Bispecific antibody comprising an anti-CLDN18.2 antibody, and its pharmaceutical composition and use

Abstract

Disclosed are bispecific antibodies comprising an anti-CLDN18.2 antibody, as well as pharmaceutical compositions and uses thereof. The bispecific antibody comprises a first protein functional region and a second protein functional region, wherein the first protein functional region is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, and the second protein functional region targets a target different from CLDN18.2 (e.g., CD47). The bispecific antibody has good biological activity and prospects for anti-tumor applications.

Description

Technical Field

[0001] Technical Field The present invention belongs to the field of biomedicine and relates to an anti-CLDN18.2 antibody, a bispecific antibody comprising an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition thereof, and its use. Specifically, the bispecific antibody is an anti-CLDN18.2 / anti-CD47 bispecific antibody.

Background Art

[0002] Background Tumors, especially malignant tumors, are currently serious health-threatening diseases worldwide and are the second most common cause of death among various diseases. In recent years, the incidence of the disease has been increasing significantly. Malignant tumors are characterized by poor treatment responses, high late metastasis rates, and poor prognoses. Currently, the conventional treatment methods clinically adopted (radiotherapy, chemotherapy, surgical treatment, etc.) can significantly relieve pain and extend the survival period, but there are significant limitations to the method, and it is difficult to further improve its effectiveness.

[0003] CD47 is also referred to as integrin-associated protein (IAP). CD47 is a five-span transmembrane protein with a molecular weight of approximately 50 kDa and belongs to the immunoglobulin superfamily. Its extracellular N-terminus is an IgV domain and binds to αvβ3 (CD51 / CD61) and αIIbβ3 (CD41 / CD61) integrins. CD47 is involved in various physiological functions such as cell migration, T cell and dendritic cell (DC) activation, and axonal development.

[0004] CD47 is expressed on all types of cells, including red blood cells, and is highly expressed on almost all types of tumor cells. It is also associated with poor prognosis. CD47 has two ligands, namely signal regulatory protein-α (SIRPα) and thrombospondin-1 (TSP1). SIRPα is a receptor transmembrane glycoprotein containing an immunoglobulin domain, belongs to the SIRP family, and is mainly expressed on phagocytes and nerve cells. In the CD47-SIRPα pathway, the CD47 protein binds to SIRPα, phosphorylates its immunoreceptor tyrosine-based inhibitory motif (ITIM), and then recruits the SHP-1 protein into the cell to cause a series of cascade reactions, inhibiting macrophage phagocytosis (Matozaki T, Murata Y, Okazawa H, et al., Functions and molecular mechanisms of the CD47-SIRPα signaling pathway. Trends in cell biology, 2009, 19(2): 72-80). Normal red blood cells are not phagocytosed due to the inhibitory signal generated by the binding of CD47 on the cell membrane surface to SIPRα of macrophages (Oldenborg P A, Zheleznyak A, Fang Y F, et al., Role of CD47 as a marker of self on red blood cells. Science, 2000, 288(5473): 2051-2054). TSP1, a homotrimer consisting of three peptide chains, is involved in cell proliferation, apoptosis, adhesion, migration, angiogenesis, and other processes through interactions with other cell surface receptors, matrix components, and growth factors (Jiang P, Lagenaur CF, Narayanan V. Integrin-associated Protein Is a Ligand for the P84 Neural Adhesion Molecule. Journal of Biological Chemistry 1999, 274: 559-62).

[0005] Macrophages are derived from monocytes, which are derived from progenitor cells in the bone marrow. Their main functions are to phagocytose cell debris and pathogens and to activate lymphocytes or other immune cells to respond to pathogens in the form of fixed or free cells. Currently, research suggests that tumor cells have a mechanism to escape macrophage phagocytosis. During the growth of tumor cells, specific proteins such as calreticulin are formed on the surface, exposing the identity of the tumor cells, and as a result, the tumor cells are phagocytosed by attracted macrophages. However, since the CD47-SIRPα pathway activates the inhibition of macrophage phagocytosis, tumor cells with highly expressed CD47 are misrecognized as normal cells by macrophages with SIRPα and thus avoid macrophage phagocytosis (CD47 is upregulated in circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Jaiswal S, Jamieson C H M, Pang W W, et al., Cell, 2009, 138(3): 271-285).

[0006] The CLDN18.2 protein is an integrin membrane protein present at epithelial and endothelial tight junctions. It consists of 261 amino acids and is one of the members of the Claudin (CLDN) family. Both its N-terminus and C-terminus are located intracellularly. The whole protein is expressed on the cell membrane. CLDN18.2 has four transmembrane domains, two extracellular loops, and one intracellular loop and is involved in the formation of tight junction structures between cells (Gunzel, D.; Yu, A. S. L. Claudins and the Modulation of Tight Junction Permeability [J]. Physiological Reviews. 2013, 93(2), 525-569).

[0007] The extracellular loop 2 of the CLDN18.2 protein has a helix-turn-helix structure and forms a close bond with the extracellular loop of the CLDN18.2 protein of adjacent cells through hydrophobic bonds between aromatic residues. The extracellular loop of the CLDN18.2 protein of adjacent cells maintains a close bond between epithelial cells and endothelial cells through interactions, regulates the intercellular osmotic pressure, maintains the polarity of epithelial cells and endothelial cells, is involved in cell proliferation, and can be involved in various signal transduction pathways through its carboxyl terminus rich in serine, threonine, and tyrosine (Cao Chenxin, Research progress of transmembrane CLDN18.2 in targeted cancer therapy [J]. International Journal of Biologicals, 2020(01): 35-36-37-38-39-40).

[0008] The expression of the CLDN18.2 protein is highly restricted in normal healthy tissues and is found only in differentiated epithelial cells of the gastric mucosa, which is beneficial for maintaining the barrier function of the gastric mucosa. However, the CLDN18.2 protein often changes abnormally during the occurrence and progression of malignant tumors. For example, when gastric epithelial tissue is subjected to malignant transformation, disruption of cell polarity will result in the exposure of epitopes of the CLDN18.2 protein on the cell surface. At the same time, the CLDN18.2 gene is also abnormally activated and is highly selectively and stably expressed in specific tumor tissues such as gastric cancer, affecting intercellular and extracellular matrix binding, affecting the maintenance of cell gate function and polarity, thereby causing changes in intercellular cytokine and ion permeability, damaging the gate function, and causing cell proliferation transformation (Hashimoto Itaru, Oshima Takashi, Claudins and Gastric Cancer: An Overview.[J]. Cancers (Basel), 2022, 14:uncertain).

[0009] Both CD47 and CLDN18.2 are tumor membrane antigens. Antibodies targeting either CD47 or CLDN18.2 have shown anti-tumor activity. However, multiple blood cells and components including platelets and red blood cells also express CD47. When CD47 antibody drugs bind to red blood cells, platelets, etc., they may cause damage to red blood cells and platelets through ADCC or ADCP, resulting in severe blood toxicity and side effects such as anemia and / or thrombocytopenia. Summary of the Invention Means for Solving the Problems

[0010] Overview Through intensive research and creative efforts, the inventors obtained an anti-CLDN18.2 antibody and, based on this, developed an anti-CLDN18.2 / anti-CD47 bispecific antibody. The inventors surprisingly found that the anti-CLDN18.2 antibody of the present invention (also abbreviated as the antibody or the antibody of the present invention) and the anti-CLDN18.2 / anti-CD47 bispecific antibody (also abbreviated as the bispecific antibody or the bispecific antibody of the present invention) have excellent affinity and / or specificity, can bind to tumor cells expressing CD47 and / or CLDN18.2 with high specificity without causing agglutination or damage to red blood cells, show good safety, and have good anti-tumor prospects. The present invention will be described in detail below.

[0011] One aspect of the present invention relates to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof. The anti-CLDN18.2 antibody includes a heavy chain variable region containing HCDR1 - HCDR3 and a light chain variable region containing LCDR1 - LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO: 31, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 32, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 33, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 34, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 35, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 36.

[0012] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, and the amino acid sequence of the heavy chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41; the amino acid sequence of the light chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO: 30, SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47.

[0013] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 47; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 47; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 45; or the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 41, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 47.

[0014] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, and the constant region of the heavy chain of the antibody is an Igγ-1 chain C region (for example, having the amino acid sequence shown in SEQ ID NO: 61) or an Igγ-4 chain C region (for example, NCBI accession: P01861.1), and the constant region of the light chain of the antibody is an Igκ chain C region (for example, NCBI accession: P01834).

[0015] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, and the anti-CLDN18.2 antibody or an antigen-binding fragment thereof is selected from Fab, Fab’, F(ab’)2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain variable fragment, humanized antibody, chimeric antibody, or diabody.

[0016] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, and the antibody contains non-CDR regions derived from non-mouse species such as human antibodies.

[0017] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, and the EC of the anti-CLDN18.2 antibody with respect to binding to cells expressing CLDN18.2 50 is 15 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, or 5 nM or less, and preferably, the EC 50 is determined by FACS (flow cytometry). In one embodiment of the present invention, the cells expressing CLDN18.2 are CHO-K1 cells expressing CLDN18.2. In one embodiment of the present invention, the cells expressing CLDN18.2 are CHO-K1 cells overexpressing CLDN18.2.

[0018] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, and the EC of the anti-CLDN18.2 antibody with respect to binding to cells expressing both CLDN18.2 and CD47 50 is 10 nM or less, 5 nM or less, or 2 nM or less, and preferably, the EC 50It is determined by FACS. In one embodiment of the present invention, the cells expressing both CLDN18.2 and CD47 are CHO-K1 cells expressing both CLDN18.2 and CD47. In one embodiment of the present invention, the cells expressing both CLDN18.2 and CD47 are CHO-K1 cells overexpressing both CLDN18.2 and CD47.

[0019] In some embodiments of the present invention, the anti-CLDN18.2 antibody is an anti-CLDN18.2 monoclonal antibody.

[0020] The present invention also relates to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, and the anti-CLDN18.2 antibody is a monoclonal antibody produced by the hybridoma cell line LT020 deposited with the China Center for Type Culture Collection (CCTCC) under the CCTCC designation CCTCC NO. C2022124.

[0021] The present invention also relates to the hybridoma cell line LT020 deposited with the China Center for Type Culture Collection (CCTCC) under the CCTCC designation CCTCC NO. C2022124.

[0022] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention.

[0023] Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.

[0024] Yet another aspect of the present invention relates to a host cell comprising the isolated nucleic acid molecule of the present invention or the recombinant vector of the present invention.

[0025] Yet another aspect of the present invention relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or the antigen-binding fragment thereof is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention, preferably, the small molecule drug is a small molecule cytotoxic drug, and more preferably, the small molecule drug is an anti-tumor chemotherapy drug.

[0026] The chemotherapy drug may be a conventional anti-tumor chemotherapy drug such as an alkylating agent, an antimetabolite, an anti-tumor antibiotic, a plant-based anti-cancer agent, a hormone, and an immunizing agent.

[0027] In one or more embodiments of the present invention, there is provided an antibody-drug conjugate in which an antibody or an antigen-binding fragment thereof is linked to a small molecule drug via a linker, and the linker may be one known to those skilled in the art. For example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond.

[0028] In one or more embodiments of the present invention, there is provided an antibody-drug conjugate in which the molar ratio of the antibody or an antigen-binding fragment thereof to the small molecule drug is 1:(2-4), for example, 1:2, 1:3, or 1:4.

[0029] Yet another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention or an antibody-drug conjugate according to any aspect of the present invention, and optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0030] In some embodiments of the present invention, the pharmaceutical composition further comprises an effective amount of an anti-CD47 antibody or an antigen-binding fragment thereof. Preferably, the anti-CD47 antibody comprises a heavy chain variable region comprising HCDR1-HCDR3 and a light chain variable region comprising LCDR1-LCDR3: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 7. The amino acid sequence of LCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 10.

[0031] In some embodiments of the present invention, a pharmaceutical composition is provided. The amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is selected from SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 24; The amino acid sequence of the light chain variable region of the anti-CD47 antibody is selected from SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26; Preferably, in the anti-CD47 antibody: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; 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: 14; 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: 18; 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: 22; 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: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; Or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26.

[0032] In some embodiments of the present invention, there is provided a pharmaceutical composition in which the heavy chain constant region of the anti-CD47 antibody is an Igγ-1 chain C region (e.g., NCBI accession: P01857) or an Igγ-4 chain C region (e.g., NCBI accession: P01861.1), and the light chain constant region of the anti-CD47 antibody is an Igκ chain C region (e.g., NCBI accession: P01834).

[0033] Yet another aspect of the present invention is: a first protein functional region targeting CLDN18.2, and a second protein functional region targeting a target other than CLDN18.2 (e.g., CD47) relates to a bispecific antibody comprising wherein: the first protein functional region is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention.

[0034] Unless otherwise specified, the bispecific antibody of the present invention is an anti-CLDN18.2 / anti-CD47 bispecific antibody.

[0035] In some embodiments of the present invention, there is provided a bispecific antibody in which the second protein functional region is an anti-CD47 antibody or an antigen-binding fragment thereof: The anti-CD47 antibody includes a heavy chain variable region containing HCDR1 to HCDR3 and a light chain variable region containing LCDR1 to LCDR3: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 7, The amino acid sequence of LCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 10.

[0036] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is selected from SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO: 24; There is provided a bispecific antibody in which the amino acid sequence of the light chain variable region of the anti-CD47 antibody is selected from SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26.

[0037] In some embodiments of the present invention, in the anti-CD47 antibody: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; 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: 14; 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: 18; 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: 22; 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: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; Or A bispecific antibody is provided, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26.

[0038] In some embodiments of the present invention, a pharmaceutical composition product is provided, wherein the heavy chain constant region of the anti-CD47 antibody is the C region of the Igγ-1 chain (e.g., NCBI accession: P01857) or the C region of the Igγ-4 chain (e.g., NCBI accession: P01861.1); and the light chain constant region of the anti-CD47 antibody is the C region of the Igκ chain (e.g., NCBI accession: P01834).

[0039] In some embodiments of the present invention, a bispecific antibody is provided in which the first protein functional region and the second protein functional region are independently a fusion protein of a single-chain variable fragment or a half-molecule monovalent antibody (IgG half-molecule, IgG-HM).

[0040] In some embodiments of the present invention, the first protein functional region is a fusion protein of a single-chain variable fragment, and the second protein functional region is a half-molecule monovalent antibody (IgG half-molecule, IgG-HM); or the first protein functional region is a half-molecule monovalent antibody (IgG half-molecule, IgG-HM), and the second protein functional region is a fusion protein of a single-chain variable fragment, and a bispecific antibody is provided.

[0041] In some embodiments of the present invention, the first protein functional region is a fusion protein of a single-chain variable fragment targeting CLDN18.2, and the second protein functional region is a half-molecule monovalent antibody (IgG half-molecule, IgG-HM) targeting CD47; or the first protein functional region is a half-molecule monovalent antibody (IgG half-molecule, IgG-HM) targeting CLDN18.2, and the second protein functional region is a fusion protein of a single-chain variable fragment targeting CD47, and a bispecific antibody is provided.

[0042] In some embodiments of the present invention, the fusion protein of a single-chain variable fragment consists of a single-chain variable fragment, a hinge region, and an Fc fragment, or consists of a single-chain variable fragment and a heavy-chain constant region. Preferably, the hinge region and the Fc fragment are the hinge region and the Fc fragment of human IgG1; preferably, the hinge region and the Fc fragment have the amino acid sequence shown in SEQ ID NO: 62; the heavy-chain constant region is the heavy-chain constant region of human IgG1, and preferably, the heavy-chain constant region has the amino acid sequence shown in SEQ ID NO: 63, and a bispecific antibody is provided.

[0043] In some embodiments of the present invention, the Fc fragment or heavy chain constant region in the fusion protein of the single-chain variable fragment has a knob mutation (e.g., S354C and T366W mutations); the heavy chain constant region of the half-molecule monovalent antibody is the heavy chain constant region of human IgG1 and has a hole mutation (e.g., Y349C, T366S, L368A, and Y407V mutations), and a bispecific antibody is provided.

[0044] In some embodiments of the present invention, the bispecific antibody consists of the peptide chain shown in SEQ ID NO: 53, the peptide chain shown in SEQ ID NO: 56, and the peptide chain shown in SEQ ID NO: 59, preferably, the peptide chain shown in SEQ ID NO: 53 and the peptide chain shown in SEQ ID NO: 56 are linked by one or more disulfide bonds in the hinge region, and the peptide chain shown in SEQ ID NO: 56 and the peptide chain shown in SEQ ID NO: 59 are linked by one or more disulfide bonds; preferably, the peptide chain shown in SEQ ID NO: 53 and the peptide chain shown in SEQ ID NO: 56 are linked by two disulfide bonds in the hinge region, and the peptide chain shown in SEQ ID NO: 56 and the peptide chain shown in SEQ ID NO: 59 are linked by one disulfide bond.

[0045] In some embodiments of the present invention, the EC of the bispecific antibody with respect to binding to cells expressing CLDN18.2 50 is 20 nM or less, 15 nM or less, or 12 nM or less, and preferably, the EC 50 is determined by FACS; and / or the EC of the bispecific antibody with respect to binding to CD47 on the surface of the erythrocyte membrane 50 is 20 nM or more, 40 nM or more, or 50 nM or more, and preferably, the EC 50A bispecific antibody determined by FACS is provided. In one embodiment of the present invention, the cells expressing CLDN18.2 are CHO-K1 cells expressing CLDN18.2. In one embodiment of the present invention, the cells expressing CLDN18.2 are CHO-K1 cells overexpressing CLDN18.2.

[0046] In some embodiments of the present invention, the EC of the bispecific antibody with respect to binding to cells expressing both CLDN18.2 and CD47 50 is 10 nM or less, 5 nM or less, or 2 nM or less, and preferably, the EC 50 A bispecific antibody determined by FACS is provided. In one embodiment of the present invention, the cells expressing both CLDN18.2 and CD47 are CHO-K1 cells expressing both CLDN18.2 and CD47. In one embodiment of the present invention, the cells expressing both CLDN18.2 and CD47 are CHO-K1 cells overexpressing both CLDN18.2 and CD47.

[0047] In some embodiments of the present invention, the bispecific antibody does not induce agglutination of red blood cells at a concentration of 3000 nM or less.

[0048] In some embodiments of the present invention, the bispecific antibody has ADCP activity, ADCC activity, and CDC activity.

[0049] In some embodiments of the present invention, the anti-CD47 antibody is an anti-CD47 monoclonal antibody.

[0050] Yet another aspect of the present invention relates to an isolated nucleic acid molecule encoding a bispecific antibody according to any aspect of the present invention.

[0051] Yet another aspect of the present invention relates to a vector containing the isolated nucleic acid molecule of the present invention.

[0052] Yet another aspect of the present invention relates to a host cell containing the isolated nucleic acid molecule of the present invention or the vector of the present invention.

[0053] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of a bispecific antibody according to any aspect of the present invention and one or more pharmaceutically acceptable excipients.

[0054] Another aspect of the present invention relates to the use of an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention or a bispecific antibody according to any aspect of the present invention in the preparation of a medicament for treating or preventing a tumor, preferably, the tumor is a CD47 and / or CLDN18.2 positive tumor; Preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0055] An anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention or a bispecific antibody according to any aspect of the present invention is used for treating or preventing a tumor, Preferably, the tumor is a CD47 and / or CLDN18.2 positive tumor; Preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0056] Another aspect of the present invention is a method for treating or preventing a tumor, comprising the step of administering an effective amount of an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention or a bispecific antibody according to any aspect of the present invention to a subject in need thereof, Preferably, the tumor is a CD47 and / or CLDN18.2 positive tumor; Preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic carcinoma, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer, and relates to a method.

[0057] In some embodiments of the present invention, a method for treating or preventing a tumor is provided, in which drug administration is performed before or after surgery and / or before or after radiotherapy.

[0058] In some embodiments of the present invention, a method for treating or preventing a tumor is provided, The anti-CLDN18.2 antibody or its antigen-binding fragment or bispecific antibody is administered at a unit dose of 0.1 to 100 mg per kg of body weight, preferably 5 to 50 mg or 5 to 15 mg per kg of body weight, Preferably, drug administration is performed once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the administration route is intravenous drip or intravenous injection.

[0059] In the present invention, unless otherwise defined, the scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In addition, the laboratory operations of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are routine procedures widely used in the corresponding fields. On the other hand, for a better understanding of the present invention, the definitions and explanations of related terms are provided below.

[0060] As used herein, the term EC 50 refers to the concentration of 50% of the maximum effect, that is, the concentration that can cause 50% of the maximum effect.

[0061] As used herein, the term "antibody" generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains are classified as κ and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε. The isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain further includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including binding to the first component (C1q) of the classical complement system of various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into hypervariable regions (termed complementarity-determining regions (CDRs)), between which conserved regions termed framework regions (FRs) are distributed. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site.The assignment of amino acids to regions or domains is based on the definitions in Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol., 1987; 196: 901-917; Chothia et al., Nature, 1989; 342: 878-883, or the IMGT numbering system. See the definitions in Ehrenmann F, Kaas Q, Lefranc M P., IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]., Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0062] The term "antibody" is not limited by any particular method of producing the antibody. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies may be antibodies of different isotypes such as IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0063] As used herein, the terms "mAb" and "monoclonal antibody" refer to antibodies or antibody fragments derived from a group of highly homologous antibodies, i.e., a group of identical antibody molecules, excluding naturally occurring spontaneous mutations. Monoclonal antibodies are highly specific for a single epitope on an antigen. Polyclonal antibodies generally contain at least two or more different antibodies that recognize different epitopes on an antigen, as compared to monoclonal antibodies. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Koehler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA technology (see, for example, U.S. Patent No. 4,816,567).

[0064] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the expected specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the receptor antibody can also be replaced by the amino acid residues of the corresponding non-human antibody or the amino acid residues of another antibody to further improve or optimize the performance of the antibody. For details of humanized antibodies, see, for example, Jones et al., Nature, 1986; 321: 522-525; Reichmann et al., Nature, 1988; 332: 323-329; Presta, Curr. Op. Struct. Biol., 1992; 2: 593-596; and Clark, Immunol. Today, 2000; 21: 397-402. In some cases, the antigen-binding fragment of the antibody is VH and V L The diabody is one in which the domains are expressed on a single polypeptide chain. However, the linker used is too short to allow the pairing of the two domains on the same chain. Thus, the domains are forced to pair with complementary domains on the other chain, generating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA, 1993; 90: 6444-6448 and Poljak R.J. et al., Structure, 1994; 2: 1121-1123).

[0065] As used herein, the term "single-chain variable fragment (ScFv)" refers to a molecule comprising an antibody heavy-chain variable region (V H ) and an antibody light-chain variable region (V L ) linked via a linker. The V L and V H domains are paired to form a monovalent molecule by a linker that allows them to form a single polypeptide chain (see, for example, Bird et al., Science, 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. USA, 1988; 85:5879-5883). Such scFv molecules have the following general structure: NH2-V L -linker fragment-V H -COOH or NH2-V H -linker fragment-V L-COOH. Suitable linkers in the prior art consist of repetitions of the GGGGS amino acid sequence or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al., Proc. Natl. Acad. Sci. USA, 1993; 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al., Protein Eng., 1995; 8: 725-731, Choi et al., Eur. J. Immunol., 2001; 31:94-106, Hu et al., Cancer Res., 1996; 56: 3055-3061, Kipriyanov et al., J. Mol. Biol., 1999; 293: 41-56 and Roovers et al., Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59).

[0066] As used herein, the term "isolated" refers to obtaining from nature by artificial means. When a particular "isolated" substance or component occurs in nature, changes may occur within the natural environment of the substance or component, or the substance or component may be isolated from the natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide occurs naturally in a particular living animal, and the same polynucleotide or polypeptide having high purity isolated from such a natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0067] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, and as a result, the genetic material elements carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage; and animal viruses. Examples of animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector can contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may further contain an origin of replication.

[0068] As used herein, the term "host cell" refers to a cell into which a vector can be introduced and includes, but is not limited to, prokaryotic cells such as Escherichia coli (E. coli) or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0069] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or is specific for an antigen) is one in which the antibody binds to a target antigen at a rate of about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K D ) means that the antibody binds to the antigen.

[0070] As used herein, "K D The term "dissociation equilibrium constant" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. A smaller dissociation equilibrium constant indicates stronger antibody-antigen binding and higher affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The dissociation equilibrium constant (K D ) binds to an antigen (e.g., CLDN18.2 protein). D can be determined using methods known to those skilled in the art, for example, using a Fortebio molecular interaction instrument.

[0071] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably, and the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably. Furthermore, as used herein, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0072] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. Such carriers and / or excipients are well known in the art (see, e.g., Remington’s Pharmaceutical Sciences, edited by Gennaro AR, 19th Ed., Pennsylvania, Mack Publishing Company, 1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0073] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, a prophylactically effective amount against a disease (e.g., a tumor) refers to an amount sufficient to prevent, arrest, or delay the onset of the disease (e.g., a tumor); a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient suffering from the disease. It is apparent that determining such an effective amount is within the ability of one of ordinary skill in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease being treated, the overall state of the patient's immune system, the general condition of the patient such as age, weight, and gender, the route of administration, and other treatments being administered concurrently.

[0074] As used herein, when referring to the amino acid sequence of the CD47 protein (NCBI GenBank: NP_001768.1), it includes the full-length CD47 protein, or the extracellular fragment of CD47, CD47 ECD, or a fragment containing CD47 ECD, and also includes a fusion protein of the full-length CD47 protein, or a fusion protein of CD47 ECD, for example, a fragment fused to the Fc protein fragment of mouse or human IgG (mFc or hFc). However, those skilled in the art will understand that in the amino acid sequence of the CD47 protein, mutations or variations (including but not limited to substitutions, deletions, and / or additions) can occur naturally or be introduced artificially without affecting its biological function. Therefore, in the present invention, the term "CD47 protein" shall include all such sequences including their natural or artificial variants. In addition, when a sequence fragment of the CD47 protein is described, the CD47 protein also includes the corresponding sequence fragment in its natural or artificial variants.

[0075] As used herein, when referring to the amino acid sequence of the CLDN18.2 protein (NCBI GenBank: NP_001002026.1), it includes the full-length CLDN18.2 protein, or the extracellular fragment of CLDN18.2, CLDN18.2 ECD, or a fragment containing CLDN18.2 ECD, and also includes a fusion protein of the full-length CLDN18.2 protein, or a fusion protein of CLDN18.2 ECD, for example, a fragment fused to the Fc protein fragment of mouse or human IgG (mFc or hFc). However, those skilled in the art will understand that in the amino acid sequence of the CLDN18.2 protein, mutations or variations (including but not limited to substitutions, deletions, and / or additions) can occur naturally or be introduced artificially without affecting its biological function. Therefore, in the present invention, the term "CLDN18.2 protein" is intended to include all such sequences including their natural or artificial variants. In addition, when a sequence fragment of the CLDN18.2 protein is described, it also includes the corresponding sequence fragment in its natural or artificial variant.

[0076] In the present invention, the term "ADCP" refers to antibody-dependent cell phagocytosis. The Fc fragment of an antibody that binds to a cell surface antigen binds to the Fc receptor of a phagocytic cell such as a macrophage, which then mediates the phagocytosis of the antibody-bound cell by the phagocytic cell.

[0077] In the present invention, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. The Fab fragment of an antibody binds to an epitope of a virus-infected cell or a tumor cell, and the Fc fragment of the antibody binds to the Fc receptor (FcR) on the surface of a killer cell (such as an NK cell, macrophage, etc.) to mediate the direct killing of the target cell by the killer cell.

[0078] In the present invention, the term "CDC" refers to complement-dependent cytotoxicity. When an antibody specifically binds to a corresponding antigen on the cell membrane surface, a complex is formed, the complement system is activated, then MAC is formed on the surface of the target cell, resulting in subsequent lysis of the target cell. Complement can cause lysis of various bacteria and other pathogenic organisms and is an important defense mechanism against pathogenic organism infections.

[0079] In the present invention, the terms "first" (e.g., the first protein functional region or the first pharmaceutical product) and "second" (e.g., the second protein functional region or the second pharmaceutical product) are used to distinguish or clarify expressions and do not have a typical consecutive meaning unless otherwise specified.

[0080] In the present invention, the term "monovalent half-antibody (IgG half-molecule, IgG-HM)" refers to an antibody molecule consisting of one heavy chain and one light chain of an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4) that is a monovalent antibody (see, for example, FENG Yi-fan et al., Construction and activity analysis of HIV specific monovalent mAb 2G12, Chinese Journal of Viral Diseases, May 2015, 5(3): 171-175).

[0081] Beneficial effects of the present invention The present invention achieves one or more of the following effects: (1) The anti-CLDN18.2 antibody of the present invention has excellent affinity and specificity; (2) The bispecific antibody of the present invention can specifically and tightly bind to, for example, CLDN18.2 and exerts an antibody-mediated cell killing effect on cells expressing CLDN18.2; (3) The bispecific antibody of the present invention can specifically and tightly bind to, for example, cells expressing both CD47 and CLDN18.2 and exerts an antibody-mediated cell killing effect on cells expressing both CD47 and CLDN18.2; (4) The bispecific antibody of the present invention can specifically bind to CD47 and effectively block the binding of CD47 to SIRPα, thereby specifically alleviating CD47 / SIRPα-mediated immunosuppression and activating the immune response; (5) The first protein functional region and the second protein functional region in the bispecific antibody of the present invention have a synergistic effect; (6) The bispecific antibody of the present invention does not induce erythrocyte aggregation, has high safety, and can have ADCP, ADCC effects, and CDC effects.

Brief Description of the Drawings

[0082] Brief Description of the Drawings

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Mode for Carrying Out the Invention

[0083] The hybridoma cell line LT012 was deposited with the China Center for Type Culture Collection (CCTCC) on June 21, 2018, under the CCTCC designation of CCTCC NO. C2018135, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0084] The hybridoma cell line LT020 was deposited with the China Center for Type Culture Collection (CCTCC) on May 19, 2022, under the CCTCC designation of CCTCC NO. C2022124, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0085] Some sequences involved in the present invention are as follows. 6F7 heavy chain variable region:

Chemical formula

[0086] 6F7 light chain variable region:

Chemical formula

[0087] 6F7CDR HCDR1: GYTFTSYW (SEQ ID NO: 5) HCDR2: IDPSDSET (SEQ ID NO: 6) HCDR3: ARLYRWYFDV (SEQ ID NO: 7) LCDR1: EIVGTY (SEQ ID NO: 8) LCDR2: GAS (SEQ ID NO: 9) LCDR3: GQSYNFPYT (SEQ ID NO: 10)

[0088] 6F7H1VH:

Chem.

[0089] 6F7L1VL:

Chem.

[0090] 6F7H2VH:

Chem.

[0091] 6F7L2VL:

Chem.

[0092] 6F7H3VH:

Chem.

[0093] 6F7L3VL:

Chem.

[0094] 6F7H4VH:

Chem.

[0095] 6F7L4VL:

Chem.

[0096] 8C5.1VH

Chem.

[0097] 8C5.1VL

Chem.

[0098] 8C5.1 CDR HCDR1: GFTFSNSA (SEQ ID NO: 31) HCDR2: ITSGVSYT (SEQ ID NO: 32) HCDR3: TRQFKGNALDY (SEQ ID NO: 33) LCDR1: QSLLNSGNQKNY (SEQ ID NO: 34) LCDR2: WAS (SEQ ID NO: 35) LCDR3: QNDYFYPLT (SEQ ID NO: 36)

[0099] 8C5.1 H1VH

Chem.

[0100] 8C5.1 H2VH

Chem.

[0101] 8C5.1 H3VH

Chem.

[0102] 8C5.1 L1VL

Chem.

[0103] 8C5.1 L2VL

Chem.

[0104] 8C5.1 L3VL

Chem.

[0105] Amino acid sequence of TEV: ENLYFQG (SEQ ID NO: 49)

[0106] Amino acid sequence of linker (GGGGS)4: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 50)

[0107] Amino acid sequence of the heavy chain constant region of human hG1WT

Chem.

[0108] Hinge region, CH2 domain, and CH3 domain (excluding knob mutation sites S354C and T366W):

Chem.

[0109] Constant region sequence (excluding whole mutation sites Y349C, T366S, L368A, and Y407V):

Chem.

[0110] Amino acid sequence of the heavy chain of Hu5F9-G4

Chem.

[0111] Amino acid sequence of the light chain of Hu5F9-G4 [Chemical formula]

[0112] Detailed description Hereinafter, embodiments of the present invention will be described in detail with reference to examples. Those skilled in the art will understand that the following examples are merely for explaining the present invention and should not be construed as limiting the scope of the present invention. Experimental procedures without specific conditions in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment used are commercially available conventional products if the manufacturer is not specified.

[0113] In the following experimental examples of the present invention, the isotype control antibodies used, namely hIgG1 and hIgG4, are antibodies targeting human anti-hen egg lysozyme (HEL). The variable region sequences of these antibodies were from the study entitled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" by Acierno et al. (Acierno et al., J Mol Biol., 2007; 374(1): 130-46). In the constant region fragment of hIgG1, the heavy chain constant region is the Igγ-1 chain C region, accession: P01857, and the light chain constant region is the Igκ chain C region, accession: P01834. In hIgG4, the heavy chain constant region is the Igγ-4 chain C region, accession: P01861.1, and the S228P mutation has been introduced to improve stability. The light chain constant region is the Igκ chain C region, accession: P01834. Both hIgG1 and hIgG4 were prepared by Akeso Biopharma Inc.

[0114] Preparation Example 1: Preparation of Anti-Human CD47 Antibody 6F7 1. Preparation of Hybridoma Cell Line LT012 The antigens used were CD47 IgV TEV-His (containing the human CD47 mature peptide at positions 19 to 141 of GenbankID: NP_942088.1 and the TEV-his tag fusion protein) and 3T3-CD47 cells (NIH / 3T3, manufacturer: ATCC, catalog number CRL-1658: A cell line stably expressing human CD47 mature peptide was constructed by transfecting NIH / 3T3 cells with the human CD47 mature peptide). Spleen cells of immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. CD47 IgV TEV-His and 3T3-CD47 cells were separately collected as antigens, and the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells that could secrete antibodies capable of specifically binding to CD47. The hybridoma cells obtained by ELISA screening were screened by competitive ELISA to obtain a hybridoma cell line that could secrete a monoclonal antibody capable of competing with receptor human SIRPαECD-hFc-biotin for binding to human CD47 IgV TEV-His (SIRPαECD refers to the extracellular region of SIRPα, positions 31 to 373 of protein GenBank accession No. NP_542970.1, and hFc refers to the human IgG Fc purification tag, specifically the C region of the Igγ-1 chain, positions 114 to 330 of GenbankID P01857). Then, this was subjected to a limiting dilution assay to obtain a stable hybridoma cell line. The above hybridoma cell line was named hybridoma cell line LT012, and the monoclonal antibody secreted from the cell line was named 6F7.

[0115] The hybridoma cell line LT012 was deposited with the China Center for Type Culture Collection (CCTCC) under the CCTCC designation of CCTCC NO.C2018135 on June 21, 2018. The deposit address is Wuhan University, Wuhan, China, postal code 430072.

[0116] 2. Preparation of anti-CD47 antibody 6F7 The cell line LT012 prepared above was cultured at 37 °C in an incubator with 5% CO2 together with a chemically defined medium (CD medium, containing 1% penicillin-streptomycin, Glutamax (Gibco 35050079)). After 7 days, the supernatant was collected, subjected to high-speed centrifugation and vacuum filtration through a precision filtration membrane, and purified using a HiTrap Protein A HP column to obtain antibody 6F7.

[0117] Preparation Example 2: Sequence analysis of anti-CD47 antibody 6F7 mRNA was extracted from the cell line LT012 cultured in Preparation Example 1 according to the method described in the manual of RNAprep pure Cell / Bacteria Kit (Tiangen, catalog number: DP430).

[0118] cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR Kit and amplified by PCR.

[0119] The PCR amplification product was directly subjected to TA cloning according to the manual of pEASY-T1 Cloning Kit (Transgen CT101).

[0120] The TA cloning product was directly sequenced. The sequencing results are as follows: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 1 and has a length of 351 bp.

[0121] The encoded amino acid sequence is shown in SEQ ID NO: 2 and has a length of 117 aa.

[0122] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 3 and has a length of 321 bp.

[0123] The encoded amino acid sequence is shown in SEQ ID NO: 4 and has a length of 107 aa.

[0124] The six CDRs of antibody 6F7 are defined by the IMGT numbering system as follows: The sequences of the heavy chain HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 5, 6, and 7, respectively.

[0125] The sequences of the light chain LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 8, 9, and 10, respectively.

[0126] Preparation Example 3: Design and Preparation of the Light and Heavy Chains of Humanized Anti-Human CD47 Antibodies 6F7H1L1(hG4), 6F7H2L2(hG4), 6F7H3L3(hG4), and 6F7H4L4(hG4) 1. Design of the Light and Heavy Chains of Humanized Anti-Human CD47 Antibodies 6F7H1L1(hG4), 6F7H2L2(hG4), 6F7H3L3(hG4), and 6F7H4L4(hG4) Based on the three-dimensional crystal structure of human CD47 protein (Hage T, Reinemer P, Sebald W., Crystals of a 1:1 Complex Between Human Interleukin-4 and the Extracellular Domain of Its Receptor Alpha Chain, Eur. J. Biochem., 1998; 258(2): 831-6) and the sequence of antibody 6F7 obtained in Preparation Example 2, the variable region sequences of antibodies 6F7H1L1, 6F7H2L2, 6F7H3L3, and 6F7H4L4 were obtained by computer modeling and mutagenesis design (antibody constant region sequences from the NCBI database: the heavy chain constant region is the Igγ-4 chain C region, accession: P01861.1; the light chain constant region is the Igκ chain C region, accession: P01834).

[0127] The designed variable region sequences are as follows: (1) Heavy and light chain variable region sequences of humanized monoclonal antibody 6F7H1L1 The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 11 and has a length of 351 bp.

[0128] The encoded amino acid sequence is shown in SEQ ID NO: 12 and has a length of 117 aa.

[0129] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 13 and has a length of 321 bp.

[0130] The encoded amino acid sequence is shown in SEQ ID NO: 14 and has a length of 107 aa.

[0131] (2) Heavy and light chain variable region sequences of humanized monoclonal antibody 6F7H2L2 The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 15 and has a length of 351 bp.

[0132] The encoded amino acid sequence is shown in SEQ ID NO: 16 and has a length of 117 aa.

[0133] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 17 and has a length of 321 bp.

[0134] The encoded amino acid sequence is shown in SEQ ID NO: 18 and has a length of 107 aa.

[0135] (3) Heavy and light chain variable region sequences of humanized monoclonal antibody 6F7H3L3 The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 19 and has a length of 351 bp.

[0136] The encoded amino acid sequence is shown in SEQ ID NO: 20 and has a length of 117 aa.

[0137] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 21 and has a length of 321 bp.

[0138] The encoded amino acid sequence is shown in SEQ ID NO: 22 and has a length of 107 aa.

[0139] (4) Heavy and light chain variable region sequences of humanized monoclonal antibody 6F7H4L4 The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 23 and has a length of 351 bp.

[0140] The encoded amino acid sequence is shown in SEQ ID NO: 24 and has a length of 117 aa.

[0141] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 25 and has a length of 321 bp.

[0142] The encoded amino acid sequence is shown in SEQ ID NO: 26 and has a length of 107 aa.

[0143] Furthermore, 6F7H1L1, 6F7H2L2, 6F7H3L3, and 6F7H4L4 have the same HCDR1 - HCDR3 and LCDR1 - LCDR3 as follows: The sequence of HCDR1 is shown in SEQ ID NO: 5, the sequence of HCDR2 is shown in SEQ ID NO: 6, and the sequence of HCDR3 is shown in SEQ ID NO: 7; The sequence of LCDR1 is shown in SEQ ID NO: 8, the sequence of LCDR2 is shown in SEQ ID NO: 9, and the sequence of LCDR3 is shown in SEQ ID NO: 10.

[0144] 2. Preparation of humanized antibodies 6F7H1L1 (hG4), 6F7H2L2 (hG4), 6F7H3L3 (hG4), and 6F7H4L4 All of the heavy chain constant regions were the Igγ-4 chain C region, accession: P01861.1; and all of the light chain constant regions were the Igκ chain C region, accession: P01834.

[0145] The heavy-chain cDNA and light-chain cDNA of 6F7H1L1(hG4), the heavy-chain cDNA and light-chain cDNA of 6F7H2L2(hG4), the heavy-chain cDNA and light-chain cDNA of 6F7H3L3(hG4), and the heavy-chain cDNA and light-chain cDNA of 6F7H4L4(hG4) were separately cloned into the pUC57simple vector (provided by Genscript) to obtain pUC57simple-6F7H1 and pUC57simple-6F7L1, pUC57simple-6F7H2 and pUC57simple-6F7L2, pUC57simple-6F7H3 and pUC57simple-6F7L3, and pUC57simple-6F7H4 and pUC57simple-6F7L4, respectively. Referring to the standard techniques described in Molecular Cloning: A Laboratory Manual (2nd Edition), the full-length genes of the heavy and light chains synthesized by EcoRI & HindIII digestion of the genes were subcloned into the expression vector pcDNA3.1 through digestion with restriction enzymes (EcoRI & HindIII) to obtain the expression plasmids pcDNA3.1-6F7H1, pcDNA3.1-6F7L1, pcDNA3.1-6F7H2, pcDNA3.1-6F7L2, pcDNA3.1-6F7H3, pcDNA3.1-6F7L3, pcDNA3.1-6F7H4, and pcDNA3.1-6F7L4. The heavy and light chain genes of the recombinant expression plasmids were further sequenced. Subsequently, designed combinations of recombinant plasmid genes containing the corresponding light and heavy chains (pcDNA3.1-6F7H1 / pcDNA3.1-6F7L1, pcDNA3.1-6F7H2 / pcDNA3.1-6F7L2, pcDNA3.1-6F7H3 / pcDNA3.1-6F7L3, and pcDNA3.1-6F7H4 / pcDNA3.1-6F7L4) were co-transfected into 293F cells separately, and then the culture supernatants were collected and purified. After confirming the sequences, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. Seven days after culturing, the cell cultures were collected and subjected to affinity purification using a Protein A column (MabSelect SURE (GE)) to obtain humanized antibodies.

[0146] Preparation Example 4: Preparation of Anti-CLDN18.2 Antibody 8C5.1 1. Preparation of Hybridoma Cell Line LT020 The immunogen used was a 3T3 cell line (Chinese Academy of Sciences) that overexpresses human Claudin18.2 (Genbank ID: NP_001002026.1) (3T3 hClaudin18.2). Spleen cells of immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. Using a CHO-K1 cell line that overexpresses human Claudin18.2 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) (CHO-K1-hClaudin18.2) as an antigen, the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells that could secrete antibodies specifically binding to CLDN18.2. The hybridoma cells obtained by screening were subjected to limiting dilution assay to obtain stable hybridoma cell lines. The above hybridoma cell line was named hybridoma cell line LT020, and the monoclonal antibody secreted by the hybridoma cell line was named 8C5.1.

[0147] The hybridoma cell line LT020 was deposited with the China Center for Type Culture Collection (CCTCC) on May 19, 2022, under the CCTCC designation of CCTCC NO. C2022124, and the deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0148] 2. Preparation of Anti-CLDN18.2 Antibody 8C5.1 The cell line LT020 prepared above was cultured in CD medium (chemically defined medium containing 4% Glutamax (Gibco 35050079) and 1% penicillin-streptomycin (Gibco 15140163)) at 5% CO2 and 37°C. After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation and vacuum filtration through a microfiltration membrane, and purified using a HiTrap Protein A HP column to obtain antibody 8C5.1.

[0149] Preparation Example 5: Sequence Analysis of Anti-CLDN18.2 Antibody 8C5.1 mRNA was extracted from the cell line LT020 cultured in Preparation Example 4 according to the method described in the manual of RNAprep pure Cell / Bacteria Kit (Tiangen, catalog number: DP430).

[0150] cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR Kit and amplified by PCR.

[0151] The PCR amplification product was directly subjected to TA cloning according to the manual of pEASY-T1 Cloning Kit (Transgen CT101).

[0152] The TA cloning product was directly sequenced. The sequencing results are as follows: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 27 and has a length of 354 bp.

[0153] The encoded amino acid sequence is shown in SEQ ID NO: 28 and has a length of 118 amino acids.

[0154] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 29 and has a length of 339 bp.

[0155] The encoded amino acid sequence is shown in SEQ ID NO: 30 and has a length of 113 amino acids.

[0156] The six CDRs of antibody 8C5.1 defined by the IMGT numbering system are as follows: The sequences of the heavy-chain HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 31, 32, and 33, respectively.

[0157] The sequences of the light-chain LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 34, 35, and 36, respectively.

[0158] Preparation Example 6: Design and Preparation of the Light Chain and Heavy Chain of a Humanized Anti-Human CLDN18.2 Antibody 1. Design of the light chain and heavy chain of humanized anti-human CLDN18.2 antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3. Based on the three-dimensional crystal structure of the human CLDN18.2 protein and the sequence of antibody 8C5 obtained in Preparation Example 5, the variable region sequences of antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 were obtained by computer modeling and mutagenesis design (antibody constant region sequences from the NCBI database: the heavy-chain constant region is the Igγ-1 chain C region, SEQ ID NO: 61; the light-chain constant region is the Igκ chain C region, accession: P01834).

[0159] The designed variable region sequences are shown in Table A below.

[0160] [Table 1]

[0161] For each of the above 7 antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3, the length of the nucleotide sequence of the heavy chain variable region was 354 bp, the length of the encoded amino acid sequence was 118 aa, the length of the nucleotide sequence of the light chain variable region was 339 bp, and the length of the encoded amino acid sequence was 113 aa.

[0162] In addition, the above 7 antibodies had the same HCDR1 - HCDR3 and LCDR1 - LCDR3 shown below: The sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 31, 32, and 22, respectively.

[0163] The sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 34, 35, and 36, respectively.

[0164] 2. Preparation of humanized antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 All heavy chain constant regions were the Igγ - 1 chain C region, SEQ ID NO: 61, and all light chain constant regions were the Igκ chain C region, Accession: P01834.

[0165] The heavy chain cDNA and light chain cDNA of each of 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 were cloned into the pUC57simple vector (provided by Genscript) to obtain pUC57simple - 8C5.1H1, pUC57simple - 8C5.1L1; pUC57simple - 8C5.1H1, pUC57simple - 8C5.1L2; pUC57simple - 8C5.1H2, pUC57simple - 8C5.1L1; pUC57simple-8C5.1H2, pUC57simple-8C5.1L2; pUC57simple-8C5.1H2, pUC57simple-8C5.1L3; pUC57simple-8C5.1H3, pUC57simple-8C5.1L2; and pUC57simple-8C5.1H3, pUC57simple-8C5.1L3 were obtained.

[0166] According to the standard techniques described in Molecular Cloning: A Laboratory Manual (2nd Edition), the synthesized full-length heavy and light chain genes were digested using EcoRI & HindIII. The genes were subcloned into the expression vector pcDNA3.1 by digestion with restriction enzymes (EcoRI & HindIII) to obtain the expression plasmids pcDNA3.1-8C5.1H1, pcDNA3.1-8C5.1L1, pcDNA3.1-8C5.1H2, pcDNA3.1-8C5.1L2, pcDNA3.1-8C5.1H3, and pcDNA3.1-8C5.1L3, and the heavy chain / light chain genes of the recombinant expression plasmids were further sequenced. Then, the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-8C5.1H1 / pcDNA3.1-8C5.1L1, pcDNA3.1-8C5.1H1 / pcDNA3.1-8C5.1L2, pcDNA3.1-8C5.1H2 / pcDNA3.1-8C5.1L1, pcDNA3.1-8C5.1H2 / pcDNA3.1-8C5.1L2, pcDNA3.1-8C5.1H2 / pcDNA3.1-8C5.1L3, pcDNA3.1-8C5.1H3 / pcDNA3.1-8C5.1L2, and (pcDNA3.1-8C5.1H3 / pcDNA3.1-8C5.1L3) were co-transfected into 293F cells respectively, and then the culture medium was collected and purified. After confirming the correct sequence by sequencing, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days, the cell culture medium was collected and affinity purified using a Protein A column to obtain the humanized antibody.

[0167] Preparation Example 7: Design and Preparation of Anti-CLDN18.2 / Anti-CD47 Bispecific Antibody 1. Structural Design The bispecific antibody in this example was named AsAb-8C4703. Its structure is shown in Table 1 below.

[0168] [Table 2]

[0169] In Table 1 above: The linker fragment, i.e., the linker, has the amino acid sequence shown in SEQ ID NO: 50.

[0170] The mutation positions of the knob and hole are numbered according to the EU numbering system.

[0171] The bispecific antibody AsAb-8C4703 contains three peptide chains named HC1, HC2, and L3 from left to right respectively, and its composition is as follows: (1) HC1 composition: ScFv (VH of 6F7H4 + linker sequence + VL of 6F7L4) + hinge region + hIgG1 CH2 + hIgG1 CH3, which contains knob mutation sites (serine S354C mutated to cysteine at position 354 and threonine T366W mutated to tryptophan at position 366).

[0172] The amino acid sequence of the scFv sequence is as follows (the underline is the linker sequence). [Chemical formula]

[0173] The hinge region, CH2 domain, and CH3 domain (double underlines indicate knob mutation sites S354C and T366W).

Chemical Structure

[0174] The full-length amino acid sequence of HC1 is as follows. (476aa, the underline indicates the linker sequence, and the double underline indicates the knob mutation site)

Chemical Structure

[0175] The full-length nucleotide sequence of HC1 is as follows.

Chemical Structure

Chemical Structure

[0176] (2) HC2 composition: VH of 8C5.1H3 + constant region of hIgG1, which includes hole mutation sites (tyrosine Y349C mutated to cysteine, threonine T366S mutated to serine, leucine L368A mutated to alanine, and tyrosine Y407V mutated to valine).

[0177] The amino acid sequence of 8C5.1H3VH is shown in SEQ ID NO: 41.

[0178] Constant region sequence (double underlines indicate hole mutation sites Y349C, T366S, L368A, and Y407V):

Chemical Structure

[0179] The full-length amino acid sequence of HC2 is as follows. (447 aa, double underlines indicate the whole mutation sites)

Chem.

[0180] The full-length nucleotide sequence of HC2 is as follows.

Chem.

Chem.

[0181] (3) L3 composition: VL of 8C5.1L3 + human Igκ constant region The amino acid sequence of 8C5.1L3VL is shown in SEQ ID NO: 47.

[0182] The amino acid sequence of human Igκ constant region

Chem.

[0183] The full-length amino acid sequence of L3 is as follows.

Chem.

[0184] The full-length nucleotide sequence of L3 is as follows.

Chem.

[0185] The peptide chains HC1 and HC2 of the bispecific antibody AsAb-8C4703 are linked by two disulfide bonds in the hinge region, and HC2 and L3 are linked by one disulfide bond.

[0186] 2. Molecular construction, expression and purification of antibody The cDNA sequences encoding the three polypeptide chains of the bispecific antibody AsAb-8C4703 were separately cloned into the pUC57simple vector (provided by Genscript) to obtain plasmids pUC57simple-HC1, pUC57simple-HC2, and pUC57simple-L3, respectively.

[0187] Plasmids pUC57simple-HC1, pUC57simple-HC2, and pUC57simple-L3 were digested with enzymes (HindIII & EcoRI), recovered by electrophoresis, and then subcloned into the pcDNA3.1 vector. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed. The supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in one step using an elution buffer. The target sample was isolated and the buffer was exchanged to PBS.

[0188] The prepared anti-CLDN18.2 / anti-CD47 bispecific antibody was used in the following experiments.

Example

[0189] Example 1: FACS assay of the binding activity of the anti-CLDN18.2 / anti-CD47 bispecific antibody 1. FACS (fluorescence-activated cell sorter) assay of the binding activity of the anti-CLDN18.2 / anti-CD47 bispecific antibody to CHO-K1-CLDN18.2-CD47 cells CHO-K1-CLDN18.2-CD47 cells (CHO-K1 cells overexpress CLDN18.2 and CD47 antigens and are obtained from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were collected in the logarithmic growth phase and seeded at 1×10 5Transferred to cells / wells. 100 μL of 1% PBSA (PBS + 1% BSA) was added, and the mixture was centrifuged at 1000×g for 5 minutes, after which the supernatant was removed. 100 μL of antibody diluted with 1% PBSA (final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM, respectively) was added. The mixture was gently mixed well and then incubated on ice for 40 minutes. 150 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 1000×g for 5 minutes. The supernatant was discarded, and the plate was washed 3 times at 200 μL per wash. 300-fold diluted PE anti-human IgG Fc antibody (Biolegend, catalog 409304) was added for resuspension, and the mixture was mixed well. The diluted antibody was added to the corresponding samples at 100 μL / well, the cell pellet was resuspended, and the mixture was incubated on ice in the dark for 0.5 hour. 150 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 1000 g for 5 minutes, after which the supernatant was removed. Then, the plate was washed 2 times with 200 μL of 1% PBSA. 200 μL of 1% PBSA was added to each well to resuspend the cell pellet, and the mixture was transferred to a flow cytometry tube. A FACS Calibur assay was performed to analyze the binding activity.

[0190] The experimental results are shown in Table 2 and Figure 1.

[0191]

Table 3

[0192] The results showed that, under the same experimental conditions, the ECs of 8C-5.1H3L3, 6F7H1L1 (hG4), and AsAb-8C4703 for binding to CHO-K1-CLDN18.2-CD47 cells 50 were 1.220 nM, 1.295 nM, and 1.245 nM, respectively.

[0193] The results showed that, under the same experimental conditions, 8C5.1H3L3, 6F7H1L1(hG4), and AsAb-8C4703 exhibited comparable binding activities to CHO-K1-CLDN18.2-CD47 cells, suggesting that AsAb-8C4703 has the activity to effectively bind to cells expressing both CLDN18.2 and CD47.

[0194] 2. FACS assay of the binding activity of anti-CLDN18.2 / anti-CD47 bispecific antibody to CHO-K1-CLDN18.2 cells CHO-K1-CLDN18.2 cells (CHO-K1 cells overexpress the CLDN18.2 antigen and are obtained from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were collected in the logarithmic growth phase and seeded at 2.5×10 5Transferred to cells / wells. 100 μL of 1% PBSA (PBS + 1% BSA) was added, and the mixture was centrifuged at 1000×g for 5 minutes, after which the supernatant was removed. 100 μL of the antibody diluted with 1% PBSA (final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM, and 0.00123 nM, respectively) was added. The mixture was gently and thoroughly mixed, then incubated on ice for 40 minutes, 150 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 1000×g for 5 minutes. The supernatant was discarded, and the plate was washed 3 times with 200 μL each time. 300-fold diluted PE anti-human IgG Fc antibody (Biolegend, catalog 409304) was added for resuspension, and the mixture was well mixed. The diluted antibody was added to the corresponding samples at 100 μL / well to resuspend the cell pellet, and the mixture was incubated on ice in the dark for 0.5 hour. 150 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 1000 g for 5 minutes, after which the supernatant was removed. Then, the plate was washed 2 times with 200 μL of 1% PBSA. 200 μL of 1% PBSA was added to each well to resuspend the cell pellet, and the mixture was transferred to a flow cytometry tube for FACS Calibur assay.

[0195] The experimental results are shown in Table 3 and Figure 2.

[0196]

Table 4

[0197] The results show that under the same experimental conditions, the ECs of 8C5.1H3L3 and AsAb-8C4703 with respect to binding to CHO-K1-CLDN18.2 cells 50 were 4.439 nM and 10.59 nM, respectively.

[0198] The results showed that under the same experimental conditions, 8C5.1H3L3 and AsAb-8C4703 exhibited comparable binding activities to CHO-K1-CLDN18.2 cells, suggesting that 8C5.1H3L3 and AsAb-8C4703 have the activity to effectively bind to CLDN18.2 on the cell membrane surface of CHO-K1-CLDN18.2.

[0199] 3. FACS assay of the binding activity of anti-CLDN18.2 / anti-CD47 bispecific antibody to red blood cells Fresh blood (voluntarily provided by healthy volunteers after informed consent) was isolated to obtain red blood cells, which were washed twice with an appropriate amount of PBS, and then the cell density was adjusted. The cell suspension was added to a V-bottom 96-well plate at 1×10 6 cells per sample, and an appropriate amount of 1% PBSA (PBS + 1% BSA) was added to each well. The mixture was centrifuged at 750×g for 5 minutes, and the supernatant was discarded. The antibody was diluted with 1% PBSA to concentrations of 900 nM, 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM, and the diluted antibody was added to the corresponding samples at 100 μL / well. The cell pellet was resuspended and incubated on ice for 40 minutes. 150 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 750×g for 5 minutes. The supernatant was discarded, and the plate was washed three times with 200 μL each time. The PE anti-human IgG Fc antibody (Biolegend, catalog 409304) was diluted with 1% PBSA, and the diluted antibody was added to the corresponding samples at 100 μL / well. The cell pellet was resuspended and incubated on ice in the dark for 30 minutes. 150 μL of 1% PBSA was added to each well, and this mixture was centrifuged at 350×g for 5 minutes. The supernatant was discarded, and the plate was washed twice with 200 μL each time. 200 μL of 1% PBSA was added to each well, the cell pellet was resuspended, and then transferred to a loading tube for testing.

[0200] The experimental results are shown in Table 4 and Figure 3.

[0201]

Table 5

[0202] The results showed that the EC 50 for the binding of the anti-CLDN18.2 / anti-CD47 bispecific antibody AsAb-8C4703 to erythrocytes was 50.68 nM.

[0203] The results indicate that the anti-CLDN18.2 / anti-CD47 bispecific antibody AsAb-8C4703 has very weak binding activity to erythrocytes.

[0204] Example 2: Competitive binding of an anti-CLDN18.2 / anti-CD47 bispecific antibody to cell membrane surface antigen CD47 An anti-CLDN18.2 / anti-CD47 bispecific antibody that competes with hSIRPαV2 for binding to cell membrane surface antigen CD47 was assayed by competitive flow cytometry. The procedure was as follows.

[0205] CHO-K1-CLDN18.2-CD47 cells were digested as usual, and 3×10 5Cells were transferred to a V-bottom 96-well plate. Then, 100 μL of 1% PBSA was added to each well, and the mixture was centrifuged and washed. 50 μL of hSIRPαV2-hFc-mFc (manufactured by Akeso Biopharma Inc., batch number: 20210120) was added to each tube, and the mixture was thoroughly mixed until the final concentration reached 3 nM and incubated on ice for 30 minutes. Corresponding serial diluted antibodies (final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM respectively) were added at 50 μL per sample, and the mixture was incubated on ice for 30 minutes. 150 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 1000×g for 5 minutes. The supernatant was discarded, and the plate was washed three more times at 200 μL per wash. APC streptavidin (Biolegend, catalog 405207) was diluted with 1% PBSA (300-fold dilution), and the diluted antibody was added to the corresponding samples at 100 μL per well. The cell pellet was resuspended and incubated on ice in the dark for 30 minutes. 150 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 1000×g for 5 minutes. The supernatant was discarded, and the plate was washed two more times at 200 μL per wash. 200 μL of 1% PBSA was added to each well, and after resuspending the cell pellet, it was transferred to loading tubes for testing.

[0206] The results are shown in Figure 4, and the EC of the sample 50 is shown in Table 5. By fluorescence quantitative analysis and curve fitting, the competitive binding EC 50 values of antibody 6F7H1L1 (hG4) and AsAb-8C4703 were calculated to be 5.003 nM and 10.05 nM respectively.

[0207]

Table 6

[0208] The results show that the antibodies 6F7H1L1 (hG4) and AsAb-8C4703 can effectively block the binding of hSIRPαV2 to CD47 on the surface of CHO-K1-CLDN18.2-CD47 host cells in a dose-dependent manner.

[0209] Example 3: Effect of anti-CLDN18.2 / anti-CD47 bispecific antibody on the aggregation of normal human red blood cells (RBCs) A 0.2 mg / mL dextran T500 solution was prepared. Fresh blood (voluntarily provided by healthy volunteers after informed consent) was isolated to obtain red blood cells, which were washed twice with an appropriate amount of PBS and then the cell density was adjusted. The cells were seeded into 96-well plates at 50 μL / well (1×10 7 / well), and 50 μL of the corresponding antibody (antibody working concentrations of 3000 nM, 1000 nM, 333.3 nM, 111.1 nM, 37.0 nM, 12.3 nM, 4.1 nM, 1.4 nM, 0.14 nM, and 0.014 nM) was added to each well. 50 μL of dextran T500 (0.1 mg / mL) was added to the positive control group, 50 μL of PBS was added to the negative control group, and hIgG1 antibody (manufactured by Akeso Biopharma Inc., batch number: 20190410) and hIgG4 antibody (manufactured by Akeso Biopharma Inc., batch number: 20190910) were added to the isotype control group. The system was incubated at 37 °C for 2 - 4 hours, and the aggregation of red blood cells was examined and photographed.

[0210] The aggregation of normal human red blood cells induced by 6F7H1L1 (hG4) and AsAb-8C4703 is shown in Figure 5.

[0211] The results showed that 6F7H1L1 (hG4) and AsAb-8C4703 had no effect on the aggregation of red blood cells at concentrations below 3000 nM and showed good safety.

[0212] Example 4: Phagocytosis of tumor cells by macrophages promoted by anti-CLDN18.2 / anti-CD47 bispecific antibody In this example, HPMM was used as an effector cell, KATOIII-CLDN18.2 was used as a target cell, and the antibody-dependent cell phagocytosis (ADCP) activity mediated by antibodies 8C5.1H3L3, 6F7H1L1 (hG4), and AsAb-8C4703 was assayed. The procedure was as follows.

[0213] KATOIII-CLDN18.2 (KATOIII cells overexpress CLDN18.2 and are obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was collected, washed once with PBS, and centrifuged at 170×g for 5 minutes. The cells were counted and analyzed for viability. CFSE (Biolegend, catalog 423801) was diluted with PBS to a concentration of 2.5 μM, and an appropriate amount of the diluted CFSE was taken to resuspend the cells (staining density: 10 million cells / mL). The cells were incubated in an incubator for 20 minutes, and 6 mL of 1640 complete medium (containing 10% FBS) was added to stop the staining. The cells were centrifuged at 170×g for 5 minutes, and the supernatant was discarded. 1 mL of 1640 complete medium was added to resuspend the cells. The cells were incubated in an incubator for 10 minutes and adjusted to a density of 150,000 cells / tube.

[0214] Antibodies (working concentrations of 1 nM, 10 nM, 100 nM, and 300 nM) were diluted with 1640 complete medium, and isotype controls hIgG1 (manufactured by Akeso Biopharma Inc., batch number: 20190410) and hIgG4 (manufactured by Akeso Biopharma Inc., batch number: 20190910) (both at a working concentration of 300 nM) were set up.

[0215] HPMM (isolated from peripheral blood of healthy volunteers who provided it voluntarily after informed consent) was collected, counted, and its viability was analyzed. The cell suspension was added to 1.5 mL EP tubes at 50,000 cells / tube, centrifuged at 1200×g for 5 minutes, and the supernatant was discarded. 50 μL of the target cell suspension and 50 μL of the antibody were placed in a 1.5 mL EP tube, and the mixture was incubated in an incubator for 2 hours.

[0216] 700 μL of 1% PBSA (PBS + 1% BSA) was added to each well, the mixture was centrifuged at 1200×g for 5 minutes, and the supernatant was discarded. The APC anti-mouse / human CD11b antibody (Biolegend, catalog 101212) was diluted with 1% PBSA (500-fold dilution), and 100 μL / well of the diluted antibody was added to the corresponding samples. The cell pellet was resuspended and incubated on ice for 30 minutes. 800 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 1200×g for 5 minutes. The supernatant was discarded, and the plate was washed 2 more times at 800 μL each time. 200 μL of 1% PBSA was added to each well, the cell pellet was resuspended, and then transferred to a loading tube for testing.

[0217] Macrophages in the system were APC-CD11b+ positive, and macrophages involved in phagocytosis were APC-CD11b+ and CFSE+ double positive. The phagocytosis index was determined as the ratio of the number of double positive cells to the number of APC positive cells, and the antibody-mediated ADCP activity was evaluated. The ADCP activity of each group represented by P% was calculated according to the following formula.

Number

[0218] The results are shown in Figure 6.

[0219] The results showed that the phagocytosis indices of 8C5.1H3L3, 6F7H1L1(hG4), and AsAb-8C4703 were significantly higher than those of the blank control and isotype control, indicating that 8C5.1H3L3, 6F7H1L1(hG4), and AsAb-8C4703 all had ADCP activity, and the ADCP activity of AsAb-8C4703 was superior to that of the 8C5.1H3L3 group, 6F7H1L1(hG4) group, and the combined group of 8C5.1H3L3 and 6F7H1L1(hG4).

[0220] Example 5: ADCC Activity Assay of Anti-CLDN18.2 / Anti-CD47 Bispecific Antibody CHO-K1-CLDN18.2-CD47 cells were collected as usual, centrifuged at 170×g for 5 minutes, and then the cells were resuspended in medium (RPMI-1640 + 1% FBS) and washed twice. The cells were resuspended in medium and then counted to adjust the cell density. The target cell suspension was added to a 96-well plate at 100 μL / well (about 3×10 4 target cells / well), and the antibodies 8C5.1H3L3, 6F7H1L1(hG4), and AsAb-8C4703 (all at working concentrations of 3 nM, 1 nM, 0.33 nM, 0.11 nM, 0.037 nM, 0.0123 nM, and 0.00123 nM) were diluted with medium (RPMI-1640 + 1% FBS), and the diluted antibodies were added to the corresponding wells at 50 μL / well. The system was incubated in an incubator at 37°C for 1 hour, and a negative control medium and an isotype control hIgG1 (manufactured by Akeso Biopharma Inc., batch: 20190410) were set up.

[0221] PBMC effector cells were collected as usual, centrifuged at 170×g for 5 minutes, and the supernatant was discarded. The cells were washed twice, resuspended in medium (RPMI-1640 + 1% FBS), and then counted. The cell density was adjusted. 50 μL of the effector cell suspension (about 9×10 5(The effector cells / wells containing) were added to the pre-incubated target cells, and the mixture was thoroughly mixed. The cells were incubated at 37 °C in a 5% CO2 incubator for 4 hours, centrifuged at 250×g for 5 minutes, and then 100 μL of the culture supernatant was carefully pipetted into a new flat-bottom 96-well plate, and 100 μL of the freshly prepared reaction solution was added to each well. The mixture was incubated at room temperature in the dark for 30 minutes, and the OD values at 490 nm and 650 nm were measured. OD value of each group = OD490nm - OD650nm.

[0222] The results are shown in Figure 7.

[0223] The results show that, compared with the isotype control, the anti-CLDN18.2 / anti-CD47 bispecific antibody AsAb-8C4703 has ADCC activity and can specifically kill CLDN18.2- and CD47-positive cells, and has better killing activity than the corresponding monoclonal antibodies 8C5.1H3L3 and 6F7H1L1 (hG4).

[0224] Example 6: CDC Activity Assay of Anti-CLDN18.2 / anti-CD47 Bispecific Antibody CHO-K1-CLDN18.2-CD47 cells were collected by conventional digestion and centrifuged at 170×g for 5 minutes. Then, the cells were resuspended in medium (RPMI-1640 + 1% FBS) and washed twice. The cells were resuspended in medium (RPMI-1640 + 1% FBS) and then counted. The cell density was adjusted. The target cell suspension was added to a 96-well plate at 100 μL / well (about 3×10 4It was added in cells / wells, and the antibodies (all at working concentrations of 100 nM, 10 nM, 1 nM, and 0.1 nM) were diluted with medium (RPMI - 1640 + 1% FBS). The diluted antibodies were added to the corresponding wells at 50 μL / well, and the system was pre - incubated at room temperature for 10 minutes. After pre - incubation, 50 μL of complement serum (final concentration 2%) was added to the target cells, and the mixture was well - mixed. The system was incubated at 37 °C for 4 hours in an incubator containing 5% CO2 and then centrifuged at 250×g for 5 minutes. 100 μL of the culture supernatant was carefully pipetted into a new flat - bottom 96 - well plate, and 100 μL of freshly prepared reaction solution was added to each well. The mixture was incubated at room temperature in the dark for 30 minutes, and the OD values at 490 nm and 650 nm were measured. OD value of each group = OD490nm - OD650nm.

[0225] As shown in Figure 8, compared with the isotype control hIgG1 (manufactured by Akeso Biopharma Inc., batch number: 20190410), the anti - CLDN18.2 / anti - CD47 bispecific antibody AsAb - 8C4703 has CDC activity, can kill tumor cells through complement - mediated means, and has equivalent activity compared with 8C5.1H3L3.

[0226] Example 7: Phagocytosis of tumor cells by macrophages promoted by anti - CLDN18.2 antibody In this example, the antibody - dependent cell - mediated phagocytosis (ADCP) activity mediated by the antibody 8C5.1H3L3 was detected using MBMM (mouse bone marrow - derived macrophages) as effector cells and CHO - K1 - CLDN18.2 as target cells.

[0227] Claudiximab (IMAB362, Zolbetuximab) is a human-mouse chimeric monoclonal antibody targeting CLDN18.2 developed by Ganymed, Germany. Claudiximab can specifically recognize and bind to the first extracellular domain (ECD1) outside the cell membrane of the CLDN18.2 protein and mediate antibody-dependent cell phagocytosis (ADCP) etc. (Singh P, Toom S, Huang Y. Anti-claudin18.2 antibody as new targeted therapy for advanced gastric cancer [J]. Journal of Hematology & Oncology, 2017, 10(1)).

[0228] The specific method of this example is as follows: CHO-K1-CLDN18.2 cells were collected as usual, centrifuged at 170×g for 5 minutes, resuspended, counted, analyzed for viability, and washed once with PBS. CFSE was diluted to 2.5 μM with PBS, and the cells were resuspended in an appropriate amount of diluted CFSE (staining density: 10 million cells / mL) and incubated in an incubator for 20 minutes.

[0229] 6 mL of DMEM complete medium (containing 10% FBS) was added to stop the staining. The cells were centrifuged at 170×g for 5 minutes, and the supernatant was discarded. 1 mL of DMEM complete medium was added, and the cells were incubated in an incubator for 10 minutes. The antibody was diluted with DMEM complete medium (10 μg / mL, 1 μg / mL, and 0.1 μg / mL), and an isotype control antibody and a reference antibody were designed.

[0230] Macrophages were collected and centrifuged at 170×g for 5 minutes. The supernatant was discarded and the cells were counted. The cells were transferred to 1.5 mL EP tubes, centrifuged at 1200×g for 5 minutes, and the supernatant was discarded. The suspension of tumor cells and antibody was incubated for 30 minutes, added to the 1.5 mL EP tubes containing macrophages, and the cells were resuspended. The mixture was thoroughly mixed and incubated in an incubator at 37°C for 2 hours. 800 μL of room temperature 1% PBSA was added to each tube, and the mixture was centrifuged at 1200×g for 5 minutes. The supernatant was discarded and the cells were washed once with 800 μL of PBSA. 100 μL / sample of 600-fold diluted APC anti-mouse / human CD11b antibody (Biolegend, catalog number: 101212) was added to the corresponding samples, the mixture was thoroughly mixed, and incubated on ice for 40 minutes. 800 μL of 1% PBSA was added to each tube, and the mixture was centrifuged at 1200×g for 5 minutes. The supernatant was discarded and each tube was washed once with 200 μL of PBSA. 200 μL of 1% PBSA was added to each tube for resuspension. Then, the mixture was transferred to flow cytometry tubes for testing.

[0231] The results are shown in Figure 9.

[0232] The results showed that the phagocytosis indices of 8C5.1H3L3 and IMAB362 (reference antibody) (manufactured by Akeso Biopharma Inc., batch number: 20190704) were significantly higher than those of the blank control and isotype control, indicating that both 8C5.1H3L3 and the reference antibody IMAB362 have ADCP activity, and 8C5.1H3L3 has better ADCP activity than IMAB362.

[0233] Example 8: Assay of the binding activity of anti-CLDN18.2 / anti-CD47 bispecific antibody to antigen by ELISA 1. The binding activities of AsAb-8C4703, 8C5.1H3L3, and IMAB362 to the antigen human CLDN18.2 were assayed separately by indirect ELISA. The procedure was as follows. The ELISA plates were coated with human CLDN18.2 at 2 μg / mL and incubated overnight at 4°C. Subsequently, the ELISA plates coated with the antigen were washed once with PBST, and then blocked with a PBST solution containing 1% BSA as a blocking solution at 37°C for 2 hours. After blocking, the ELISA plates were washed three times with PBST. Antibodies serially diluted with the PBST solution (the antibody dilution gradients are shown in Table 6) were added. The ELISA plates containing the test antibodies were incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a working solution of the HRP-labeled goat anti-human IgG FC (H+L) (Jackson, catalog number: 109-035-098) secondary antibody diluted at a ratio of 1:5000 was added, and then the plates were incubated at 37°C for 30 minutes. After the incubation was completed, the plates were washed three times with PBST, TMB (Neogen, 308177) was added for color development in the dark for 5 minutes, and then a stop solution was added to stop the color reaction. The ELISA plates were immediately placed in a microplate reader, and the OD values of each well in the ELISA plates were read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.

[0234] The assay results are shown in Table 6 and Figure 10.

[0235]

Table 7

[0236] Figure 10 shows that AsAb-8C4703, 8C5.1H3L3, and IMAB362 can effectively bind to human CLDN18.2 in a dose-dependent manner. By quantitative analysis of the absorbance of the binding antibodies, the binding efficiencies EC of the antibodies AsAb-8C4703, 8C5.1H3L3, and IMAB362 (as a control) obtained by curve fitting calculation 50 were 0.021 nM, 0.011 nM, and 0.014 nM, respectively.

[0237] The results show that under the same experimental conditions, AsAb-8C4703 and 8C5.1H3L3 exhibit effective activity in binding to human CLDN18.2.

[0238] 2. The binding activities of AsAb-8C4703 and 6F7H1L1(hG4) to the antigen CD47-Igv-TEV-his were separately assayed by indirect ELISA. The procedure was as follows. The ELISA plates were coated with 2 μg / mL of CD47-Igv-TEV-his and incubated overnight at 4°C. Subsequently, the ELISA plates coated with the antigen were washed once with PBST and then blocked with a PBST solution containing 1% BSA as a blocking solution for 2 hours at 37°C. After blocking, the ELISA plates were washed three times with PBST. Antibodies serially diluted with PBST solution (the dilution gradients of the antibodies are shown in Table 7) were added. The ELISA plates containing the test antibodies were incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a working solution of the HRP-labeled goat anti-human IgG FC (H+L) (Jackson, catalog number: 109-035-098) secondary antibody diluted at a ratio of 1:5000 was added, and then the plates were incubated at 37°C for 30 minutes. After the incubation was completed, the plates were washed three times with PBST, TMB (Neogen, 308177) was added for 5 minutes in the dark for color development, and then a stop solution was added to stop the color reaction. The ELISA plates were immediately placed in a microplate reader, and the OD values of each well in the ELISA plates were read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.

[0239] The assay results are shown in Table 7 and Figure 11.

[0240]

Table 8

[0241] Figure 11 shows that AsAb-8C4703 and 6F7H1L1 (hG4) can effectively bind to CD47-Igv-TEV-his in a dose-dependent manner. By quantitative analysis of the absorbance of the binding antibody, the binding efficiency EC of the antibodies AsAb-8C4703 and 6F7H1L1 (hG4) obtained by curve fitting calculation 50 was 0.164 nM and 0.012 nM, respectively.

[0242] The results show that under the same experimental conditions, AsAb-8C4703 and 6F7H1L1 (hG4) exhibit effective activity with respect to the binding to CD47-Igv-TEV-his.

[0243] Example 9: Determination of kinetic parameters of humanized antibody-CLDN18.2 / anti-CD47 bispecific antibody The affinity constants of antibodies against human CLDN18.2 and CD47 ECD-His were determined using a Fortebio Octer molecular interaction instrument. The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). The method for detecting the affinity of the antibody against human CLDN18.2 is as follows. 20 nM of the antibody was immobilized on the AHC sensor for 60 seconds. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized antibody on the sensor to CLDN18.2 His at concentrations of 6.25 - 100 nM (2-fold dilution) was measured for 180 seconds. The protein was dissociated in the buffer for 360 seconds. The method for detecting the affinity of the antibody against human CD47 ECD-His is as follows. 30 nM of the antibody was immobilized on the AHC sensor for 120 seconds. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized antibody on the sensor to the protein CD47 ECD-His at concentrations of 4.94 - 400 nM (3-fold dilution) was measured for 50 seconds. The protein was dissociated in the buffer for 120 seconds. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed by 1:1 model fitting to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 12.0, and the data analysis software was Fortebio Data Analysis HT 12.0.

[0244] The kinetic parameters of antibodies 8C5.1H3L3 and AsAb-8C4703 for binding to CLDN18.2 His are shown in Table 8, and the detection results of the kinetic characteristic parameters are shown in Figures 12 and 13, respectively.

[0245]

Table 9

[0246] K D is the affinity constant, kon is the binding rate of the antigen and the antibody, kdis is the dissociation rate of the antigen and the antibody, K D = kdis / kon.

[0247] The results indicate that both antibody 8C5.1H3L3 and AsAb-8C4703 have good affinity for antigen CLDN18.2 His.

[0248] Table 9 shows the kinetic parameters of antibody 6F7H1L1 (hG4) and AsAb-8C4703 for binding to CD47 ECD-His, and the detection results of the kinetic characteristic parameters are shown in Figures 14 and 15 respectively.

[0249]

Table 10

[0250] K D is the affinity constant, kon is the binding rate of the antigen and the antibody, kdis is the dissociation rate of the antigen and the antibody, and K D = kdis / kon.

[0251] The results indicate that both antibody 6F7H1L1 (hG4) and AsAb-8C4703 have good affinity for the antigen.

[0252] Example 10: Binding of anti-CLDN18.2 / anti-CD47 bispecific antibody to human immune cells PBMC cells were collected as usual, washed twice with an appropriate amount of PBS, counted, and analyzed for viability. The PBMC suspension was added to a 96-well plate at 3×10 5Added to the cells / samples, centrifuged at 750×g for 5 minutes, and the supernatant was discarded. Mouse IgG isotype control (Thermofisher, catalog 10400C) at a final concentration of 5 μg / mL was added, the mixture was blocked on ice for 20 minutes, and centrifuged at 750×g for 5 minutes. The supernatant was discarded. 100 μL of serial diluted antibodies (working concentrations 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 0.37 nM, 0.037 nM, and 0.0037 nM) were added, and isotype control hIgG1 (manufactured by Akeso Biopharma Inc., batch number: 20170424) and hIgG4 (manufactured by Akeso Biopharma Inc., batch number: 20190910) (both at working concentration 900 nM) were designed. The cell pellet was resuspended, incubated on ice for 40 minutes, and 1% PBSA (PBS + 1% BSA) was added. The mixture was centrifuged at 750×g for 5 minutes, and the supernatant was discarded. The plate was washed three more times. 100 μL of a suspension of FITC anti-human CD3 (Biolegend, catalog 344804), Brilliant Violet 421™ anti-human CD19 antibody (Biolegend, catalog 302234), and mouse anti-human IgG Fc-AF647 (Southern Biotech, catalog 9040-31) was added, the cell pellet was resuspended, and incubated in the dark on ice for 30 minutes. 1% PBSA was added to each well, and the mixture was centrifuged at 750×g for 5 minutes. The supernatant was discarded, and the plate was washed two more times. 1% PBSA was added to each well, the cell pellet was resuspended, and then transferred to a loading tube for FACS Calibur assay. The reference antibody Hu5F9-G4 is a humanized IgG4 antibody drug developed by Forty Seven that targets CD47 with high affinity.

[0253] The results are shown in Table 10 and Figures 16 and 17.

[0254]

Table 11

[0255] The experimental results showed that the binding MFI values of AsAb-8C4703 to CD3+ CD19- T cells and CD3- CD19+ B cells were both lower compared to the reference antibody Hu5F9-G4 (manufactured by Akeso Biopharma Inc., batch number: 20220303), and their corresponding EC 50 values were both high, indicating that the binding activity of AsAb-8C4703 to CD3+ CD19- T cells and CD3- CD19+ B cells was much weaker than that of Hu5F9-G4.

[0256] Example 11: Experiment on the inhibition of tumor growth in vivo by an anti-CLDN18.2 / anti-CD47 bispecific antibody To detect the in vivo tumor inhibitory activity of the anti-CLDN18.2 / anti-CD47 bispecific antibody, MC38-CD47-CLDN18.2 cells (MC38 mouse colon cancer cell line transfected with human CD47 and human CLDN18.2 prepared by Akeso Biopharma Inc., and the MC38 is from Shanghai Model Organisms Center, Inc.) were subcutaneously inoculated on the right side of 5- to 7-week-old female C57BL / 6 mice (purchased from GemPharmatech). The modeling and specific dosing regimens are shown in Table 11. After dosing, the length and width of the tumors in each group were measured, and the tumor volume was calculated.

[0257] After grouping, the tumor size was measured twice a week using calipers, and the tumor volume was calculated according to the formula TV = 0.5 × ab 2 (where a is the major axis of the tumor, b is the minor axis of the tumor, and TV is the tumor volume). TGI (%) (tumor growth inhibition rate) was calculated from the tumor volume according to the formula: TGI (%) = (1 - average RTV treat / average RTV vehicle ) × 100%, where RTV = Vt / V0, RTV is the relative tumor volume, average RTV treat and average RTV vehicleare the average relative tumor volumes of the treatment group and the model group respectively, and Vt and V0 are the tumor volumes on the t-th day and the 0-th day respectively. GraphPad prism software was used for the plot, and the drug efficacy results were evaluated according to TGI.

[0258]

Table 12

[0259] The results are shown in Figures 18, 19, 20, and 21.

[0260] The results are: On the 21st day of administration after grouping, the tumor growth inhibition TGI (%) of 17 mg / kg of AsAb-8C4703 was 85.43%, which was better at equimolar doses than the single-drug group of 6F7H1L1(hG4) 20 mg / kg (TGI = 18.28%), the single-drug group of 8C5.1H3L3 20 mg / kg (TGI = 23.09%), and the combined group of 6F7H1L1(hG4) 20 mg / kg + 8C5.1H3L3 20 mg / kg (TGI = 23.78%); The tumor growth inhibition TGI (%) of 5.7 mg / kg of AsAb-8C4703 was 83.62%, which was better at equimolar doses than the combined group of 6F7H1L1(hG4) 6.7 mg / kg + 8C5.1H3L3 6.7 mg / kg (TGI = 51.22%); The tumor growth inhibition TGI (%) of 1.9 mg / kg of AsAb-8C4703 was 44.26%, indicating that it was better at equimolar doses than the combined group of 6F7H1L1(hG4) 2.2 mg / kg + 8C5.1H3L3 2.2 mg / kg (TGI = 3.74%).

[0261] In addition, as shown in Figures 20 and 21, the test drug was well tolerated by the mice with tumors, and no effect on the body weight of the mice with tumors was found in the group, indicating good safety.

[0262] While specific embodiments of the present invention have been described in detail, those skilled in the art will be able to make various modifications and substitutions to those details in accordance with all the disclosed teachings, and it will be understood that all these changes fall within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

**Claim 1** A bispecific antibody comprising a first protein functional region and a second protein functional region, wherein: The first protein functional region is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof; The second protein functional region targets a target other than CLDN18.2 (e.g., CD47), The anti-CLDN18.2 antibody comprises a heavy chain variable region comprising HCDR1-HCDR3 and a light chain variable region comprising LCDR1-LCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 31, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 32, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 33, The amino acid sequence of LCDR1 is shown in SEQ ID NO: 34, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 35, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 36, a bispecific antibody. **Claim 2** The amino acid sequence of the heavy chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41; The amino acid sequence of the light chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO: 30, SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO:

47. The bispecific antibody according to claim 1. **Claim 3** In the anti-CLDN18.2 antibody: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 45; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 47; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 45; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 47; The amino acid sequence of the VH domain is shown in SEQ ID NO: 41, and the amino acid sequence of the VL domain is shown in SEQ ID NO: 43; The amino acid sequence of the VH domain is shown in SEQ ID NO: 41, and the amino acid sequence of the VL domain is shown in SEQ ID NO: 45; or The amino acid sequence of the VH domain is shown in SEQ ID NO: 41, and the amino acid sequence of the VL domain is shown in SEQ ID NO:

47. The bispecific antibody according to claim 1 or 2.

4. The heavy chain constant region of the anti-Claudin 18.2 antibody is an Igγ-1 chain C region or an Igγ-4 chain C region, and the light chain constant region of the anti-Claudin 18.2 antibody is an Igκ chain C region. The bispecific antibody according to any one of claims 1 to 3.

5. The anti-CLDN18.2 antibody or the antigen-binding fragment thereof is selected from Fab, Fab', F(ab') ２ , Fd, Fv, dAb, complementarity-determining region fragment, single-chain variable fragment, humanized antibody, chimeric antibody, or diabody, the bispecific antibody according to any one of claims 1 to 4.

6. The anti-Claudin 18.2 antibody contains a non-CDR region derived from a non-mouse species such as a human antibody. The bispecific antibody according to any one of claims 1 to 5.

7. The EC of the anti-CLDN18.2 antibody with respect to binding to cells expressing CLDN18.2 ５０ is 15 nM or less, 10 nM or less, or 5 nM or less; and / or The EC of the anti-CLDN18.2 antibody with respect to binding to cells expressing both CLDN18.2 and CD47 ５０ is 10 nM or less, 5 nM or less, or 2 nM or less, Preferably, the EC ５０ is determined by FACS respectively, The bispecific antibody according to any one of claims 1 to 6.

8. The anti-Claudin 18.2 antibody is a monoclonal antibody produced by the hybridoma cell line LT020 deposited with the China Center for Type Culture Collection (CCTCC) under the CCTCC designation of CCTCC NO. C2022124. The bispecific antibody according to any one of claims 1 to 7.

9. The second protein functional region is an anti-CD47 antibody or an antigen-binding fragment thereof: The anti-CD47 antibody contains a heavy chain variable region containing HCDR1 to HCDR3 and a light chain variable region containing LCDR1 to LCDR3: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

7. The amino acid sequence of LCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

10. The bispecific antibody according to any one of claims 1 to 8.

10. The amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is selected from SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, and SEQ ID NO:

24. The amino acid sequence of the light chain variable region of the anti-CD47 antibody is selected from SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO:

26. The bispecific antibody according to claim 9.

11. In the anti-CD47 antibody: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; 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: 14; 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: 18; 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: 22; 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: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; Or The bispecific antibody according to claim 9 or 10, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

26.

12. The bispecific antibody according to any one of claims 9 to 11, wherein the heavy chain constant region of the anti-CD47 antibody is an Igγ-1 chain C region or an Igγ-4 chain C region, and the light chain constant region of the anti-CD47 antibody is an Igκ chain C region.

13. The bispecific antibody according to any one of claims 1 to 12, wherein the first protein functional region and the second protein functional region are independently a fusion protein of a single-chain variable fragment or a half-molecule monovalent antibody (IgG half-molecule, IgG-HM).

14. The first protein functional region is a fusion protein of a single-chain variable fragment, and the second protein functional region is a half-molecule monovalent antibody (IgG half-molecule, IgG-HM) or The bispecific antibody according to any one of claims 1 to 13, wherein the first protein functional region is a half-molecule monovalent antibody (IgG half-molecule, IgG-HM), and the second protein functional region is a fusion protein of a single-chain variable fragment.

15. The first protein functional region is a fusion protein of a single-chain variable fragment targeting CLDN18.2, and the second protein functional region is a half-molecule monovalent antibody targeting CD47 or The bispecific antibody according to any one of claims 1 to 13, wherein the first protein functional region is a half-molecule monovalent antibody targeting CLDN18.2, and the second protein functional region is a fusion protein of a single-chain variable fragment targeting CD47.

16. The fusion protein of the single-chain variable fragment consists of a single-chain variable fragment, a hinge region, and an Fc fragment, or consists of a single-chain variable fragment and a heavy chain constant region, preferably, the hinge region and the Fc fragment are the hinge region and the Fc fragment of human IgG1; preferably, the hinge region and the Fc fragment have the amino acid sequence shown in SEQ ID NO: 62; The heavy chain constant region is the heavy chain constant region of human IgG1, and preferably, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO:

63. The bispecific antibody according to any one of claims 13 to 15.

17. The heavy chain constant region of the Fc fragment or the single-chain variable fragment in the fusion protein has a knob mutation (for example, S354C and T366W mutations), The heavy chain constant region of the half-molecule monovalent antibody is the heavy chain constant region of human IgG1 and has a hole mutation (for example, Y349C, T366S, L368A, and Y407V mutations). The bispecific antibody according to any one of claims 13 to 16.

18. It consists of the peptide chain shown in SEQ ID NO: 53, the peptide chain shown in SEQ ID NO: 56, and the peptide chain shown in SEQ ID NO: 59, Preferably, the peptide chain shown in SEQ ID NO: 53 and the peptide chain shown in SEQ ID NO: 56 are linked by one or more disulfide bonds in the hinge region, and the peptide chain shown in SEQ ID NO: 52 and the peptide chain shown in SEQ ID NO: 59 are linked by one or more disulfide bonds; Preferably, the peptide chain shown in SEQ ID NO: 53 and the peptide chain shown in SEQ ID NO: 56 are linked by two disulfide bonds in the hinge region, and the peptide chain shown in SEQ ID NO: 56 and the peptide chain shown in SEQ ID NO: 59 are linked by one disulfide bond. The bispecific antibody according to any one of claims 1 to 17.

19. The EC of the bispecific antibody with respect to binding to cells expressing CLDN18.2 ５０ is 20 nM or less, 15 nM or less, or 12 nM or less; EC of the bispecific antibody with respect to binding to CD47 on the surface of erythrocyte membranes ５０ is 20 nM or more, 40 nM or more, or 50 nM or more; and / or EC of the bispecific antibody with respect to binding to cells expressing both CLDN18.2 and CD47 ５０ is 10 nM or less, 5 nM or less, or 2 nM or less, Preferably, the EC ５０ is determined by FACS respectively, The bispecific antibody according to any one of claims 1 to 18.

20. The bispecific antibody according to any one of claims 1 to 19, wherein the bispecific antibody does not induce agglutination of red blood cells at a concentration of 3000 nM or less.

21. The bispecific antibody according to any one of claims 1 to 19, wherein the bispecific antibody has ADCP activity, ADCC activity, and CDC activity.

22. An isolated nucleic acid molecule encoding the bispecific antibody according to any one of claims 1 to 21.

23. A vector containing the isolated nucleic acid molecule according to claim 22.

24. A host cell containing the isolated nucleic acid molecule according to claim 22 or the vector according to claim 23.

25. A pharmaceutical composition comprising an effective amount of the bispecific antibody according to any one of claims 1 to 21 and one or more pharmaceutically acceptable adjuvants.

26. Preferably, the tumor is a CD47 and / or CLDN18.2 positive tumor; Preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer. Use of a bispecific antibody according to any one of claims 1 to 21 in the preparation of a medicament for treating or preventing a tumor.

27. Preferably, the tumor is a CD47 and / or CLDN18.2 positive tumor. Preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer. A bispecific antibody according to any one of claims 1 to 21 for use in the treatment or prevention of a tumor.

28. A method for treating or preventing a tumor, comprising the step of administering an effective amount of a bispecific antibody according to any one of claims 1 to 21 to a subject in need thereof. Preferably, the tumor is a CD47 and / or CLDN18.2 positive tumor. Preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer. A method.

29. The method for treating or preventing a tumor according to claim 28, wherein the drug administration is performed before or after surgery and / or before or after radiotherapy.

30. The bispecific antibody is administered at a unit dose of 0.1 to 100 mg per kg of body weight, preferably 5 to 50 mg or 5 to 15 mg per kg of body weight. Preferably, the drug administration is performed once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks. Preferably, the administration route is intravenous drip or intravenous injection. The method for treating or preventing a tumor according to claim 28 or 29.

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