Anti-CLDN18.2 antibody, its pharmaceutical composition, 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 This invention belongs to the field of biomedicine and relates to an anti-CLDN18.2 antibody, its pharmaceutical composition, and its use. [Background technology]

[0002] background Tumors, particularly malignant tumors, are currently a serious health threat worldwide and the second leading cause of death among various diseases. In recent years, the incidence of these diseases has increased dramatically. Malignant tumors are characterized by poor treatment response, a high rate of late metastasis, and a poor prognosis. While conventional treatments currently used clinically (radiotherapy, chemotherapy, surgery, etc.) significantly alleviate pain and extend survival, they have significant limitations, and further improvement in their effectiveness remains challenging.

[0003] CLDN18.2 is an integrin membrane protein found in epithelial and endothelial tight junctions. It consists of 261 amino acids and is a member of the Claudin (CLDN) family, with both its N-terminus and C-terminus located intracellularly. The entire protein is expressed on the cell membrane. CLDN18.2 has four transmembrane domains, two extracellular loops, and one intracytoplasmic loop, and is involved in the formation of intercellular tight junction structures (Gunzel, D.; Yu, ASL 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 connection with the extracellular loop of the CLDN18.2 protein of adjacent cells via hydrophobic bonds between aromatic residues. The extracellular loop of the CLDN18.2 protein of adjacent cells maintains close junctions between epithelial and endothelial cells through interactions, regulates intercellular osmotic pressure, maintains the polarity of epithelial and endothelial cells, is involved in cell proliferation, and can participate in diverse signaling pathways via serine, threonine, and tyrosine-rich carboxyl terminus (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 is often abnormally altered during the development and progression of malignant tumors. For example, when gastric epithelial tissue is subjected to malignant transformation, disruption of cell polarity will lead to exposure of the CLDN18.2 protein epitope on the cell surface. Simultaneously, the CLDN18.2 gene is also abnormally activated and expressed with high selectivity and stability in certain tumor tissues such as gastric cancer, affecting intercellular and extracellular matrix junctions, influencing cell barrier function and polarity maintenance, thereby causing changes in intercellular cytokine and ion permeability, damaging barrier function, and inducing 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, Germany. Claudiximab specifically recognizes and binds to the first extracellular domain (ECD1) outside the cell membrane of the CLDN18.2 protein with high affinity, 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. [Overview of the Initiative] [Means for solving the problem]

[0008] overview Through intensive research and creative efforts, the inventors obtained an anti-CLDN18.2 antibody. Surprisingly, the inventors found that the anti-CLDN18.2 antibody of the present invention (also referred to simply as the antibody or the antibody of the present invention) possesses excellent affinity and / or specificity, can bind to CLDN18.2-expressing tumor cells with high specificity, and offers promising antitumor prospects. The present invention is described in detail below.

[0009] One aspect of the present invention relates to 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 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.

[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 50 of the anti-CLDN18.2 antibody regarding binding to cells expressing CLDN18.2 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 its antigen-binding fragment, 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 CTCCC 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 CTCCC 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 an isolated nucleic acid molecule of the present invention.

[0021] A further aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule or a recombinant vector of the present invention.

[0022] A further aspect of the present invention relates to an antibody-drug conjugate comprising an antibody or its antigen-binding fragment and a small molecule drug, wherein the antibody or its antigen-binding fragment is an anti-CLDN18.2 antibody or its antigen-binding fragment 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 antitumor chemotherapy agent.

[0023] The chemotherapeutic agent may be a conventional antitumor chemotherapeutic agent such as alkylating agents, antimetabolites, antitumor antibiotics, plant-based anticancer agents, hormones, and immunotherapies.

[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, the linker may be 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 the antibody or its antigen-binding fragment to the small molecule drug is 1:(2-4), for example, 1:2, 1:3, or 1:4.

[0026] A further aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of an anti-CLDN18.2 antibody or its antigen-binding fragment according to any aspect of the present invention or an antibody-drug conjugate according to any aspect of the present invention, wherein the pharmaceutical composition optionally further comprises one or more pharmaceutically acceptable auxiliary substances.

[0027] A further aspect of the present invention relates to the use of an anti-CLDN18.2 antibody or its antigen-binding fragment according to any aspect of the present invention in the preparation of a pharmaceutical 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 biliary tract cancer, bronchogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0028] An anti-CLDN18.2 antibody or its antigen-binding fragment is provided according to any aspect of the present invention 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 the following: biliary tract cancer, bronchogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0029] A further aspect of the present invention relates to a method for treating or preventing a tumor, the method comprising the step of administering an effective amount of an anti-CLDN18.2 antibody or its antigen-binding fragment according to any aspect of the present invention to a subject in need; Preferably, the tumor is a CLDN18.2-positive tumor; Preferably, the tumor is selected from one or more of the following: biliary tract cancer, bronchogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach 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, wherein the drug is administered before or after surgery and / or before or after radiotherapy.

[0031] In some embodiments of the present invention, a method for treating or preventing tumors is provided. The dose of each 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, the drug is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the route of administration is intravenous drip infusion or intravenous injection.

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

[0033] When used herein, EC 50 The term refers to a concentration that is 50% of the maximum effect, i.e., a concentration that can produce 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 into κ and λ light chains. Heavy chains are classified into μ, δ, γ, α, or ε. Antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable and constant regions are linked by “J” regions of about 12 or more amino acids, and the heavy chain further includes “D” regions 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 immunoglobulin to host tissue or factors, including binding to the first component (C1q) of the classical complement system of various cells in 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)), with conserved regions called framework regions (FRs) distributed between them. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding site.The assignment of amino acids to regions or domains is based on the definitions in Bethesda Md, 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 antibody production. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies may also 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 an antibody or antibody fragment derived from a group of highly homologous antibodies, i.e., a group of identical antibody molecules, excluding spontaneous mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies, compared to monoclonal antibodies, generally contain at least two different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using hybridoma technology, first reported by Koehler et al. (Koehler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but they can also be obtained using recombinant DNA technology (see, for example, 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 region of a human immunoglobulin (receptor antibody) is replaced by the CDR region of a non-human antibody (donor antibody), where the donor antibody may be a non-human (e.g., mouse, rat, or rabbit) antibody having the expected specificity, affinity, or reactivity. In addition, several amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody or other antibodies to further improve or optimize the performance of the antibody. For details on 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 a variable region of an antibody heavy chain (V H ) and a variable region of an antibody light chain (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” means obtained from nature by artificial means. Where a particular “isolated” substance or component exists in nature, it may have undergone a change in its natural environment, or it may have been isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide may occur 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 preclude 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 polynucleotides can be inserted. If a 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, thereby enabling the expression of the genetic material elements contained within the vector 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 phages or M13 phages; 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 a variety of elements that control expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may further contain replication initiation sites.

[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, e.g., Escherichia coli or Bacillus subtilis; fungal cells, e.g., yeast or Aspergillus; insect cells, e.g., Drosophila S2 or Sf9; or animal cells, e.g., 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 a 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 to an antigen) is one in which the antibody has a binding rate of approximately 10 -5 Less than M, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 Affinity (K) less than or equal to M D This means that it binds to the antigen.

[0043] When 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 express the binding affinity between the antibody and the antigen. A smaller dissociation equilibrium constant indicates stronger antibody-antigen binding and a higher affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of approximately 10. -5 Less than M, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 Dissociation equilibrium constant (K) less than or equal to M D ) binds to the antigen (e.g., CLDN18.2 protein). K D This can be determined using methods known to those skilled in the art, for example, using the Fortebio molecular interaction instrument.

[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. Furthermore, as used herein, amino acids are generally represented by one- and three-letter abbreviations 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” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and 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 modifiers, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers 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.

[0046] As used herein, the term “effective dose” means an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactic effective dose for a disease (e.g., a tumor) means an amount sufficient to prevent, halt, or delay the onset of the disease (e.g., a tumor); a therapeutic effective dose means an amount sufficient to cure or at least partially halt the disease and its complications in a patient suffering from the disease. It is clear that determining such an effective dose is within the capabilities of those skilled in the art. For example, an effective dose for therapeutic purposes will depend on the severity of the disease being treated, the overall state of the patient’s own immune system, the patient’s overall condition such as age, weight, and sex, the route of administration, and other treatments given concurrently.

[0047] When 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 fragments containing CLDN18.2 ECD, as well as fusion proteins of the full-length CLDN18.2 protein, or fusion proteins of CLDN18.2 ECD, such as fragments fused to an Fc protein fragment of mouse or human IgG (mFc or hFc). However, those skilled in the art will understand that mutations or alterations (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of the CLDN18.2 protein can occur naturally or be artificially introduced without affecting its biological function. Accordingly, in this invention, the term “CLDN18.2 protein” includes all such sequences, including their natural or artificial variants. In addition, if 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 This 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 drawing]

[0049] Brief explanation of the drawing [Figure 1] Determination of the binding activity of anti-CLDN18.2 specific antibody 8C5.1 to antigens on the surface of CHO-K1-CLDN18.2 cells by ELISA. [Figure 2] ELISA determination of the binding activity of anti-CLDN18.2 specific antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, and 8C5.1H2L2 to antigens on the surface of CHO-K1-CLDN18.2 cells. [Figure 3] ELISA determination of the binding activity of anti-CLDN18.2 specific antibodies 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 to antigens on the surface of CHO-K1-CLDN18.2 cells. [Figure 4] FACS assay for the binding activity of anti-CLDN18.2 antibodies 8C5.1 and IMAB362 to CHO-K1-CLDN18.2 cells. [Figure 5] FACS assay of the binding activity of anti-CLDN18.2 antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, 8C5.1H3L3, and IMAB362 to CHO-K1-CLDN18.2 cells. [Figure 6] Assay on the ADCC activity of anti-CLDN18.2 antibodies against CHO-K1-CLDN18.2 cells. [Figure 7] Assay on the CDC activity of anti-CLDN18.2 antibodies against CHO-K1-CLDN18.2 cells. [Figure 8]Phagocytosis of MBMM in CHO-K1-CLDN18.2 cells mediated by 8C5.1H3L3 and IMAB362. [Modes 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 CCTCC No. C2022124. The depositary address is Wuhan University, Wuhan, China, postal code 430072.

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

[0052] 8C5.1VL [ka]

[0053] 8C5.1 CDR HCDR1:GFTFSNSA (Sequence ID 5) HCDR2:ITSGVSYT(Sequence ID 6) HCDR3:TRQFKGNALDY (Sequence ID 7) LCDR1:QSLLNSGNQKNY (Sequence ID 8) LCDR2:WAS (Sequence ID 9) LCDR3:QNDYFYPLT(Sequence ID 10)

[0054] 8C5.1 H1VH [ka]

[0055] 8C5.1 H2VH [ka]

[0056] 8C5.1 H3VH [ka]

[0057] 8C5.1 L1VL [ka]

[0058] 8C5.1 L2VL [ka]

[0059] 8C5.1 L3VL [ka]

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

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

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

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

[0064] Detailed explanation Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Experimental procedures in the examples that do not involve specific conditions are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or equipment used are all commercially available conventional products unless the manufacturer is 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 lentivirus expression vector used was pCDH-hCLDN18.2FL-Puro (CLDN18.2, GenBankID: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 to prepare the anti-CLDN18.2 antibody was a 3T3 cell line overexpressing human Claudin18.2 (GenBankID:NP-001002026.1) (Chinese Academy of Sciences) (3T3 hCLDN18.2). Hybridoma cells were prepared by fusing spleen cells from immunized mice with mouse myeloma cells. Using a CHO-K1 cell line overexpressing human Claudin18.2 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) (CHO-K1-hCLDN18.2) as an antigen, hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies that specifically bind to CLDN18.2. The hybridoma cells obtained by screening were subjected to limiting dilution cloning to obtain stable hybridoma cell lines. The hybridoma cell line described above 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 CCTCC No. C2022124. The depositary 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 5% CO2 and 37°C in CD medium (a chemically defined medium containing 4% Glutamax (Gibco 35050079) and 1% penicillin-streptomycin (Gibco 15140163)). After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation and vacuum filtration using a microfiltration 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 cell line LT020 cultured in Preparation Example 1, according to the instructions in the RNAprep pure Cell / Bacteria Kit (Tiangen, catalog number: DP430) manual.

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

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

[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 Sequence 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, as defined by the IMGT numbering system, are as follows: Regarding 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; Regarding the light chains, the sequence of LCDR1 is shown in sequence number 8, the sequence of LCDR2 is shown in sequence number 9, and the sequence of LCDR3 is shown in sequence number 10.

[0077] Preparation Example 3: Design and preparation of light and heavy chains of a humanized anti-human CLDN18.2 antibody 1. Design of light and heavy chains for 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 on a computer, and mutations were designed according to the model to obtain the variable region sequences of antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 (for all antibodies, the heavy chain constant region is the Igγ-1 chain C region, SEQ ID NO: 23, and the light chain constant region is the Igκ chain C region, SEQ ID NO: 24).

[0078] The designed variable region array is shown in Table 1 below.

[0079] [Table 1]

[0080] For each of the seven antibodies listed above—8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3—the length of the nucleotide sequence in the heavy chain variable region was 354 bp, and the length of the encoded amino acid sequence was 118 aa. The length of the nucleotide sequence in the light chain variable region was 339 bp, and the length of the encoded amino acid sequence was 113 aa.

[0081] Furthermore, the seven antibodies listed above had the same HCDR1-HCDR3 and LCDR1-LCDR3 as shown below: The sequence of HCDR1 is shown in sequence number 5, the sequence of HCDR2 is shown in sequence number 6, and the sequence of HCDR3 is shown in sequence number 7; The sequence of LCDR1 is shown in sequence number 8, the sequence of LCDR2 is shown in sequence number 9, and the sequence of LCDR3 is shown in sequence number 10.

[0082] 2. Preparation of humanized antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 The heavy chain constant region was entirely the Igγ-1 chain C region, sequence number 23, and the light chain constant region was entirely the Igκ chain C region, accession: P01834, sequence number 24.

[0083] The heavy 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 (provided by Genscript) vector to create pUC57simple-8C5.1H1 and pUC57simple-8C5.1L1, respectively. pUC57simple-8C5.1H1, pUC57simple-8C5.1L2; pUC57simple-8C5.1H2, pUC57simple-8C5.1L1; pUC57simple-8C5.1H2, pUC57simple-8C5.1L2; pUC57simple-8C5.1H2, pUC57simple-8C5.1L3; We obtained pUC57simple-8C5.1H3, pUC57simple-8C5.1L2, and pUC57simple-8C5.1H3 and pUC57simple-8C5.1L3.

[0084] Following 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 restriction enzyme digestion (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. The heavy / light chain genes of the recombinant expression plasmids were then 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 simultaneously transfected into 293F cells, and the culture medium was then collected and purified. After sequencing confirmed the correctness of the sequences, an endotoxin-free expression plasmid was prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days, the cell culture medium was collected and affinity purification was performed using a protein A column to obtain humanized antibodies. [Examples]

[0085] Example 1: Determination of the binding activity of anti-CLDN18.2 specific antibody to the antigen by ELISA. CHO-K1-CLDN18.2 cells (CHO-K1 Chinese hamster ovary cells overexpressing CLDN18.2, purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: 3111C0001CCC000004) were divided into 2 × 10⁻¹⁴ cells. 4 Cells were seeded in wells on cell plates (Corning, 3599) and incubated overnight in 5% CO2 at 37°C. The incubated cell plates were then washed once with PBST, and blocked at 37°C for 2 hours using PBST containing 1% BSA as a blocking solution. After blocking, the plates were washed three times with PBST. Antibodies, serially diluted in PBST solution (the antibody dilution gradient is shown in Table 2), were added. ELISA plates containing the test antibodies were incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, for 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. For 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. The plates were then incubated at 37°C for 30 minutes. After incubation, the plates were washed four times with PBST, TMB (Neogen, 308177) was added for color development in the dark for 5 minutes, and then the color reaction was stopped by adding a stop solution. The ELISA plates were immediately placed in a microplate reader, and the OD values ​​of each well in the ELISA plate were read at 450 nm. The data were analyzed and processed using 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 antibodies 8C5.1, 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 were all able to effectively bind to the antigen on the surface of CHO-K1-CLDN18.2 cells, with binding efficiency being dose-dependent; and the binding activity of the antibodies was stronger than that of the positive control antibody IMAB362, which has the same target.

[0091] Example 2: Flow cytometry determination of the binding activity of anti-CLDN18.2 specific antibody to the antigen. 1. Determination of the binding activity of the anti-CLDN18.2 specific antibody 8C5.1 to the antigen by flow cytometry. CHO-K1-CLDN18.2 cells were collected during the logarithmic growth phase and placed in 1.5 mL EP tubes in groups of 3 × 10⁶. 5Cells were transferred to wells, and then 1% PBSA was added. The mixture was centrifuged at 1000×g for 5 minutes, and the supernatant was removed. 100 μL of antibody 8C5.1 was diluted 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), and 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) were added, and the mixtures were gently mixed thoroughly and incubated on ice for 1 hour. Add 1000 μL of 1% PBSA and centrifuge the mixture at 1000 × g for 5 minutes. Remove the supernatant and wash twice with 1% PBSA. For 8C5.1, add 300-fold diluted PE goat anti-mouse IgG (minimum x-reactivity) antibody (Biolegend, catalog number: 405308); for 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 on ice in the dark for 40 minutes. Add 1000 μL of 1% PBSA and centrifuge the mixture at 1000 × g for 5 minutes. Remove the supernatant and wash 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 the 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. CHO-K1-CLDN18.2 cells were collected during the logarithmic growth phase and placed in a 96-well plate with a V-bottom in 3 × 10⁶ cells. 5 Add the antibody to the cells / well, then centrifuge at 700×g for 5 minutes, remove the supernatant; add the antibody diluted to 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) in 100 μL / tube, 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, wash three 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 on ice in the dark for 40 minutes; add 200 μL of 1% PBSA was added, the mixture was centrifuged at 700 × g for 5 minutes, the supernatant was removed and washed three times; the cells were resuspended with 200 μL of 1% PBSA, and the turbidity was transferred 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 were obtained under the same experimental conditions: EC regarding the binding of 8C5.1 and IMAB362 to CHO-K1-CLDN18.2 cells 50 The values ​​were 0.2136 μg / mL and 0.6873 μg / mL, respectively; EC regarding the binding of 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, 8C5.1H3L3, and IMAB362 to CHO-K1-CLDN18.2 cells 50The 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 experimental results described above indicate that 8C5.1, 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, 8C5.1H3L3, and IMAB362 have the activity to effectively bind to CLDN18.2 on the cell membrane surface of CHO-K1-CLDN18.2, and that 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 binding CLDN18.2 on the cell membrane surface of CHO-K1-CLDN18.2.

[0099] Example 3: Assay on the ADCC activity of anti-CLDN18.2 antibody CHO-K1-CLDN18.2 cells were collected as usual, centrifuged at 170×g for 5 minutes, resuspended in medium (RPMI-1640 + 1% FBS), then washed twice; the cells were resuspended in medium (RPMI-1640 + 1% FBS), counted, cell density adjusted, and target cell turbidity was collected in 100 μL / well (approximately 3 × 10⁴). 4Target cells were added to a 96-well plate; antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, 8C5.1H3L3, and IMAB362 were diluted in culture medium (RPMI-1640 + 1% FBS) (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); 50 μL / well of each diluted antibody was added to the corresponding well, and the mixture was incubated in an incubator at 37°C for 1 hour; during this time, negative control medium and isotype control hIgG1 (Akeso Biopharma) were used. Set up a batch number (20190410) manufactured by Inc.

[0100] Effector cells (PBMCs) (from healthy donors) were collected as usual, centrifuged at 170 × g for 5 minutes, the supernatant was removed, and the cells were washed twice; the cells were resuspended in culture medium (RPMI-1640 + 1% FBS), counted, and the cell density adjusted; and target cells (approximately 6 × 10) were pre-incubated in 50 μL of effector cell turbidity. 5 Add the mixture to the effector cells (including the wells), mix well, incubate in an incubator with 5% CO2 and 37°C for 4 hours, then centrifuge at 250×g for 5 minutes; carefully pipette 100 μL of the culture supernatant into a new 96-well flat-bottom plate, add 100 μL of the freshly prepared reaction solution to each well, incubate the mixture in the dark at room temperature for 30 minutes; measure the OD values ​​at 490 nm and 650 nm, and the OD value for each group = OD 490nm -OD 650nm That was the case.

[0101] The results are shown in Figure 6.

[0102] The results show that, compared to isotype controls, 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, possess ADCC activity and can specifically kill CLDN18.2-positive cells, and that they have comparable killing activity.

[0103] Example 4: Assay on the CDC activity of anti-CLDN18.2 antibody 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), then washed twice; the cells were resuspended in medium (RPMI-1640 + 1% FBS), counted, cell density adjusted, and target cell turbidity was dispensed in 100 μL / well (approximately 3 × 10⁶). 4 Add cells (per well) to a 96-well plate; dilute the antibody in medium (RPMI-1640 + 1% FBS) (working concentrations were all 30 μg / mL, 3.33 μg / mL, 0.11 μg / mL, 0.001 μg / mL, and 0.0001 μg / mL); add 50 μL of the diluted antibody to the corresponding well; pre-incubate the mixture at room temperature for 10 minutes; add 50 μL of complement serum (final concentration: 2%) to the pre-incubated target cells; mix the mixture well; and incubate in an incubator for 5% Incubate in CO2 at 37°C for 4 hours, then centrifuge at 250×g for 5 minutes; carefully pipette 100 μL of culture supernatant into a new 96-well flat-bottom plate, add 100 μL of freshly prepared reaction solution to each well, and incubate the mixture in the dark at room temperature for 30 minutes; measure the OD values ​​at 490 nm and 650 nm, and the OD value for each group = OD 490nm -OD 650nm That was the case.

[0104] The results are shown in Figure 7.

[0105] The results showed that, compared to the isotype control (Akeso Biopharma Inc., batch number: 20190410), the anti-CLDN18.2 antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 all possessed CDC activity and were capable of killing tumor cells under complement-mediated conditions. Their activity was also superior to that of the positive control antibody IMAB362.

[0106] Example 5: Anti-CLDN18.2 antibody that promotes phagocytosis of tumor cells by macrophages. In this example, antibody-dependent phagocytic activity (ADCP) 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 embodiment 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] Staining was stopped by adding 6 mL of DMEM complete medium (containing 10% FBS). The cells were centrifuged at 170 × g for 5 minutes, and the supernatant was removed. 1 mL of DMEM complete medium was added, and the cells were incubated in an incubator for 10 minutes. Antibodies were diluted in DMEM complete medium (10 μg / mL, 1 μg / mL, and 0.1 μg / mL) to design isotype control antibodies and reference antibodies.

[0109] Macrophages were collected, centrifuged at 170×g for 5 minutes, the supernatant was removed, and the cells were counted; the cells were transferred to 1.5 mL EP tubes, centrifuged at 1200×g for 5 minutes, the supernatant was removed; the tumor cells and antibody turbidity incubated for 30 minutes was added to 1.5 mL EP tubes containing macrophages, resuspended, 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, the mixture was centrifuged at 1200×g for 5 minutes, the supernatant was removed, and the mixture was 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 sample, the mixture was mixed well, and the mixture was 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 then removed; each tube was washed once with 200 μL of PBSA, and 200 μL of 1% PBSA was added to each tube for resuspension. The mixture was then transferred to flow cytometry tubes for testing.

[0110] The results are shown in Figure 8.

[0111] The results showed that the phagocytic 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. This indicates that both 8C5.1H3L3 and the reference antibody IMAB362 possess ADCP activity, and that 8C5.1H3L3 exhibits better ADCP activity than IMAB362.

[0112] While specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all disclosed teachings, and that all such modifications fall within the scope of protection of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-CLDN18.2 antibody or its antigen-binding fragment, wherein the anti-CLDN18.2 antibody comprises 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. An anti-CLDN18.2 antibody or its antigen-binding fragment, wherein 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.

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 its antigen-binding fragment according to claim 1.

3. The amino acid sequence of the heavy chain variable region is shown in Sequence ID No. 2, and the amino acid sequence of the light chain variable region is shown in Sequence ID No. 4; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 11, and the amino acid sequence of the light chain variable region is shown in Sequence ID 17; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 11, and the amino acid sequence of the light chain variable region is shown in Sequence ID 19; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 11, and the amino acid sequence of the light chain variable region is shown in Sequence ID 21; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 13, and the amino acid sequence of the light chain variable region is shown in Sequence ID 17; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 13, and the amino acid sequence of the light chain variable region is shown in Sequence ID 19; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 13, and the amino acid sequence of the light chain variable region is shown in Sequence ID 21; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 15, and the amino acid sequence of the light chain variable region is shown in Sequence ID 17; The amino acid sequence of the heavy chain variable region is shown in Sequence ID 15, and the amino acid sequence of the light chain variable region is shown in Sequence ID 19; or The amino acid sequence of the heavy chain variable region is shown in Sequence ID 15, and the amino acid sequence of the light chain variable region is shown in Sequence ID 21. The anti-CLDN18.2 antibody or its antigen-binding fragment according to claim 1.

4. The anti-CLDN18.2 antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain constant region of the antibody 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.

5. The anti-CLDN18.2 antibody or its antigen-binding fragment is Fab, Fab', F(ab') 2 An anti-CLDN18.2 antibody or antigen-binding fragment thereof according to claim 1, selected from Fv, scFv, humanized antibody, or chimeric antibody.

6. The antibody includes a non-CDR region derived from a human antibody. The anti-CLDN18.2 antibody or its antigen-binding fragment according to claim 1.

7. EC regarding the binding of the anti-CLDN18.2 antibody to cells expressing CLDN18.2 50 However, it is 0.5 μg / mL or less, 0.4 μg / mL or less, or 0.3 μg / mL or less; The EC 50 This is measured by FACS. The anti-CLDN18.2 antibody or its antigen-binding fragment according to claim 1.

8. The antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 23, and the light chain constant region has the amino acid sequence shown in SEQ ID NO:

24.

9. An isolated nucleic acid molecule encoding an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A recombinant vector comprising an isolated nucleic acid molecule as described in claim 9.

11. A host cell comprising an isolated nucleic acid molecule or a recombinant vector as described in claim 9, wherein the recombinant vector comprises the isolated nucleic acid molecule as described in claim 9.

12. Hybridoma cell line LT020, deposited with the China Center for Type Culture Collection (CCTCC) under CCTCC No. C2022124.

13. 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 the anti-CLDN18.2 antibody or the antigen-binding fragment thereof as described in any one of claims 1 to 8.

14. The following: (1) The small molecule drug is a small molecule cytotoxic drug or an antitumor chemotherapy drug; (2) The antibody or its antigen-binding fragment is linked to the small molecule drug via a linker; (3) The antibody or its antigen-binding fragment is linked to the small molecule drug via a hydrazone bond, disulfide bond, or peptide bond; (4) The molar ratio of the antibody or its antigen-binding fragment to the small molecule drug is 1:(2-4), The antibody-drug conjugate according to claim 13, characterized by one or more of the above.

15. A pharmaceutical composition comprising an effective amount of an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, and further comprising one or more pharmaceutically acceptable auxiliary substances.

16. A pharmaceutical composition comprising an effective amount of the antibody-drug conjugate described in claim 13, and further comprising one or more pharmaceutically acceptable auxiliary substances.

17. Use of an anti-CLDN18.2 antibody or its antigen-binding fragment according to any one of claims 1 to 8 in the preparation of a pharmaceutical product for the treatment or prevention of tumors.

18. The tumor is a CLDN18.2-positive tumor; or The use according to claim 17, wherein the tumor is one or more selected from biliary tract cancer, bronchogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

19. An anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, for use in the treatment or prevention of tumors.

20. The tumor is a CLDN18.2-positive tumor; or The anti-CLDN18.2 antibody or antigen-binding fragment thereof according to claim 19, wherein the tumor is one or more selected from biliary tract cancer, bronchogenic 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.

21. A pharmaceutical composition for treating or preventing a tumor, comprising an effective amount of an anti-CLDN18.2 antibody or its antigen-binding fragment according to any one of claims 1 to 8.

22. A pharmaceutical composition for treating or preventing tumors, the following: (1) The drug is administered before or after surgery and / or before or after radiation therapy; (2) 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; (3) The drug is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; (4) The route of administration is intravenous infusion or intravenous injection; (5) The tumor is a CLDN18.2-positive tumor; or The tumor is one or more selected from biliary tract cancer, bronchogenic lung cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer. The pharmaceutical composition according to claim 21, characterized by one or more of the above.

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