined by flow cytometry. As a comparison, the ability of antibodies 1F8 (1F8 antibody in WO2018075857A1), F4AM4-IgG1 (referred to as F4AM4 in the accompanying drawings; the amino acid sequence of the heavy chain is SEQ ID NO: 19, and the amino acid sequence of the light chain is SEQ ID NO: 20) and A7H3L3 to block the binding of CD47 to SIRPα on NUGC-4 and hCLDN18.2-NUGC-4 tumor cells was also determined.

[0176] The specific method is as follows: Take 1×10 5NUGC-4 cells or hCLDN18.2-NUGC-4 cells were centrifuged at low speed (300 g) and the supernatant was removed. The cells at the bottom of the centrifuge tube were rinsed once with the prepared FACS buffer (1×PBS buffer containing 2% FBS); then, the gradient dilution of the test antibody was added to the rinsed cells and incubated for 1 hour; after rinsing the cells twice with FACS buffer, 100 μL of 1 μg / mL SIRPα-mFc (ACRO, SIA-H52A8) was added and incubated at 4°C for 1 hour; after rinsing three times with FACS buffer, 100 μL of 1:200 diluted PE-labeled goat anti-mouse Fc secondary antibody (Abcam, ab98742) was added, and the supernatant was removed after incubation at 4°C for 1 hour, and 200 μL of FACS buffer was added to the cells to resuspend the cells. Finally, the amount of SIRPα-mFc bound to the cells (expressed as mean fluorescence intensity (MFI)) was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0177] The results on NUGC-4 cells are as follows Fig.22a As shown: Antibody F4AM4-IgG1 can effectively block the binding of CD47 and SIRPα, with an IC50 of 0.033μg / mL (0.226nM); Antibody 1F8 has a weaker blocking effect, with an IC50 of 15.36μg / mL (105.6nM); and dual antibody A has almost no blocking ability.

[0178] The results on hCLDN18.2-NUGC-4 cells are as follows Figure 22b As shown: Antibody F4AM4-IgG1 still has a strong blocking ability on this tumor cell, with an IC50 of 0.056μg / mL (0.383nM); compared with the blocking ability on NUGC-4 cells, the blocking ability of dual antibody A on hCLDN18.2-NUGC-4 cells is significantly improved, and is better than antibody 1F8; the IC50s of dual antibody A and 1F8 blocking the binding of CD47 and SIRPα on hCLDN18.2-NUGC-4 are 0.765 μg / mL (4.476nM) and 11.98μg / mL (82.34nM), respectively; in addition, the anti-CLDN18.2 single-domain antibody NA3S-H1 has no blocking effect because it only binds to CLDN18.2. Based on this, although the blocking activity of bispecific antibody A on cells expressing CD47 single target is weaker than that of antibody 1F8, its blocking activity on cells expressing both CD47 and CLDN18.2 dual targets is significantly better than that of antibody 1F8, and it will exert a stronger ability to block the binding of CD47 to the receptor SIRPα.

[0179] Example 8 In vivo tumor inhibition experiment of anti-CD47-CLDN18.2 bispecific antibody

[0180] 8.1 In vivo tumor inhibition experiment 1

[0181] Female nude mice (16-18 g) aged 6-7 weeks were housed in an independent ventilation box with constant temperature and humidity. The temperature in the room was 21-24°C and the humidity was 30-53%. 6 hCLDN18.2-NUGC-4 cells were injected subcutaneously into the left axilla of nude mice (day 0). When the subcutaneous tumor volume of the mice reached 300-400mm 3 Around 20 days after the operation (day 20), the samples of mice with large differences in tumor volume were removed, and then they were randomly divided into groups according to the tumor volume (8 mice in each group): PBS treatment group, NA3S-H1 monoclonal antibody administration group, A7H3L3 monoclonal antibody administration group, NA3S-H1+A7H3L3 combined administration group and dual antibody A administration group. Taking NA3S-H1 monoclonal antibody 5mg / kg as the standard, all other drugs were administered in equimolar doses, namely A7H3L3 monoclonal antibody 9.4mg / kg, NA3S-H1+A7H3L3 combined 5mg / kg+9.4mg / kg, dual antibody A10.6mg / kg. The drugs were administered twice a week, alternating between intraperitoneal injection (ip) and intravenous injection (iv). The length (mm) and width (mm) of the tumor were observed and recorded at any time, and the tumor volume (V) was calculated as follows: V = (length × width 2 ) / 2, tumor inhibition rate TGI (%) = (1-average tumor volume of drug administration group / average tumor volume of PBS treatment group) × 100%.

[0182] Antibody tumor inhibition results Fig.23a As shown in Table 13, it can be seen that: at this equimolar dose, the NA3S-H1 monoclonal antibody administration group showed almost no tumor inhibitory effect, and the other groups showed a certain tumor inhibitory effect, among which dual antibody A had the best effect, reaching a tumor inhibition rate of nearly 54.54% before the 39th day, and the tumor size was close to the initial tumor volume on the 20th day, and was better than the result of the combination drug (A7H3L3+NA3S-H1(9.4+5mpk)), which indicates that the binding of dual antibody A to CLDN18.2 can enhance its blocking effect on the binding of CD47 and SIRPα.

[0183] Table 13 Tumor inhibition rate of bispecific antibodies in mice

[0184] Number of days NA3S-H1 A7H3L3 Double Antibody A A7H3L3+NA3S-H1 25 2.96％ -1.31％ 3.41％ 12.73％ 28 8.76％ 2.17％ 27.69％ 18.40％ 32 -5.33％ 3.73％ 40.26％ 19.30％ 35 -6.94％ 2.10％ 44.31％ 19.49％ 39 2.01％ 15.00％ 54.54％ 33.71％ 42 -7.26％ 14.65％ 51.45％ 30.84％ 46 -8.69％ 24.65％ 48.33％ 32.59％ 49 -5.94％ 25.18％ 45.12％ 25.62％

[0185] 8.2 In vivo tumor inhibition experiment 2

[0186] The mice were randomly divided into groups (8 mice per group): PBS treatment group, monoclonal antibody NA3S-H1 administration group, and dual antibody A group. The first administration was performed on the day of inoculation (day 0), and the administration was performed twice a week, with NA3S-H1 monoclonal antibody 2.5 mg / kg as the standard, and dual antibody A was administered at an equimolar dose, i.e. 5.3 mg / kg. The rest was consistent with Example 8.1.

[0187] The results are as follows Figure 23b As shown, at this equimolar dose, the NA3S-H1 monoclonal antibody administration group showed a certain tumor inhibition effect, with a tumor inhibition rate of about 54.99% (day 34); the tumors in all mice in the dual-antibody A group were completely inhibited, with a tumor inhibition rate close to 100% (day 34). This result proves that the tumor inhibition effect of dual-antibody A is significantly better than that of the anti-CLDN18.2 monoclonal antibody NA3S-H1.

[0188] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. SEQUENCE LISTING <110> Baochuan Biopharmaceutical Technology (Shanghai) Co., Ltd. <120> Use of a bispecific antibody against CLDN 18.2 and CD47 for treating diseases <130> P22010370C <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 Antibody LCDR1 <400> 1 Arg Ala Ser Gln Asp Ile Ser Asn His Leu Asn 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 Antibody LCDR2 <400> 2 Tyr Thr Ser Arg Ile His Ser 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 Antibody LCDR3 <400> 3 Gln Gln Gly Tyr Thr Leu Pro Phe 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 Antibody HCDR1 <400> 4 Asp Ile Tyr Ile Tyr 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 Antibody HCDR2 <400> 5 Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe Gln 1 5 10 15 Gly <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 Antibody HCDR3 <400> 6 Gly Tyr Gly Ser Gly Phe Ala Tyr 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 Antibody HCDR1 <400> 7 Ile Pro Val Met Gly 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 Antibody HCDR2 <400> 8 Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys Gly 1 5 10 15 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 Antibody HCDR3 <400> 9 Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val 1 5 10 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 antibody light chain variable region <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser Ser Phe Arg Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 antibody light chain constant region <400> 11 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Variable region of anti-CD47 antibody heavy chain <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 13 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 antibody heavy chain constant region <400> 13 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 14 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLDN18.2 antibody heavy chain variable region amino acid sequence <400> 14 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 antibody light chain amino acid sequence (double anti-A short chain) <220> <221> SIGNAL <222> (1)..(20) <223> Signal peptide <400> 15 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Ile Ser Asn His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Ile His Ser Gly Val Pro Ser 65 70 75 80 Ser Phe Arg Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr 100 105 110 Thr Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 16 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Anti-CD47 antibody heavy chain amino acid sequence <220> <221> SIGNAL <222> (1)..(20) <223> Signal peptide <400> 16 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Ile Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60 Leu Glu Trp Ile Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr 65 70 75 80 Asp Gln Lys Phe Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr 85 90 95 Asn Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 17 <211> 597 <212> PRT <213> Artificial Sequence <220> <223> Double antibody A heavy chain <220> <221> SIGNAL <222> (1)..(20) <223> Signal peptide <400> 17 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Ile Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60 Leu Glu Trp Ile Gly Lys Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr 65 70 75 80 Asp Gln Lys Phe Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr 85 90 95 Asn Thr Ala Tyr Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Ser Gly Phe Ala Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 465 470 475 480 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 485 490 495 Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro Val Met Gly 500 505 510 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Gly Ile 515 520 525 Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys Gly Arg Phe 530 535 540 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn 545 550 555 560 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Leu Val 565 570 575 Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln Gly Thr Leu 580 585 590 Val Thr Val Ser Ser 595 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 18 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 19 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> F4AM4-IgG1 heavy chain <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Thr Asp Gly Thr Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Pro Tyr Tyr Gly Thr Arg Tyr Gly Ser Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 20 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> F4AM4-IgG1 light chain <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Lys Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. Use of a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 in the preparation of a medicament for preventing and / or treating pancreatic cancer and / or colorectal cancer, wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to CD47 and a second antigen-binding domain that specifically binds to CLDN 18.2; The amino acid sequence of LCDR1 of the first antigen binding domain is shown in SEQ ID NO:1, the amino acid sequence of LCDR2 is shown in SEQ ID NO:2, the amino acid sequence of LCDR3 is shown in SEQ ID NO:3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:6; The second antigen binding domain comprises an immunoglobulin single variable domain VHH; The amino acid sequence of HCDR1 of the second antigen binding domain is shown in SEQ ID NO:7, the amino acid sequence of HCDR2 is shown in SEQ ID NO:8, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:9; The heavy chain of the first antigen-binding domain and the heavy chain variable region of the second antigen-binding domain are connected by a linker; The heavy chain variable region of the second antigen-binding domain is connected to the C-terminus of the heavy chain of the first antigen-binding domain via a linker.

2. The use according to claim 1, characterized in that The framework regions of the light chain variable regions and / or heavy chain variable regions of the first and second antigen-binding domains are human framework regions.

3. The use according to claim 2, characterized in that The amino acid sequence of the light chain variable region of the first antigen binding domain is shown in SEQ ID NO: 10; and / or, The amino acid sequence of the heavy chain variable region of the first antigen binding domain is shown in SEQ ID NO: 12; and / or, The amino acid sequence of the heavy chain variable region of the second antigen binding domain is shown in SEQ ID NO:

14.

4. The use according to claim 3, characterized in that The light chain constant region of the first antigen-binding domain is a human κ light chain constant region, and / or the heavy chain constant region of the first antigen-binding domain is a human IgG1 heavy chain constant region.

5. The use according to claim 4, characterized in that The amino acid sequence of the light chain constant region of the first antigen binding domain is shown in SEQ ID NO:11; and / or the amino acid sequence of the heavy chain constant region of the first antigen binding domain is shown in SEQ ID NO:

13.

6. The use according to claim 5, characterized in that The amino acid sequence of the heavy chain of the first antigen binding domain is shown in SEQ ID NO:16, and / or the amino acid sequence of the linker is shown in SEQ ID NO:

18.

7. The use according to claim 6, characterized in that The bispecific antibody comprises a long chain and a short chain, the short chain comprises the amino acid sequence shown in SEQ ID NO:15, and the long chain comprises the amino acid sequence shown in SEQ ID NO:

17.

8. Use of a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 in combination with a platinum drug in the preparation of a drug for preventing and / or treating tumors, characterized in that: The bispecific antibody or antigen-binding fragment thereof is defined in the use according to any one of claims 1 to 7, wherein the tumor is a CLDN 18.2 and / or CD47 positive tumor; the tumor is pancreatic cancer, colorectal cancer or gastric cancer.

9. The use according to claim 8, characterized in that The platinum drug is selected from cisplatin, carboplatin and oxaliplatin.

10. The use according to claim 9, characterized in that The platinum drug is oxaliplatin.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 as defined in the use according to any one of claims 1 to 7, and a platinum drug.

12. The pharmaceutical composition according to claim 11, characterized in that The platinum drug is selected from cisplatin, carboplatin and oxaliplatin.

13. The pharmaceutical composition according to claim 12, characterized in that The platinum drug is oxaliplatin.

14. A medicine kit, characterized in that: The kit comprises kit A and kit B, wherein: The drug kit A comprises a bispecific antibody or an antigen-binding fragment thereof targeting CLDN 18.2 and CD47 as defined in the application according to any one of claims 1 to 7; The drug kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.

15. The kit of claim 14, wherein: The chemotherapeutic agent is a platinum drug.

16. The kit of claim 15, wherein: The platinum drug is selected from cisplatin, carboplatin and oxaliplatin.

17. The kit of claim 16, wherein: The platinum drug is oxaliplatin.

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