Anti-CLDN18.2 Antibody, Pharmaceutical Composition Containing the Same, and Use

Abstract

Anti-CLDN18.2 antibodies, as well as pharmaceutical compositions and uses thereof are provided. In particular, anti-CLDN18.2 antibodies or antigen-binding fragments thereof are provided. The anti-CLDN18.2 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3. The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 7; the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 9, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 10. The anti-CLDN18.2 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, its pharmaceutical composition and use.

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 response, high late metastasis rate, and poor prognosis. Currently, the conventional treatment methods clinically adopted (radiation therapy, chemotherapy, surgical treatment, etc.) can significantly relieve pain and prolong the survival period, but there are significant limitations to these methods and it is difficult to further improve their effectiveness.

[0003] 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. Its N-terminal and C-terminal are both 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 the tight junction structure between cells (Gunzel, D.; Yu, A. S. L. Claudins and the Modulation of Tight Junction Permeability [J]. Physiological Reviews. 2013, 93(2), 525-569).

[0004] 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).

[0005] 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 lead to 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).

[0006] Claudiximab (IMAB362, Zolbetuximab) is a human-mouse chimeric monoclonal antibody targeting CLDN18.2 developed by Ganymed in Germany. Claudiximab can specifically recognize and bind with high affinity to the first extracellular domain (ECD1) outside the cell membrane of the CLDN18.2 protein, but does not bind to any other Claudin family members. Claudiximab eliminates tumor cells by mediating antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). (Singh P, Toom S, Huang Y. Anti-claudin 18.2 antibody as new targeted therapy for advanced gastric cancer[J]. Journal of Hematology & Oncology, 2017, 10(1)).

[0007] Currently, there is a need to develop novel anti-CLDN18.2 antibody drugs.

Summary of the Invention

Means for Solving the Problems

[0008] Summary Through intensive research and creative efforts, the inventors obtained an anti-CLDN18.2 antibody. The inventors surprisingly found that the anti-CLDN18.2 antibody of the present invention (hereinafter also abbreviated as the antibody or the antibody of the present invention) has excellent affinity and / or specificity, can bind to CLDN18.2-expressing tumor cells with high specificity, and has good anti-tumor prospects. The present invention will be described in detail below.

[0009] One aspect of the present invention relates to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, and the anti-CLDN18.2 antibody comprises 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: 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.

[0010] 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: 2, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15; The amino acid sequence of the light chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

[0011] 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: 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: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21.

[0012] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided. For this antibody, the heavy chain constant region is the Igγ-1 chain C region (for example, having the amino acid sequence shown in SEQ ID NO: 23) or the Igγ-4 chain C region (for example, NCBI accession: P01861.1), and the light chain constant region is the Igκ chain C region (for example, having the amino acid sequence shown in SEQ ID NO: 24).

[0013] 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, or chimeric antibody.

[0014] In some embodiments of the present invention, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof is provided, and the antibody contains a non-CDR region derived from a non-mouse species such as a human antibody.

[0015] 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 CLDN 50 is 0.5 μg / mL or less, 0.4 μg / mL or less, 0.35 μg / mL or less, 0.3 μg / mL or less, or 0.25 μg / mL or less, and preferably, the EC 50 is measured 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.

[0016] In some embodiments of the present invention, the anti-CLDN18.2 antibody is an anti-CLDN18.2 monoclonal antibody.

[0017] The present invention also relates to an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein 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.

[0018] 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.

[0019] 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.

[0020] Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.

[0021] 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.

[0022] 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 an 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.

[0023] The chemotherapy drug may be a conventional anti-tumor chemotherapy drug such as an alkylating agent, an antimetabolite, an antitumor antibiotic, a plant-based anti-cancer agent, a hormone, and an immunizing agent.

[0024] In one or more embodiments of the present invention, an antibody-drug conjugate is provided 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.

[0025] In one or more embodiments of the present invention, an antibody-drug conjugate is provided in which the molar ratio of an antibody or an antigen-binding fragment thereof to a small molecule drug is 1:(2 to 4), for example, 1:2, 1:3, or 1:4.

[0026] 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.

[0027] Yet 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 in the preparation of a medicament for the treatment or prevention of tumors; Preferably, the tumor is a 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. I

[0028] An anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention is provided for use in the treatment or prevention of tumors; Preferably, the tumor is a CLDN18.2-positive tumor; Preferably, the tumor is selected from one or more of 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.

[0029] Yet another aspect of the present invention relates to a method for treating or preventing a tumor, the method comprising administering to a subject in need thereof an effective amount of an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention; Preferably, the tumor is a CLDN18.2-positive tumor; Preferably, the tumor is selected from one or more of 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.

[0030] In some embodiments of the present invention, a method for treating or preventing a tumor is provided, and the drug administration is performed before or after surgery and / or before or after radiotherapy.

[0031] In some embodiments of the present invention, a method for treating or preventing a tumor is provided, The dose of each administration of the anti-CLDN18.2 antibody or an antigen-binding fragment thereof is 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.

[0032] In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In addition, laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are routine procedures widely used in the corresponding fields. On the other hand, to better understand the present invention, definitions and explanations of related terms are provided below.

[0033] As used herein, EC 50 refers to the concentration at 50% of the maximum effect, i.e., the concentration capable of causing 50% of the maximum effect.

[0034] 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 kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon. 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 (referred to as complementarity determining regions (CDRs)), between which conserved regions called 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 of 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 definition 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.

[0035] 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.

[0036] 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, e.g., U.S. Patent No. 4,816,567).

[0037] 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.

[0038] 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 enables them to generate 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 GGGGS amino acid sequence repeats 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).

[0039] As used herein, the term "isolated" refers to obtaining from nature by artificial means. When a particular "isolated" substance or component exists in nature, changes may occur in 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.

[0040] 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. The vector can be introduced into a host cell by transformation, transduction, or transfection, and as a result, the genetic 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. 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). The vector may contain various elements for controlling 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.

[0041] 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.

[0042] 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 the antigen it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) binds to the antigen with an affinity (K -5 less than about 10 -6 M, for example, about 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less. D )

[0043] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to represent the binding affinity between an antibody and an antigen. A smaller dissociation equilibrium constant indicates a stronger antibody-antigen binding and a higher affinity between the antibody and the antigen. Generally, an antibody binds to an antigen (e.g., the CLDN18.2 protein) with a dissociation equilibrium constant (K -5 less than about 10 -6 M, for example, about 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less. K D can be determined using methods known to those skilled in the art, for example, using a Fortebio molecular interaction instrument. D

[0044] 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. Further, as used herein, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0045] 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, for example, 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, the pH adjuster includes, but is not limited to, phosphate buffer; the surfactant includes, but is not limited to, cationic, anionic, or nonionic surfactants such as Tween-80; and the ionic strength enhancer includes, but is not limited to, sodium chloride.

[0046] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, a prophylactically effective amount against a disease (e.g., a tumor) refers to an amount sufficient to prevent, halt, 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 halt 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 those skilled 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 simultaneously.

[0047] 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 of the CLDN18.2 protein, or the extracellular fragment CLDN18.2 ECD of CLDN18.2, or a fragment containing CLDN18.2 ECD, and also includes a fusion protein of the full length of the 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" shall 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.

[0048] 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 for CLDN18.2; (2) The anti-CLDN18.2 antibody of the present invention has high ADCC activity, CDC activity, and / or ADCP activity; (3) The anti-CLDN18.2 antibody of the present invention is capable of specifically killing CLDN18.2-positive cells. [Brief explanation of the drawings]

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0050] 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.

[0051] Some sequences involved in the present invention are as follows: 8C5.1VH

Chemical Formula

[0052] 8C5.1VL

Chemical Formula

[0053] 8C5.1 CDR HCDR1: GFTFSNSA (SEQ ID NO: 5) HCDR2: ITSGVSYT (SEQ ID NO: 6) HCDR3: TRQFKGNALDY (SEQ ID NO: 7) LCDR1: QSLLNSGNQKNY (SEQ ID NO: 8) LCDR2: WAS (SEQ ID NO: 9) LCDR3: QNDYFYPLT (SEQ ID NO: 10)

[0054] 8C5.1 H1VH

Chemical Formula

[0055] 8C5.1 H2VH [Chemistry]

[0056] 8C5.1 H3VH [Chemistry]

[0057] 8C5.1 L1VL [Chemistry]

[0058] 8C5.1 L2VL [Chemistry]

[0059] 8C5.1 L3VL [Chemistry]

[0060] Amino acid sequence of the heavy chain constant region of hG1WT [Chemistry]

[0061] Amino acid sequence of the light chain constant region [Chemistry]

[0062] Amino acid sequence of the heavy chain of the positive control antibody IMAB362: [Chemistry]

[0063] Amino acid sequence of the light chain of the positive control antibody IMAB362: [Chemistry]

[0064] 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.

[0065] The cell line CHO-K1-CLDN18.2 was constructed by Akeso Biopharma Inc. The cell line CHO-K1-CLDN18.2 was prepared by viral infection of CHO-K1 cells using a third-generation lentivirus system (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentiviral expression vector used was pCDH-hCLDN18.2FL-Puro (CLDN18.2, GenBank ID: NP_001002026.1, vector pCDH-CMV-MCS-EF1-Puro, purchased from Youbio, catalog number: VT1480).

[0066] Preparation Example 1: Preparation of Anti-CLDN18.2 Antibody 8C5.1 1. Preparation of Hybridoma Cell Line LT020 The immunogen used for the preparation of the anti-CLDN18.2 antibody was a 3T3 cell line that overexpressed human Claudin18.2 (GenBank ID: NP-001002026.1) (Chinese Academy of Sciences) (3T3 hCLDN18.2). Spleen cells from immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. A CHO-K1 cell line that overexpressed human Claudin18.2 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) (CHO-K1-hCLDN18.2) was used as an antigen, and 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 cloning 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.

[0067] 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.

[0068] 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 precision filtration membrane, and purified using a HiTrap protein A HP column to obtain antibody 8C5.1.

[0069] Preparation Example 2: Sequence Analysis of Anti-CLDN18.2 Antibody 8C5.1 mRNA was extracted from the cell line LT020 cultured in Preparation Example 1 according to the method described in the manual of RNAprep pure Cell / Bacteria Kit (Tiangen, catalog number: DP430).

[0070] cDNA was synthesized and amplified by PCR according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR kits.

[0071] The PCR amplification product was directly subjected to TA cloning according to the manual of pEASY-T1 Cloning Kit (Transgen CT101).

[0072] 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 354 bp.

[0073] The encoded amino acid sequence is shown in SEQ ID NO: 2 and has a length of 118 amino acids.

[0074] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 3 and has a length of 339 bp.

[0075] The encoded amino acid sequence is shown in SEQ ID NO: 4 and has a length of 113 amino acids.

[0076] The six CDRs of antibody 8C5.1 defined by the IMGT numbering system are as follows: For the heavy chain, 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; For the light chain, 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.

[0077] Preparation Example 3: 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 sequence of antibody 8C5 obtained in Preparation Example 2, an antibody model was simulated by computer, mutations were designed according to the model, and the sequences of the variable regions of antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 were obtained (for the antibodies, the heavy chain constant region is all the C region of the Igγ-1 chain, SEQ ID NO: 23, and the light chain constant region is all the C region of the Igκ chain, SEQ ID NO: 24).

[0078] The designed variable region sequences are shown in Table 1 below.

[0079] [Table 1]

[0080] 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.

[0081] Furthermore, the above 7 antibodies had the same HCDR1-HCDR3 and LCDR1-LCDR3 as shown below: 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.

[0082] 2. Preparation of humanized antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 All of the heavy chain constant regions were the Igγ-1 chain C region, SEQ ID NO: 23, and all of the light chain constant regions were the Igκ chain C region, Accession: P01834, SEQ ID NO: 24.

[0083] The respective heavy chain cDNAs and light chain cDNAs 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.

[0084] 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 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. Furthermore, the heavy chain / light chain genes of the recombinant expression plasmids were sequenced. Subsequently, 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, then the culture medium was collected and purified. After confirming that the sequence was correct 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.

Example

[0085] Example 1: Determination of the binding activity of an anti-CLDN18.2 specific antibody to an antigen by ELISA CHO-K1-CLDN18.2 cells (Chinese hamster ovary cells CHO-K1 overexpressing CLDN18.2, purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: 3111C0001CCC000004) were seeded at 2×10 4 cells / well in a cell plate (Corning, 3599), cultured overnight at 5% CO2 and 37 °C, then the cell plate with the cells incubated was washed once with PBST, and blocked at 37 °C for 2 hours using PBST containing 1% BSA as a blocking solution. After blocking, the plate was washed 3 times with PBST. Antibodies serially diluted with PBST solution (the dilution gradients of the antibodies are shown in Table 2) were added. The ELISA plate containing the test antibodies was incubated at 37 °C for 30 minutes and then washed 3 times with PBST. After washing, in the case of mouse antibodies, a working solution of HRP-labeled goat anti-mouse FC (H+L) (Jackson, catalog number: 109-035-098) secondary antibody diluted at a ratio of 1:5000 was added, and in the case of humanized antibodies and IMAB362, a working solution of HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog number: 109-035-088) secondary antibody diluted at a ratio of 1:5000 was added. Then, the plate was incubated at 37 °C for 30 minutes. After incubation, the plate was washed 4 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 plate was immediately placed in a microplate reader, and the OD value of each well in the ELISA plate was read at 450 nm. The data were analyzed and processed by SoftMax Pro 6.2.1.

[0086] The assay results are shown in Tables 2 - 4 and Figures 1 - 3.

[0087]

Table 2

[0088]

Table 3

[0089]

Table 4

[0090] The results show that all of antibodies 8C5.1, 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 can effectively bind to the antigen on the surface of CHO-K1-CLDN18.2 cells, and the binding efficiency is dose-dependent; the binding activity of the antibodies is stronger than that of the positive control antibody IMAB362 having the same target.

[0091] Example 2: Determination of the binding activity of anti-CLDN18.2 specific antibodies to the antigen by flow cytometry 1. Determination of the binding activity of anti-CLDN18.2 specific antibody 8C5.1 to the antigen by flow cytometry Collect CHO-K1-CLDN18.2 cells in the logarithmic growth phase and place 3×10 in a 1.5 mL EP tube 5Transfer to cells / wells and then add 1% PBSA. After centrifuging the mixture at 1000×g for 5 minutes, the supernatant was removed. Dilute 100 μL of antibody 8C5.1 with 1% PBSA (final concentrations: 30 μg / mL, 10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.11 μg / mL, 0.011 μg / mL, 0.0011 μg / mL, and 0.00011 μg / mL), add 100 μL of antibody IMAB362 (final concentrations: 100 μg / mL, 33.3 μg / mL, 11.1 μg / mL, 3.7 μg / mL, 0.37 μg / mL, 0.037 μg / mL, 0.003 μg / mL, and 0.0003 μg / mL) respectively, gently mix the mixture well, and incubate on ice for 1 hour. Add 1000 μL of 1% PBSA, after centrifuging the mixture at 1000×g for 5 minutes, the supernatant was removed and washed twice with 1% PBSA. In the case of 8C5.1, add 300-fold diluted PE-goat anti-mouse IgG (minimal x-reactivity) antibody (Biolegend, catalog number: 405308); in the case of antibody IMAB362, add 300-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-098). Resuspend the mixture, mix well, and incubate in the dark on ice for 40 minutes. Add 1000 μL of 1% PBSA, after centrifuging the mixture at 1000×g for 5 minutes, the supernatant was removed and washed twice with 1% PBSA. Add 200 μL of 1% PBSA to resuspend the cell pellet, and transfer the mixture to a flow cytometry tube for FACSCalibur assay.

[0092] The experimental results are shown in Table 5 and Figure 4.

[0093]

Table 5

[0094] 2. Determination of the binding activity of anti-CLDN18.2 specific antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 to the antigen by flow cytometry Collect CHO-K1-CLDN18.2 cells in the logarithmic growth phase, add them to a V-bottom 96-well plate at 3×10 5 cells / well, then centrifuge at 700×g for 5 minutes, remove the supernatant; add the antibody diluted at the corresponding concentration according to the experimental design (30 μg / mL, 10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.11 μg / mL, 0.011 μg / mL, 0.001 μg / mL, and 0.0001 μg / mL) at 100 μL / tube respectively, incubate the mixture on ice for 1 hour; add 200 μL of 1% PBSA, centrifuge the mixture at 700×g for 5 minutes, remove the supernatant, and wash 3 times; add 100 μL of 400-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-098), mix the mixture well, and incubate in the dark on ice for 40 minutes; add 200 μL of 1% PBSA, centrifuge the mixture at 700×g for 5 minutes, remove the supernatant, and wash 3 times; add 200 μL of 1% PBSA to resuspend the cells, and transfer the suspension to a flow cytometry tube for testing.

[0095] The experimental results are shown in Table 6 and Figure 5.

[0096]

Table 6

[0097] The results are under the same experimental conditions: The EC 50 values for the binding of 8C5.1 and IMAB362 to CHO-K1-CLDN18.2 cells are 0.2136 μg / mL and 0.6873 μg / mL respectively; The EC 50It shows that the values were 0.2164 μg / mL, 0.2417 μg / mL, 0.2668 μg / mL, 0.2345 μg / mL, 0.2432 μg / mL, 0.2135 μg / mL, 0.2808 μg / mL, and 0.3581 μg / mL, respectively.

[0098] The above experimental results show that 8C5.1, 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, 8C5.1H3L3, and IMAB362 have the activity of effectively binding to CLDN18.2 on the cell membrane surface of CHO-K1-CLDN18.2, and 8C5.1, 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 have stronger activity than the control antibody IMAB362 in the binding of CLDN18.2 on the cell membrane surface of CHO-K1-CLDN18.2.

[0099] Example 3: Assay for ADCC Activity of Anti-CLDN18.2 Antibody Collect CHO-K1-CLDN18.2 cells as usual, centrifuge at 170×g for 5 minutes, resuspend in medium (RPMI-1640 + 1% FBS), then wash twice; resuspend the cells in medium (RPMI-1640 + 1% FBS), count, adjust the cell density, and prepare the target cell suspension at 100 μL / well (about 3×10 4Added to a 96-well plate in the target cells / well); antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, 8C5.1H3L3, and IMAB362 were diluted with medium (RPMI-1640 + 1% FBS) (the working concentrations were all 30 μg / mL, 10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.11 μg / mL, 0.011 μg / mL, 0.001 μg / mL, and 0.0001 μg / mL), and the diluted antibodies were added to the corresponding wells at 50 μL / well. After that, they were incubated at 37 °C for 1 hour in an incubator; during this time, negative control medium and isotype control hIgG1 (manufactured by Akeso Biopharma Inc., batch number: 20190410) were set up.

[0100] Effector cells PBMC (from healthy donors) were collected as usual, centrifuged at 170×g for 5 minutes, then the supernatant was removed and washed twice; the cells were resuspended in medium (RPMI-1640 + 1% FBS), counted, and the cell density was adjusted; 50 μL of the effector cell suspension was added to the pre-incubated target cells (about 6×10 5 effector cells / well), the mixture was well mixed, incubated at 5% CO2 and 37 °C for 4 hours in an incubator, and then centrifuged at 250×g for 5 minutes; 100 μL of the culture supernatant was carefully pipetted into a new 96-well flat-bottom plate, 100 μL of the newly prepared reaction solution was added to each well, and the mixture was incubated in the dark at room temperature for 30 minutes; the OD values were measured at 490 nm and 650 nm, and the OD value of each group = OD 490nm -OD 650nm [[ID=IO]]was.

[0101] The results are shown in Figure 6.

[0102] The results show that, compared with the isotype control, the anti-CLDN18.2 antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2 and 8C5.1H3L3, as well as the positive control antibody IMAB362, have ADCC activity and can specifically kill CLDN18.2-positive cells, and they have comparable killing activities.

[0103] Example 4: Assay for CDC activity of anti-CLDN18.2 antibodies CHO-K1-CLDN18.2 cells were digested and collected as usual, centrifuged at 170×g for 5 minutes, resuspended in medium (RPMI-1640 + 1% FBS), and then washed twice; the cells were resuspended in medium (RPMI-1640 + 1% FBS), counted, the cell density was adjusted, and the target cell suspension was added to a 96-well plate at 100 μL / well (about 3×10 4 cells / well); the antibodies were diluted with medium (RPMI-1640 + 1% FBS) (the working concentrations were all 30 μg / mL, 3.33 μg / mL, 0.11 μg / mL, 0.001 μg / mL, and 0.0001 μg / mL), and the diluted antibodies were added to the corresponding wells at 50 μL / well, and the mixture was pre-incubated at room temperature for 10 minutes; 50 μL of complement serum (final concentration: 2%) was added to the pre-incubated target cells, the mixture was well mixed, incubated in an incubator at 5% CO2 and 37 °C for 4 hours, and then centrifuged at 250×g for 5 minutes; 100 μL of the culture supernatant was carefully pipetted into a new 96-well flat-bottom plate, 100 μL of the newly prepared reaction solution was added to each well, and the mixture was incubated in the dark at room temperature for 30 minutes; the OD values were measured at 490 nm and 650 nm, and the OD value of each group = OD 490nm -OD 650nm was.

[0104] The results are shown in Figure 7.

[0105] The results showed that compared with the isotype control (manufactured by Akeso Biopharma Inc., batch number: 20190410), all of the anti-CLDN18.2 antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 had CDC activity and were able to kill tumor cells under the mediation of complement, and their activities were better than those of the positive control antibody IMAB362.

[0106] Example 5: Anti-CLDN18.2 Antibody Promoting Phagocytosis of Tumor Cells by Macrophages In this example, the antibody-dependent cell 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.

[0107] The specific method of this example is as follows: CHO-K1-CLDN18.2 was 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.

[0108] 6 mL of complete DMEM medium (containing 10% FBS) was added to stop the staining. After the cells were centrifuged at 170×g for 5 minutes, the supernatant was removed. 1 mL of complete DMEM medium was added, and the cells were incubated in an incubator for 10 minutes. The antibodies were diluted with complete DMEM medium (10 μg / mL, 1 μg / mL, and 0.1 μg / mL), and isotype control antibodies and reference antibodies were designed.

[0109] Macrophages were collected and centrifuged at 170×g for 5 minutes. After removing the supernatant, the cells were counted; the cells were transferred to a 1.5 mL EP tube and centrifuged at 1200×g for 5 minutes, then the supernatant was removed; the tumor cells and antibody suspension incubated for 30 minutes were added to the 1.5 mL EP tube containing macrophages, then resuspended and mixed well, and the mixture was incubated in an incubator at 37°C for 2 hours; 800 μL of room temperature 1% PBSA was added to each tube, and after centrifuging the mixture at 1200×g for 5 minutes, the supernatant was removed and washed once with 800 μL of PBSA; 600-fold diluted APC anti-mouse / human CD11b antibody (Biolegend, catalog number: 101212) was added to the corresponding samples at 100 μL / sample, the mixture was mixed well and incubated on ice for 40 minutes. 800 μL of 1% PBSA was added to each tube, and after centrifuging the mixture at 1200×g for 5 minutes, the supernatant was removed; each tube was washed once with 200 μL of PBSA, and 200 μL of 1% PBSA was added to each tube for resuspension. Then, the mixture was transferred to a flow cytometry tube for testing.

[0110] The results are shown in Figure 8.

[0111] 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.

[0112] Although specific embodiments of the present invention have been described in detail, those skilled in the art can 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 protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

**Claim 1** An anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the anti-CLDN18.2 antibody comprises a heavy chain variable region containing HCDR1-HCDR3 and a light chain variable region containing LCDR1-LCDR3, and: 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, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof. **Claim 2** the amino acid sequence of the heavy chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15; the amino acid sequence of the light chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, the anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to claim 1. **Claim 3** 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: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

21. The anti-CLDN18.2 antibody or antigen-binding fragment thereof according to claim 1 or 2. **Claim 4** For the antibody, the heavy chain constant region is the Igγ-1 chain C region or the Igγ-4 chain C region, and the light chain constant region is the Igκ chain C region. The anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. **Claim 5** The anti-CLDN18.2 antibody or antigen-binding fragment thereof is Fab, Fab', F(ab') ２ , Fd, Fv, dAb, complementarity-determining region fragment, single-chain variable fragment, humanized antibody, or chimeric antibody, and the anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. **Claim 6** The antibody contains a non-CDR region derived from a non-mouse species such as a human antibody. The anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. **Claim 7** EC regarding the binding of the anti-CLDN18.2 antibody to cells expressing CLDN18.2 ５０ is 0.5 μg / mL or less, 0.4 μg / mL or less, or 0.3 μg / mL or less; Preferably, the EC ５０ is measured by FACS, The anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. **Claim 8** The 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 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7. **Claim 9** An isolated nucleic acid molecule encoding the anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. **Claim 10** A recombinant vector containing the isolated nucleic acid molecule according to claim 9. **Claim 11** A host cell containing the isolated nucleic acid molecule according to claim 9 or the recombinant vector according to claim 10. **Claim 12** The hybridoma cell line LT020 deposited with the China Center for Type Culture Collection (CCTCC) under the CCTCC designation of CCTCC NO. C2022124. **Claim 13** An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is the anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an anti-tumor chemotherapy drug. **Claim 14** The antibody or antigen-binding fragment thereof is linked to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond. The antibody-drug conjugate according to claim 13, wherein preferably, the molar ratio of the antibody or its antigen-binding fragment to the small molecule drug is 1:(2-4).

15. A pharmaceutical composition comprising an effective amount of the anti-CLDN18.2 antibody or its antigen-binding fragment according to any one of claims 1 to 8, or the antibody-drug conjugate according to claim 13 or 14, optionally further comprising one or more pharmaceutically acceptable excipients.

16. Use of the anti-CLDN18.2 antibody or its antigen-binding fragment according to any one of claims 1 to 8 in the preparation of a medicament for the treatment or prevention of tumors, wherein preferably, the tumor is a CLDN18.2-positive tumor; wherein preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, 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.

17. The anti-CLDN18.2 antibody or its antigen-binding fragment according to any one of claims 1 to 8 for use in the treatment or prevention of tumors, wherein preferably, the tumor is a CLDN18.2-positive tumor; wherein preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, 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.

18. A method for treating or preventing tumors, comprising the step of administering an effective amount of the anti-CLDN18.2 antibody or its antigen-binding fragment according to any one of claims 1 to 8 to a subject in need thereof, wherein preferably, the tumor is a CLDN18.2-positive tumor; wherein preferably, the tumor is one or more selected from cholangiocarcinoma, bronchiogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, 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.

19. The method for treating or preventing tumors according to claim 18, wherein the drug administration is performed before or after surgery and / or before or after radiotherapy.

20. The dose of each administration of the anti-CLDN18.2 antibody or its antigen-binding fragment is 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 carried out 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. A method for treating or preventing a tumor according to claim 18 or 19.

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