Anti-CLDN18.2 Antibody, Drug Conjugate, Method for Producing the Same, and Use

Abstract

The present invention provides anti-CLDN18.2 antibodies and antibody-drug conjugates made with the antibodies that are effective in treating CLDN18.2-positive tumors, including, but not limited to, cancers such as gastric cancer and pancreatic cancer.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the priority of Chinese Patent Application No. 202011385844.4, filed on November 30, 2020, with the invention title "Anti - CLDN18.2 Antibody, Drug Conjugate (also called drug complex), Its Manufacturing Method and Use". The full text of the patent application is incorporated herein by reference and used for all purposes.

[0002] The present invention generally relates to the field of biopharmaceuticals, and in particular, to anti - CLDN18.2 antibodies, related conjugates (also called complexes) (e.g., antibody - drug conjugates), and their use in the treatment or prevention of tumors.

Background Art

[0003] Tight junctions (TJs) are important functional components of the adhesion between normal epithelial cells. They bind cells in a mechanical manner, form an epithelial barrier, prevent macromolecular transport between cells, and maintain the polarity of epithelial cells. The proteins that make up tight junctions are mainly Claudin (also called claudin, and for the sake of avoiding any ambiguity, the English name Claudin or the abbreviation CLDN is used in this specification), Occludin, ZO - 1, ZO - 2, ZO - 3, cingulin, Pals1, MUPP1. Among them, Claudin protein and Occludin protein are the most important proteins.

[0004] Claudin proteins are skeletal proteins that form tight junctions. Their abnormal expression may lead to structural destruction and functional damage of epithelial and endothelial cells, and may play an important role in the development of various diseases. To date, the Claudin gene family has been shown to contain 24 members, and it has been revealed that there is a high degree of evolutionary and functional conservation among the members. The molecular mass of Claudin is 22-27 kD. Each Claudin molecule has the same structure, and Claudin is widely distributed in tumor tissues different from normal tissues, with differences in expression. Abnormal expression of several Claudin proteins has also been observed in precancerous gastric lesions and gastric cancer, and is involved in the prognosis.

[0005] Since the humanized Claudin18 gene has two different first exons, it can produce two splice variants, Claudin18.1 (hereinafter abbreviated as CLDN18.1) and Claudin18.2 (hereinafter abbreviated as CLDN18.2). The lengths of the amino acid sequences of human CLDN18.1 and CLDN18.2 are both 261 amino acid residues, and 21 of the amino acid residues are different among the amino acid residues at positions 0-70. The two subtypes of CLDN18 perform transcriptional amplification in different tissues. Among them, CLDN18.1 is selectively expressed in normal lung cells, while CLDN18.2 is localized in short-lived cells of the differentiated gastric epithelium with expression in normal stomach.

[0006] Scientific research has shown that CLDN18.1 and CLDN18.2 are structurally very similar, but their expression in tumors is very different, for example, in normal tissues, CLDN18.1 is only expressed in the lungs, while CLDN18.2 is limitedly expressed in the stomach, and in tumor tissues, CLDN18.1 does not have obvious high expression in the lungs, but CLDN18.2 is upregulated in cancers such as gastric cancer, esophageal cancer, and pancreatic cancer. For example, when gastric epithelial tissue undergoes malignant transformation, the disturbance of cell polarity leads to the exposure of the CLDN18.2 protein epitope on the cell surface. In addition, the CLDN18.2 gene is also abnormally activated and highly selectively and stably expressed in certain tumor tissues, and is involved in the proliferation, differentiation, and migration of tumor cells, making it an effective molecular target for potential antitumor drugs.

[0007] The annual incidence of gastric cancer worldwide is 13.86 / 100,000, and many patients are already in the advanced stages at the time of diagnosis, with poor recovery after surgery. The incidence rate is high among the elderly, with the average overall survival time being less than one year, and the five-year survival rate being less than 20%. Pancreatic cancer is also currently one of the most aggressive tumors, with a median survival time of less than six months and an overall five-year survival rate of less than 6%.

[0008] Research and development of antitumor drugs targeting CLDN18.2 is actively being carried out worldwide. At present, there are many projects targeting CLDN18.2 (about 28 projects), and the project types include monoclonal antibodies, bispecific antibodies and CAR-T targeting CLDN18.2. Among all the projects, the one with the fastest research and development progress is the monoclonal antibody Claudiximab (named Zolbetuximab after acquisition) from Ganymed (already acquired by Astellas), whose gastric cancer indication test has already reached the third clinical stage. A total of nine projects have entered the clinical stage, most of which are in the preclinical stage, and there is a great deal of uncertainty about the therapeutic effect. Based on the current data, no reports of clinical studies on antibody-drug conjugates (ADCs) targeting CLDN18.2 have been found yet.

[0009] CLDN18.2 is an excellent target for gastrointestinal cancer and pancreatic cancer. However, since there are differences in 7 amino acid residues in the extracellular domain ECD1 of approximately 50 amino acid sequences between CLDN18.1 and CLDN18.2, how to design an antibody that specifically recognizes only CLDN18.2 and does not recognize CLDN18.1 has become a difficult problem in the development of this target monoclonal antibody drug.

[0010] Overall, monoclonal antibody-based therapy has characteristics such as high target specificity and low side effects. However, the therapeutic effect alone is limited. Therefore, most monoclonal antibody drugs are used in combination with chemotherapy drugs. Currently, the main route to enhance the therapeutic effect of monoclonal antibodies is antibody-drug conjugates. Antibody-drug conjugates (also called antibody-drug conjugates) are novel anti-cancer biomissile drugs, mainly composed of three parts: an antibody, a drug molecule, and a linker that connects the two. When a monoclonal antibody and a drug are chemically conjugated, the antibody-drug conjugate utilizes the targeting property of the monoclonal antibody to specifically recognize the receptor targeted by the antibody on the cancer cell surface. After binding to the receptor, it enters the cell and uses intracellular proteases to release the drug, preventing the growth of cancer cells and killing cancer cells. Antibody-drug conjugate technology integrates small molecule drugs and biological proteins, combines the advantages of both, enhances the drug efficacy, reduces toxicity and side effects, and manufactures next-generation therapeutic products.

[0011] By September 2020, the FDA had approved the marketing of 9 types of ADC drugs, such as Adcetris from Seattle Genetics, Kadcyla and Polivy from Genentech, Besponsa and Mylotarg from Wyeth, Lumoxiti and Enhertu from AstraZeneca, and Trodelvy from Immunomedics. No ADC drugs developed in China have been marketed yet.

[0012] Both antibody drugs alone and antibody-drug conjugates are drugs with promising future, and further research and development in the pharmaceutical field are urgently needed.

Summary of the Invention

Means for Solving the Problems

[0013] According to a first aspect, the present invention provides a conjugate comprising the anti-CLDN18.2 antibody or an antigen-binding fragment of the antibody of the present invention conjugated to one or more drug molecules. According to a second aspect, the present invention provides a pharmaceutical composition comprising the conjugate according to the first aspect and a pharmaceutically acceptable carrier. According to a third aspect, the present invention provides the use of the conjugate according to the first aspect or the pharmaceutical composition according to the second aspect in the manufacture of a drug for treating or preventing cancer.

[0014] According to a fourth aspect, the present invention provides a method for treating cancer in an individual, comprising administering to the individual suffering from cancer a therapeutically effective amount of the conjugate according to the first aspect or the pharmaceutical composition according to the second aspect. According to a fifth aspect, the present invention provides a medical product (for example, a kit) comprising the conjugate according to the first aspect or the pharmaceutical composition according to the second aspect. According to a sixth aspect, the present invention provides the use of the conjugate according to the first aspect and an anti-proliferative agent in the manufacture of an anti-tumor drug.

[0015] According to a seventh aspect, the present invention provides a pharmaceutical composition comprising the conjugate according to the first aspect and an anti-proliferative agent. According to an eighth aspect, the present invention provides a method for treating a tumor in an individual, comprising administering to the individual suffering from the tumor a therapeutically effective amount of the conjugate according to the first aspect or the pharmaceutical composition according to the second aspect and an anti-proliferative agent. According to the ninth aspect, the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment of said antibody, a pharmaceutical composition comprising said antibody or antigen-binding fragment, the pharmaceutical use of said antibody or antigen-binding fragment, and a method for treating tumors / cancers using said antibody or antigen-binding fragment.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0017] 〔Definitions〕 Unless otherwise defined, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, experts can refer in particular to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art that refer to one of the 20 common L-amino acids.

[0018] Notwithstanding the numerical ranges and parameter approximations set forth in the broad scope of the present invention, the numerical values set forth in the specific embodiments are described as accurately as possible. However, any numerical value inherently contains a certain error due to the standard deviation existing in each measurement. Also, all ranges disclosed herein should be understood to cover all sub-ranges included within that range. For example, the described range of "1 to 10" includes any and all sub-ranges between the minimum value of 1 and the maximum value of 10 (including the endpoints), i.e., all sub-ranges starting with a minimum value of 1 or more, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. Also, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0019] As used herein, the terms "pharmaceutical composition", "combination medicine", and "pharmaceutical combination" are used interchangeably and refer to a combination of at least one drug combined together to achieve a particular purpose and any carrier or adjuvant substance useful as a medicine. In certain embodiments, the pharmaceutical composition includes combinations that are separated temporally and / or spatially as long as they can act together to achieve the objectives of the present invention. For example, the components included in the pharmaceutical composition (e.g., the antibodies, nucleic acid molecules, combinations of nucleic acid molecules, and / or conjugates described herein) can be administered to a subject either as a whole or separately. When the components included in the pharmaceutical composition are administered to a subject separately, the components may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline solution such as PBS (phosphate buffered saline), glucose, mannitol, dextroglucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, or polyalkylene glycol such as polypropylene glycol, triglyceride, etc. The type of pharmaceutically acceptable carrier used depends, in particular, on whether the composition described herein is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions of the present invention may contain wetting agents, emulsifying agents or buffering substances as additives.

[0020] The pharmaceutical composition, vaccine or pharmaceutical preparation according to the present invention can be administered by any suitable route, such as oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration or intravenous administration. As used herein, "therapeutically effective amount" or "effective amount" means an amount sufficient to demonstrate its benefit to the subject being administered. The actual amount administered, the rate and duration of administration vary depending on the condition and severity of the subject being treated. The prescription for treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of the specialist and other physicians, determined by the specialist and other physicians, and generally the disease being treated, the individual condition of the patient, the site of delivery, the method of administration, and other factors known to the physician are considered.

[0021] As used herein, the terms "subject" or "individual" mean mammals such as humans, but may also be other animals such as wild animals (e.g., egrets, cranes, etc.), domestic animals (e.g., ducks, geese, etc.), or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, squirrels, etc.).

[0022] The term "antibody" generally encompasses intact antibodies and any antigen-binding fragments ("antigen-binding portions") or single-chain forms thereof. "Full-length / intact antibody" refers to a protein comprising at least two heavy chains (H) and two light chains (L) interconnected via disulfide bonds. Each heavy chain comprises a heavy-chain variable region (abbreviated as VH) and a heavy-chain constant region, and the heavy-chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light-chain variable region (abbreviated as VL) and a light-chain constant region containing the CL domain. The VH and VL regions may further be subdivided into a plurality of hypervariable regions called complementarity-determining regions (CDRs) interspersed with a plurality of more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. These variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (Clq). Chimeric antibodies or humanized antibodies are also included in the antibodies according to the present invention. The full-length / intact antibody may be any type of antibody such as IgD, IgE, IgG, IgA, or IgM (or the above subclasses), but the antibody does not have to belong to any particular class. Immunoglobulins can be designated into different classes based on the amino acid sequence of the constant region of the heavy chain. Usually, immunoglobulins have five major classes, IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are known.

[0023] Complementary determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions within the variable regions that have the greatest influence on the affinity and specificity of the antibody for its target. There are many common definition methods for CDR sequences in VH or VL, such as IMGT, Chothia definition, Kabat definition, etc. For a given antibody variable region sequence, the CDR sequences in the VH and VL sequences can be determined according to the IMGT, Chothia definition, or Kabat definition.

[0024] The term "humanized antibody" can include CDR regions derived from human-derived antibodies, and means an antibody in which other parts of the antibody molecule are derived from one (or more) human antibodies. Furthermore, in order to retain the binding affinity, some residues in the framework (referred to as FR) section can be modified, and the humanized antibody or its fragment according to the present invention can be produced by techniques well known to those skilled in the art. The term "semi-humanized antibody" means, with respect to a humanized antibody or a fully humanized antibody, an antibody in which one antibody chain contains a variable region derived from a mouse (like a chimeric antibody) and the other antibody chain contains a humanized variable region.

[0025] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. The chimeric antibody or its fragment according to the present invention can be produced using recombinant DNA technology. For example, the chimeric antibody according to the present invention can be produced by cloning recombinant DNA containing a promoter, a sequence encoding the variable region of a non-human, particularly a mouse monoclonal antibody according to the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by such a recombinant gene is, for example, a mouse-human chimera, and the specificity of this antibody is determined by the variable region derived from mouse DNA, and its isotype is determined by the constant region derived from human DNA. For methods of producing chimeric antibodies, reference can be made to, for example, the literature Verhoeyn et al. (BioEssays, 8:74, 1988).

[0026] The term "monoclonal antibody" refers to a product of antibody molecules having a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope. The term "bispecific antibody" means an antibody having the ability to bind simultaneously to two antigen epitopes. The two types of antigen epitopes may be on different antigens or on the same antigen. Bispecific antibodies can have multiple structural arrangements. For example, a bispecific antibody may be composed of two Fc fragments and two binding portions (similar to a natural antibody except that the two arms bind to different antigen targets or epitopes) that are separately fused thereto, and the antigen-binding portion may be a single-chain antibody (scfv) or a Fab fragment.

[0027] As used herein, the term "antigen-binding fragment" particularly refers to Fv, scFv (sc means single chain), Fab, F(ab’)2, Fab’, scFv-Fc fragment, or a bispecific antibody (diabody), or any fragment that can extend the half-life by chemical modification or by incorporation into liposomes, such as obtained by adding a poly(alkylene) glycol such as polyethylene glycol ("PEGylation") (Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)2-PEG or Fab’-PEG, which are called polyethylene glycolylated fragments of the antibody) (where "PEG" is polyethylene glycol). The antigen-binding fragment of the anti-CLDN18.2 antibody of the present invention has CLDN18.2 binding activity. For example, the antigen-binding fragment is composed of or contains a partial sequence of the heavy chain or light chain variable chain of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, and such an antigen-binding fragment contains at least 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived.

[0028] Examples of antigen-binding fragments include: (1) a Fab fragment which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) an F(ab')2 fragment which may be a divalent fragment having two Fab' fragments linked by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv) which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) a (scFv)2 which can include two VH domains linked via a peptide linker and two VL domains combined with these two VH domains via disulfide bridges, but are not limited thereto.

[0029] The terms "Fc fragment", "Fc domain", "Fc portion" or similar terms mean a part of the constant region of an antibody heavy chain including the hinge region, the CH2 fragment and the CH3 fragment of the constant region.

[0030] Generally, for producing a monoclonal antibody or its antigen-binding fragment, particularly a monoclonal antibody or its antigen-binding fragment derived from a mouse, reference can be made to the techniques described in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988), or the techniques for producing from hybridoma cells described by Kohler and Milstein (Nature, 256: 495-497, 1975).

[0031] Based on the structural information of the anti-CLDN18.2 monoclonal antibody described in the present specification, it can be prepared in cells of CHO-K1 (ATCC Number: CCL-61, Lot No.: 59965043) using methods known in the art. The term "homology / identity / identity" of an amino acid or nucleic acid sequence is the proportion of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and gap introduction, and is defined as reaching the maximum proportion of homology as necessary. Alignment methods and computer programs are known in the art.

[0032] The term "specific binding" means a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0033] The inventors of the present application first obtained an anti-CLDN18.2 monoclonal antibody that can bind to CLDN18.2 on CLDN18.2-positive cells and can be internalized with high efficiency. This antibody is very suitable for the development of ADCs. In the subsequent research and development of ADCs, when the inventors of the present application used NH2-PEG3-Val-Cit as a linker, good stability of the binding between the linker and the drug molecule was achieved. Thereby, a humanized antibody and a small molecule drug (for example, MMAE) were bound via the linker, and the obtained ADC drug has extremely strong killing effects on CLDN18.2-highly expressing cancer cells, particularly pancreatic cancer, gastric cancer, and lung cancer cells, and has good stability. Specifically, by in vivo experiments, when an antibody-drug conjugate was intravenously administered to nude mice having CLDN18.2-positive gastric or pancreatic xenograft tumors, the growth of the tumors was inhibited in a dose-dependent manner, and a significant therapeutic effect was observed with a single-dose intravenous administration of about 1 to 8 mg / kg. The optimal therapeutic effect was obtained at 8 mg / kg, and the individuals showed good tolerance. Its overall therapeutic effect was remarkable, and moreover, the ADC drug obtained in the present invention can produce a bystander effect and further enhanced the therapeutic effect.

[0034] According to one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CLDN18.2. Specifically, the antibody includes a heavy chain and a light chain, wherein (i) the heavy chain includes three CDR regions, and at least one amino acid sequence of the CDR regions has an amino acid sequence shown in SEQ ID NO: 1, 2, or 3, or a sequence having at least 80% (preferably 85%, 90%, 95%, 98%, or 99%) sequence identity therewith, and / or (ii) the light chain includes three CDR regions, and at least one amino acid sequence of the CDR regions has an amino acid sequence shown in SEQ ID NO: 4, 5, or 6, or a sequence having at least 80% (preferably 85%, 90%, 95%, 98%, or 99%) sequence identity therewith.

[0035] In some specific embodiments, the antibody includes a heavy chain and a light chain, wherein (i) the heavy chain includes three CDR regions, and the CDR regions respectively have amino acid sequences shown in SEQ ID NO: 1, 2, and 3, and / or (ii) the light chain includes three CDR regions, and the CDR regions respectively have amino acid sequences shown in SEQ ID NO: 4, 5, and 6. In some specific embodiments, the antibody or the antigen-binding fragment thereof according to the present invention is isolated.

[0036] In some specific embodiments, the heavy chain includes a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain includes a light chain variable region of the amino acid sequence shown in SEQ ID NO: 8. In some specific embodiments, the heavy chain includes a heavy chain of the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain includes a light chain of the amino acid sequence shown in SEQ ID NO: 10. In some specific embodiments, the antibody according to the present invention is a monoclonal antibody.

[0037] In some specific embodiments, the antibody according to the present invention is a bispecific antibody. For example, one arm of the bispecific antibody may be an antigen-binding fragment (e.g., Fab or scfv) of the anti-CLDN18.2 antibody according to the present invention, and the other arm may be an antigen-binding fragment (e.g., Fab or scfv) targeting another antigen (e.g., another antigen target that can be used for ADC construction) or another CLDN18.2 antigen epitope (different from the CLDN18.2 binding epitope of the anti-CLDN18.2 antibody according to the present invention).

[0038] In some specific embodiments, the antibody according to the present invention is a humanized antibody including a semi-humanized antibody and a fully humanized antibody. In some specific embodiments, the antibody or its antigen-binding fragment according to the present invention has ADCC activity. In some specific embodiments, the antibody or its antigen-binding fragment according to the present invention has CDC activity.

[0039] In some specific embodiments, the antibody or its antigen-binding fragment according to the present invention specifically binds to CLDN18.2 and does not substantially bind to CLDN18.1. In a specific embodiment, the antibody includes a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype, or IgG4 subtype. In some specific embodiments, the heavy chain constant region of the antibody may be a human IgG1 subtype, human IgG2 subtype, human IgG4 subtype, mouse IgG1 subtype, or mouse IgG2a subtype.

[0040] In some specific embodiments, the heavy chain constant region is the IgG1 subtype, that is, the antibody is an IgG1-type antibody. In some specific embodiments, the antibody includes a light chain constant region selected from the κ subtype or the λ subtype. In some specific embodiments, the light chain constant region of the antibody may be a human κ subtype, a human λ subtype, a mouse κ subtype, or a mouse λ subtype.

[0041] In some specific embodiments, the antibody according to the present invention is an IgG1κ antibody. In some specific embodiments, the antibody according to the present invention or its antigen-binding fragment is for use in the treatment or prevention of cancers overexpressing CLDN18.2. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the native epitope of CLDN18.2 present on the surface of living cells. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the extracellular domain of CLDN18.2. In one embodiment, an antibody having the ability to bind to CLDN18.2 binds to the first extracellular region of CLDN18.2.

[0042] According to another aspect, the present invention provides an isolated polynucleotide encoding the antibody according to the present invention. According to yet another aspect, the present invention provides a combination of isolated polynucleotides comprising a polynucleotide encoding the light chain of the antibody according to the present invention or its antigen-binding fragment and a polynucleotide encoding the heavy chain of the antibody according to the present invention or its antigen-binding fragment. According to another aspect, the present invention provides an expression vector comprising the polynucleotide according to the present invention or a combination of the polynucleotides according to the present invention, wherein the polynucleotide is effectively linked to regulatory sequences for expressing the polypeptide encoded thereby in a host cell or a cell-free expression system.

[0043] In some embodiments of the present invention, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a phage. The prokaryotic host cell may be Escherichia coli, Bacillus subtilis, Streptomyces, Proteus mirabilis, etc. The eukaryotic host cell may be a fungus (e.g., Pichia pastoris, Saccharomyces cerevisiae, fission yeast, Trichoderma, etc.), an insect cell (e.g., Spodoptera frugiperda, etc.), a plant cell (e.g., tobacco, etc.), a mammalian cell (e.g., BHK cell, CHO cell, COS cell, myeloma cell, etc.), etc. In some embodiments, the host cell according to the present invention is preferably a mammalian cell, more preferably a BHK cell, a CHO cell, an NSO cell or a COS cell.

[0044] According to another aspect, the present invention provides an antibody-drug conjugate comprising the anti-CLDN18.2 antibody of the present invention or an antigen-binding fragment thereof conjugated to one or more drug molecules. For the embodiments and technical features of the anti-CLDN18.2 antibody of the present invention or an antigen-binding fragment thereof, refer to the above. Since CLDN18.2 is mainly a molecular target of cancer / tumor cells, in some embodiments, the drug molecule is an anti-cancer drug. However, those skilled in the art will understand that when CLDN18.2 becomes a target for other diseases in non-cancer cells / non-tumor cells, the drug molecule can be selected according to the target disease. The types of anti-cancer drugs include, but are not limited to, cytotoxic drugs, immunopotentiators or radioisotopes.

[0045] In some embodiments, the types of cytotoxic drugs include tubulin inhibitors (e.g., alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites, or antitumor antibiotics. In some embodiments, the tubulin inhibitor includes, but is not limited to, auristatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, Ansamitocin, Mertansine or dolastatin, and their derivatives).

[0046] In some embodiments, the DNA topoisomerase inhibitor includes camptothecin analogs or DNA topoisomerase I inhibitors and their derivatives, such as DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyacuminatin, topotecan, larototecan, velotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0047] In some embodiments, the DNA damaging agent includes, but is not limited to, calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine). In some embodiments, the immunopotentiator includes, but is not limited to, levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic-polycytidylic acid or polyinosinic-polyuridylic acid. In some embodiments, the antimetabolite includes, but is not limited to, methotrexate, 6-mercaptopurine or 5-fluorouracil.

[0048] In some embodiments, the antitumor antibiotic includes, but is not limited to, polypeptide antibiotics (such as actinomycin D or bleomycin) or anthraquinone drugs (such as doxorubicin or mitoxantrone hydrochloride). In some embodiments, the radioisotope is 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 Co or 177 Lu, and is not limited thereto.

[0049] In some embodiments, an antibody having the ability to bind to CLDN18.2 is covalently attached to a drug moiety via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker can be cleaved under intracellular conditions. In one embodiment, the linker is hydrolyzable at a pH less than 5.5. In some embodiments, the linker may be cleaved by an intracellular protease. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the linker contains a dipeptide. In some embodiments, the dipeptide is valine (Val)-citrulline (Cit). In some embodiments, the antibody is linked to the linker via a cysteine mercapto of the antibody. In one embodiment, the antibody is linked to the linker via an amino (particularly the amino of a glutamine residue) of the antibody. Non-limiting examples of linkers include mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, NH2-(PEG) m -Val-Cit, NH2-(PEG) m -Val-Cit-pAB, where m is an integer from 1 to 8.

[0050] In some specific embodiments, the antibody-drug conjugate according to the present invention has the following general formula Ab-(L-U)n, where Ab represents the antibody according to the present invention that targets CLDN18.2, L is a linker (for example, NH2-(PEG)m-Val-Cit, NH2-(PEG)m-Val-Cit-pAB, NH2-(PEG)m-Val-Cit-pABC, mc-Val-Cit-pAB or Val-Cit, where m represents the number of PEGs and may be an integer from 1 to 8), U is a drug (for example, DM1, DM4, MMAE, MMAF, DXD and SN38), n represents the drug antibody ratio (DAR), the DAR value may be an average value, or may be any numerical value from 1 to 8 (not limited to integers, may be decimals), preferably an integer from 1 to 8, more preferably 2, 4, 6, 8, and even more preferably 2.

[0051] According to another aspect, the present invention provides a pharmaceutical preparation (for example, a pharmaceutical composition) comprising the antibody-drug conjugate according to the present invention and a pharmaceutically useful diluent, carrier or excipient. According to another aspect, the present invention provides a medical preparation comprising the antibody-drug conjugate according to the present invention. In some embodiments, the medical preparation exists in the form of a kit comprising a container containing the antibody-drug conjugate according to the present invention. In one embodiment, the medical preparation further comprises printed instructions for using the product in a method for treating or preventing cancer (especially cancer expressing CLDN18.2).

[0052] The antibody-drug conjugate according to the present invention is effective for the treatment and / or prevention of cancers associated with the expression of CLDN18.2 (CLDN18.2 positive) cells. As non-limiting examples, the cancers may be gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer, and metastases of these cancers, particularly gastric metastasis, peritoneal metastasis and lymph node metastasis. The cancers suitable for treatment with the antibody-drug conjugate according to the present invention are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung and ovary, and the antibody-drug conjugate according to the present invention is particularly suitable for the treatment of gastric cancer and pancreatic cancer.

[0053] Accordingly, the present invention also provides a plurality of inventions related to the above-mentioned therapeutic uses. According to one aspect, the present invention provides the use of the above-mentioned antibody conjugate in the manufacture of a drug for treating or preventing cancer. According to another aspect, the present invention provides a method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the above-mentioned antibody-conjugate or a pharmaceutical preparation or pharmaceutical composition comprising said conjugate.

[0054] According to another aspect, the present invention provides the use of the above-mentioned antibody-conjugate and an anti-proliferative agent in the manufacture of a drug for treating a tumor (e.g., the above-mentioned cancer). According to another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody conjugate and an anti-proliferative agent. According to another aspect, the present invention provides a method for treating a tumor in an individual, comprising administering to the individual a therapeutically effective amount of the above-mentioned antibody conjugate or a pharmaceutical preparation or pharmaceutical composition comprising said antibody conjugate together with an anti-proliferative agent.

[0055] In certain embodiments, the anti-proliferative agent includes, but is not limited to, paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, and iproplatin. In certain embodiments, the anti-proliferative agent may be other antibodies, antibody-drug conjugates, or fusion proteins.

Examples

[0056] The following examples are used for illustrative purposes only and are not intended to limit the scope of the present invention.

[0057] Experimental equipment and materials

Table 1A

[0058] Cell lines used in the experiments

Table 1B

[0059] Example 1: Production of a fully humanized anti-CLDN18.2 monoclonal antibody The anti-CLDN18.2 antibody used in the present invention is produced by immunizing human Ig transgenic mice. The transgenic mice were immunized with CHO cells or 3T3 cells transfected with human CLDN18.2. The immunogen was injected intraperitoneally (IP), subcutaneously (SC), into the footpad (fp), or into the tail of the mouse. The immune response was tested by periodically measuring the titer of mouse plasma anti-CLDN18.2. Mice with plasma having a sufficient titer against CLDN18.2 were used for hybridoma fusion. Finally, immune enhancement was also performed by injecting the immunogen into the peritoneal cavity, footpad, or tail vein of the mouse before removing the spleen and lymph nodes of the mouse.

[0060] The sera of immunized mice were screened by fluorescence-activated cell sorting (FACS), and mice producing antibodies conjugated to CLDN18.2 were selected. Cell lines expressing CLDN18.2 (CHO or 3T3) were incubated with serially diluted immunized mouse sera, and then specific antibody binding was detected using a PE-fluorescently labeled anti-mouse IgG Ab and detected with a fluorescence-activated cell sorter (iQue plus, Sartorius). Mouse sera were also confirmed by imaging tests. CHO (or 3T3 cells) expressing CLDN18.2 were diluted with the sera of immunized mice and incubated, the cells were washed, fixed with formaldehyde, washed again, and then specific antibody binding was detected using an Alexa488-fluorescently labeled goat anti-mouse antibody and scanned and analyzed with a cell imager (Cytation 5, Biotek). After confirming the mice producing antibodies conjugated to CLDN18.2, spleens and lymph nodes were collected from the immunized mice, lymphocytes were isolated, and fused with mouse myeloma cells Sp2 / 0 (ATCC, CRL1581) by electrofusion, and the resulting hybridomas were screened as anti-CLDN18.2 specific antibodies. The cells were seeded in flat-bottom 96-well tissue culture plates and then incubated in selective medium (HAT medium) for 2 weeks and then switched to hybridoma culture medium. Approximately 10 - 14 days after seeding the cells on the plates, screening for anti-CLDN18.2 specific binding imaging of the hybridoma supernatants from single wells was performed by the above cell imaging method. The relevant hybridomas were screened from the hybridomas produced from the immunized groups of 3 mice. The hybridoma cells produced by immunizing mice and electrofusion were seeded in flat-bottom 96-well tissue culture plates such that each well contained one or more hybridoma cells. The hybridoma supernatants from single wells were detected by the cell imaging method, and the cloning supernatants secreting positive antibodies specifically bound to CHO cells transfected with CLDN18.2 but did not bind to CHO cells transfected with CLDN18.1 and CHO cells.Hybridomas secreting positive antibodies were transferred to 24-well plates and confirmed by rescreening. The confirmed positive antibody hybridomas were sorted into single clones using a single cell sorter. For each positive hybridoma, 96 subclones were sorted and confirmed by rescreening. Positive subclones producing preliminary candidate molecules were subjected to in vitro amplification culture and used for sequencing, and a small amount of antibody from the positive subclones was used for purification and antibody characterization and verification. The related sequences of the obtained antibody SYJS001 are as follows.

[0061]

Table 1

[0062] SEQ ID NO:7 (Heavy chain variable region) EVQLSESGGALVQPGESLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSSLSGSGRSTYYAASIKGRFTISRDNSKNTLYLQMSSLRAEDTAIYYCAKSLSYYHYYFDYWGQGTLVTVSS SEQ ID NO:8 (Light chain variable region) DIQLTQSPSFLSASVGDRVPITCRASQDISNYLAWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFASYHCQQVKTYPLTFGGGTKVEIK SEQ ID NO:9 (Heavy chain) EVQLSESGGALVQPGESLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSSLSGSGRSTYYAASIKGRFTISRDNSKNTLYLQMSSLRAEDTAIYYCAKSLSYYHYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:10 (Light chain) DIQLTQSPSFLSASVGDRVPITCRASQDISNYLAWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFASYHCQQVKTYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0063] Example 2: Vector construction and antibody expression 2.1 Vector design The plasmid vector pGenHT1.0-DGV used for the expression of SYJS001 monoclonal antibody was provided by Nanjing Genscript Biotech Co., Ltd. (abbreviated as "Genscript"). The vector map is referred to Figure 3, and the information of each element is shown in Table 2.

[0064]

Table 2

[0065] In this study, the heavy chain, light chain DNA sequences of SYJS001 and the expression vector pGenHT1.0-DGV were all artificially synthesized by GenScript.

[0066] Design and synthesis of the heavy chain The artificially synthesized heavy chain was named SYJS001-HC. An NruI endonuclease site was introduced at the 5'-end, a PmLI endonuclease site was introduced at the 3'-end, and at the same time, a Kozak sequence and a signal peptide sequence (19 amino acids): MGWSCIILFLVATATGVHS (SEQ ID NO:19) were introduced after the 5'-end NruI endonuclease site. The expression frame of the heavy chain was designed as follows. NruI-Kozak sequence-signal peptide-SYJS001-HC-stop codon-PmLI

[0067] Design and synthesis of the light chain The artificially synthesized light chain was named SYJS001-LC. During the synthesis process, an AscI endonuclease site was introduced at the 5'-end of the light chain, an FseI endonuclease site was introduced at the 3'-end, and at the same time, a Kozak sequence and a signal peptide sequence (19 amino acids): MGWSCIILFLVATATGVHS (SEQ ID NO:19) were introduced after the 5'-end AscI endonuclease site. The expression frame of the light chain was designed as follows. ASCI-Kozak sequence-signal peptide-SYJS001-LC-stop codon-FseI

[0068] 2.2 Construction of recombinant vectors The insertion site of SYJS001-HC is the polyclonal NruI / PmLI site downstream of the pGenHT1.0-DGV vector, and the insertion site of SYJS001-LC is the upstream polyclonal AscI / FseI site. The promoters of the two polyclonal sites are all CMV, and the heavy chain and light chain are constructed on the same empty vector. Double enzyme digestion with NruI / PmLI and ligation transformation were performed on the PCR amplification product SYJS001-HC and the plasmid vector pGenHT1.0-DGV, and positive clones were screened by the Kan+ resistance label to obtain a correctly constructed recombinant heavy chain expression vector, named SYJS001-HC in pGenHT1.0-DGV. Next, double enzyme digestion, ligation transformation and clone screening were performed on the light chain SYJS001-LC and SYJS001-HC in pGenHT1.0-DGV using AscI / FseI. Double enzyme digestion identification with AscI / PmLI (see Figure 4) and sequencing were performed on the positive clones to obtain a correctly constructed recombinant heavy chain and light chain expression vector, named SYJS001 in pGenHT1.0-DGV, and its structural schematic diagram is shown in Figure 5. As a result of the double enzyme digestion identification of SYJS001 in pGenHT1.0-DGV, the size of the target region sequence was 4527bp. The obtained recombinant plasmid was electroporated into the host cell CHOK1 to obtain a stable cell line highly expressing the SYJS001 antibody protein.

[0069] 2.3 Antibody expression and purification The stable cell line highly expressing the SYJS001 antibody was placed in serum-free CD FortICHO and cultured in a shaking flask. After a certain period of time, the culture supernatant was collected. The HiTrap MabSelect SuRe 1 mL column (product of GE Healthcare Life Sciences, part number: 11-0034-93) was equilibrated with PBS solution at pH = 7.4 for 10 column volumes, and the flow rate was set at 0.5 mL / min. The culture supernatant was injected through a 0.45 μm filtration membrane, and the flow rate was set at 0.5 mL / min. It was then rewashed with PBS solution at pH 7.4 for 5 - 10 column volumes, and the flow rate was set at 0.5 mL / min. Elution was carried out at a flow rate of 0.5 mL / min using 100 mM citrate buffer (pH 3.6), and the elution peak was collected to obtain the SYJS001 antibody with a purity higher than 95%.

[0070] Example 3: Production of SYJS001 ADC Appropriate volumes of L&D (whose structure is shown in Figure 2), reaction buffer, SYJS001 antibody, mTGase (transglutaminase), and H2O were transferred into a disposable reaction bag made of elastic ethylene-vinyl acetate using a peristaltic pump in an appropriate order. The reaction bag was sealed, uniformly mixed, and then placed at 30°C, with the reaction time set at 24 - 144 h. Sampling was carried out every 24 h during the reaction, and the coupling rate was measured by C4-HPLC analysis. When the coupling rate was 95% or higher, the reaction was terminated and purified immediately.

[0071] The amino acid sequence of the transglutaminase actually used is as follows. DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP(SEQ ID NO:20)

[0072] Example 4: Analysis and identification of the physical and chemical properties of SYJS001 ADC 1. Identification of the modification rate of SYJS001 ADC Experimental steps 1) Injection: 10 μL of the supernatant of the ADC reaction solution sample after reduction was injected into a chromatography column (Waters XBridge C4, 3.5 μm, 4.6 mm × 250 mm). 2) Elution: Mobile phase A was 0.1% aqueous TFA solution, and mobile phase B was 0.1% acetonitrile solution. The ratio of mobile phase A:B was adjusted to 9:1, 7:3, 6.5:3.5, 6:4, 5.5:4.5, 5:5, 1:9, 9:1 at 0, 5, 8, 15, 20, 22, 25, 30 min respectively for elution. The flow rate was controlled at 0.8 mL / min, and the detection wavelength was 280 nm. From the experimental results shown in Figure 6, the modification rate of SYJS001 ADC was 95.11%.

[0073] 2. Detection of the DAR value of SYJS001 ADC Experimental steps 1. Injection: 10 μg of the SYJS001 ADC sample was injected into a chromatography column (Agilient PLRP-S, 5 μm, 2.1 mm * 50 mm). 3. Dissolution: Mobile phase A was 0.1% aqueous TFA solution, and mobile phase B was 0.1% acetonitrile solution. The ratio of mobile phase A to B was adjusted to 7.3:2.1, 6.5:3.5, 5.7:4.3, 0.5:9.5, and 7.3:2.7 at 0, 8, 25, 26, and 31.5 min, respectively, for dissolution. The flow rate was controlled at 0.25 mL / min, and the detection wavelength was set at 280 nm. From the results shown in Figure 7, the average DAR was 2.

[0074] Example 5: Flow assay of the binding ability of SYJS001 ADC to CLDN18.2 protein In this experiment, flow cytometry technology was used to detect the binding ability of SYJS001 ADC to CLDN18.2 proteins of different species.

[0075] The overexpressing cell lines (HEK293-human CLDN18.2, HEK293 mouse CLDN18.2, cynomolgus monkey (crab-eating macaque) GLDN18.2, CHO-K1) were incubated with SYJS001 ADC samples at each concentration, and then incubated with a secondary antibody that binds to IgG (goat anti-human IgG (H+L) cross-adsorbed secondary antibody). The fluorescence signal values at each concentration were detected by flow cytometry, and the binding ability of the samples to CLDN18.2 of different species was analyzed.

[0076] Specific experimental steps The starting concentration of the protein sample SYJS001 ADC was set at 45 μg / mL and diluted by a 3-fold gradient, for a total of 11 gradients. 100 μL of each concentration of the antibody was taken, and cells expressing human, mouse, and cynomolgus monkey CLDN18.2 (1×10 6 cells / mL, 100 μL / well) were incubated at 4°C for 1.5 h. The cells were washed to remove unbound samples, and goat anti-human IgG (H+L) cross-adsorbed secondary antibody (1:1000 dilution) was added and incubated at 4°C for 1 h. After washing, the fluorescence signal values of the corresponding wells were detected using a flow cytometer. The data were analyzed using GraphPad Prism 5 software. A regression model of a four-parameter equation was selected to create an "S" curve, and the half-maximal effective dose ED 50(C value) was generated. From the experimental results shown in Fig. 8 and Table 3, SYJS001 ADC showed good affinity for all of human, mouse, and cynomolgus monkey CLDN18.2.

[0077]

Table 3

[0078] Example 6: Specific binding of SYJS001 ADC and CLDN18.2 6.1. Specificity and cross-reactivity between SYJS001 monoclonal antibody and CLDN18 family members CHOK1-CLDN18.2 and HEK293-CLDN18.1 cells were cultured in the corresponding complete medium and passaged for 2 - 3 days. When the cell confluence reached 90%, the cell density was adjusted to 2 - 3×10 6 cells / mL using the complete medium, and the cell suspension was added to a flow cytometry 96-well assay plate at 100 μL per well using a multi-channel pipette. After centrifugation at 2500 rpm for 5 min, the supernatant was discarded. The cells were washed twice with 2% FBS / PBS. The SYJS001 monoclonal antibody was diluted with 2% FBS / PBS buffer, with the starting concentration of the naked antibody set at 6 μg / mL and diluted in a 3-fold gradient to a total of 8 gradients. Two duplicate wells were set up for each drug concentration, and corresponding blank controls were set up. It was set at 100 μL per well and incubated at 4°C for 2 h. After washing three times with 2% FBS / PBS, 488-labeled sheep anti-human IgG (diluted 1:5000) was added and incubated at 4°C for 1 h. After washing three times with 2% FBS / PBS, the corresponding fluorescence channel was selected for reading.

[0079] 6.2 Specific binding between SYJS001 ADC and CLDN18.2 HEK293-CLDN18.2 cells were cultured in the corresponding complete medium and passaged for 2 - 3 days. When the cell confluence reached 90%, the cell density was adjusted to 2 - 3×10 6Adjusted to cells / mL, and the cell suspension was added to a flow cytometry 96-well assay plate at 100 μL per well using a multi-channel pipette. Centrifuged at 2500 rpm for 5 min, and the supernatant was discarded. Washed twice with 2% FBS / PBS. The SYJS001 monoclonal antibody and SYJS001 ADC were each diluted with 2% FBS / PBS buffer, with the starting concentration of ADC set at 15 μg / mL, diluted in a 3-fold gradient, for a total of 11 gradients. Two duplicate wells were set up for each drug concentration, and corresponding blank controls were set up. At 100 μL per well, incubated at 4 °C for 2 h. Washed three times with 2% FBS / PBS, added 488-labeled sheep anti-human IgG (1:1000 dilution), and incubated at 4 °C for 1 h. After washing three times with 2% FBS / PBS, the corresponding fluorescence channel was selected for reading.

[0080] According to the results (see Figures 9 and 10), the SYJS001 monoclonal antibody obtained in the present invention can specifically bind to CLDN18.2, and it was shown that there is no obvious cross-reaction with CLDN18.1. Compared with the naked antibody, SYJS001 ADC has the same specific binding as CLDN18.2, and the binding of the toxin did not obviously affect the affinity.

[0081] Example 7: Verification experiment of the endocytosis of SYJS001 ADC Experimental steps HEK293-CLDN18.2 cells were collected and the cells were resuspended using DMEM complete medium. The resuspended target cells were gently pipetted several times to a single cell suspension, the cell viability was identified using the trypan blue staining method, and the cells were counted. The cell density was adjusted to 1×10 5 cells / mL. Inoculated into a confocal 96-well plate cell culture dish at 100 μL per well, with the number of cells inoculated per well set at 1×10 4 , and SYJS001 labeled with Zenon TM pHrodo TM iFL was added to the 96-well plate, with its final concentration set at 2 μg / mL, and continuously cultured for 24 h under the conditions of a 37 °C, 5% CO2 incubator. All images were observed with a 20X objective lens of a laser confocal microscope and the images were taken.

[0082] From the experimental results shown in Figure 11, SYJS001 ADC causes endocytosis in HEK293-CLDN18.2 cells and localizes in lysosomes (Invitrogen's Zenon TM pHrdo TM iFL IgG Labeling Reagents (Z25611) shows fluorescence only under the acidic environment of lysosomes. For example, the three parts surrounded by squares in the rightmost HEK293-CLDN18.2 figure in Figure 11 show green fluorescent dots under the microscope), while in the HEK293 and HEK293-CLDN18.1 cell figures, endocytosis did not occur and no green fluorescent dots were observed.

[0083] Example 8: Inhibitory effects of SYJS001 naked antibody and ADC on the growth of different cells Experimental steps Target cells were collected and resuspended in a single-cell suspension. The cell viability was identified using the trypan blue staining method, and the cells were counted. The cell density was adjusted to 1×10 5 cells / mL. 100 μL per well was added to a 96-well black flat-bottom cell culture plate. Diluted test articles were added 20 μL each to the 96-well black flat-bottom cell culture plate inoculated with cells. The cells were placed in a cell incubator (37 °C, 5% CO2) and incubated for 66 ± 3 h. Resazurin sodium solution (w / v 0.03%) was added to make it 20 μL per well. It acted at 37 °C for 3 - 4 h, and the fluorescence value was read at 550 nm / 610 nm using a microplate reader. Figures were created using Magellan6 or similar figure-making software, and the half-inhibitory concentration IC 50 was fitted. The output parameter C was IC 50 (unit ng / mL).

[0084] From the experimental results shown in FIGS. 12 to 17 and Table 4, SYJS001 ADC was shown to be able to inhibit the growth of NCI-N87-CLDN18.2 (gastric cancer cell line), KATOIII (gastric cancer cell line), NCI-H460-CLDN18.2 (lung cancer cell line), NUGC4-CLDN18.2 (gastric cancer cell line), PATU8988S (pancreatic cancer cell line), and BxPC-3-CLDN18.2 (pancreatic cancer cell line) cancer cells in vitro.

[0085]

Table 4

[0086] From this, SYJS001 ADC has a significant inhibitory effect on gastric cancer cells, lung cancer cells, and human pancreatic cancer cells that overexpress CLDN18.2, and the inhibitory effect on human gastric cancer cells KATOIII and human pancreatic cancer cells PATU8988S that do not express or have low expression of CLDN18.2 was relatively weak.

[0087] Example 9: In vivo efficacy evaluation of SYJS001 ADC 9.1 Comparative data on drug efficacy with gemcitabine and cisplatin as controls 1) In this experiment, a human pancreatic cancer Bxpc3-18.2 nude mouse xenograft tumor model was used. When the tumor volume reached about 100 mm 3 (on the 39th day after inoculation), 48 animals with good tumor growth were selected. According to the tumor volume, the animals were divided into 6 groups of 8 each (day 0), and 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), 2, 4, and 8 mg / kg of SYJS001-ADC (single administration), 8 mg / kg of SYJS001-mAb (SYJS001 naked antibody) (single administration), and gemcitabine (Gemcitabine, GEM) 50 mg / kg (biw×4, twice a week for a total of 4 weeks) were intravenously administered. The tumor diameter was measured twice a week, the body weight of the mice was weighed, the data were recorded, and the tumor growth was dynamically observed by measuring the tumor diameter at different times after administration. The experiment was terminated on the 28th day. After the mice were asphyxiated with carbon dioxide, the tumors were removed and the tumor weights were measured.

[0088] Results: In this experiment, in the 2, 4, and 8 mg / kg (single-dose) groups of SYJS001-ADC, the 8 mg / kg (single-dose) group of SYJS001-mAb, and the 50 mg / kg (biw×4) group of Gemcitabine, the tumor weight inhibition rates were 56.6%, 94.8%, 97.8%, -36.2%, and 51.0% respectively. Compared with the solvent control group (0.9% sodium chloride injection), the 2 mg / kg, 4 mg / kg, and 8 mg / kg (single-dose) groups of SYJS001-ADC and the 50 mg / kg (biw×4: administered twice a week for a total of 4 doses) group of GEM could all significantly inhibit tumor growth (P<0.01). Compared with the 50 mg / kg (biw×4) group of the positive control GEM, the 4 mg / kg and 8 mg / kg (single-dose) groups of SYJS001-ADC had a significant tumor inhibitory effect, especially in the 8 mg / kg group, which had an inhibitory effect nearly twice as high (p<0.001) (see Figure 18).

[0089] 2) In this experiment, a nude mouse xenograft tumor model was constructed using human gastric cancer NUGC-4-18.2. When the tumor volume reached approximately 120 mm 3 (on the 6th day after inoculation), 64 animals with good tumor growth were selected. According to the tumor volume, the animals were divided into 8 groups of 8 each (on the 0th day), and 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), 1 mg / kg, 2 mg / kg, 4 mg / kg (qw×3), 4 mg / kg, 8 mg / kg (single-dose) of SYJS001-ADC, 4 mg / kg (qw×3) of SYJS001-mAb, and cisplatin 6 mg / kg (qw×3, once a week for a total of 3 weeks) were administered intravenously respectively. The tumor diameter was measured twice a week, the body weight of the mice was weighed, the data was recorded, and the growth changes of the tumor were dynamically observed by measuring the tumor diameter at different times after administration. The experiment was terminated on the 20th day. After the animals were asphyxiated with carbon dioxide, the tumors were removed and the tumor weights were measured.

[0090] Results: In this experiment, the tumor weight inhibition rates of SYJS001-ADC at 1 mg / kg, 2 mg / kg, 4 mg / kg (qw×3), 4 mg / kg, 8 mg / kg (single administration), SYJS001-mAb at 4 mg / kg (qw×3), and cisplatin at 6 mg / kg (qw×3) were 98.0%, 100%, 100%, 100%, 100%, -1.2%, and 66.5% respectively. Compared with the solvent control group, each group except the 4 mg / kg (qw×3) group of SYJS001-mAb could significantly inhibit tumor growth (P<0.001). The groups of SYJS001-ADC at 1 mg / kg, 2 mg / kg, 4 mg / kg (qw×3), 4 mg / kg, and 8 mg / kg (single administration) were all significantly superior to the positive control group Cisplatin 6 mg / kg (qw×3) in terms of efficacy and toxicity (P<0.05) (see Figure 19).

[0091] 9.2 Comparative pharmacodynamic data with IMAB362-ADC as the control IMAB362 (also known as Zolbetuximab), a reported IgG1 subtype chimeric monoclonal antibody that selectively targets the first extracellular domain of CLDN18.2 and has slight activity against CLDN18.1, was selected as the control antibody in this experiment.

[0092] 1) In this experiment, a human pancreatic cancer Bxpc3-18.2 nude mouse transplanted tumor model was used, and when the tumor volume was approximately 100 mm 3On day 39 after inoculation, 64 animals with good tumor growth were selected. According to the tumor volume, the animals were divided into 8 groups of 8 each (day 0), and 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), 2 mg / kg, 4 mg / kg, and 8 mg / kg of SYJS001-ADC (single dose), IMAB362-ADC (the IMAB362 sequence refers to Patent CN201680021997.6, gene synthesis was performed by Nanjing Genscript Biotech Co., Ltd., protein expression was transiently expressed using the KOP293 transient transfection protein expression system of Zhuhai Guirui Biotech Co., Ltd., and the specific steps refer to its official website. The production of IMAB362-ADC refers to the production method of Example 3 SYJS001 ADC) 2 mg / kg, 4 mg / kg, and 8 mg / kg (single dose), and 8 mg / kg of SYJS001-mAb (single dose) were each intravenously administered. The tumor diameter was measured twice a week, the body weight of the mice was measured, the data were recorded, and the tumor diameter of the tumor at different times after administration was measured to dynamically observe the tumor growth. The experiment was terminated on day 28. After the mice were asphyxiated with carbon dioxide, the tumors were removed and the tumor weights were measured.

[0093] Results: In this experiment, in the 2 mg / kg, 4 mg / kg, and 8 mg / kg (single-dose) groups of SYJS001-ADC, the 8 mg / kg (single-dose) group of SYJS001-mAb, and the 2 mg / kg, 4 mg / kg, and 8 mg / kg (single-dose) groups of IMAB362-ADC, the tumor volumes were 35.9%, 6.0%, 3.4%, 116.2%, 47.0%, 6.8%, and 5.1% of the vehicle control group, respectively. Compared with the vehicle control group, both the 2 mg / kg, 4 mg / kg, and 8 mg / kg (single-dose) groups of SYJS001-ADC and the 2 mg / kg, 4 mg / kg, and 8 mg / kg (single-dose) groups of IMAB362-ADC could significantly inhibit tumor growth (p<0.01). Compared with the positive control IMAB362-ADC group, at high doses, there was little difference in efficacy, but under low-dose (effective dose) conditions, the in vivo tumor inhibitory effect of SYJS001 ADC was significantly superior to that of IMAB362-ADC. Specifically, refer to Figure 20 (relative tumor volume, RTV).

[0094] According to the data published by Chunze Li in 2019 (Clinical pharmacology of vc-MMAE antibody-drug conjugates in cancer patients :learning from eight first-in-human Phase 1 studies 2019 vol.12,No.1,MABS), the clinical dosage range of ADCs based on VC-MMAE is generally 0.1 - 3.2 mg. Therefore, the low dose in this experiment is a more promising dosage for clinical application, and the clinical potential and formulation properties of SYJS001-ADC were superior to those of IMAB362-ADC.

[0095] 2) In this experiment, a nude mouse xenograft tumor model was constructed using human gastric cancer NUGC-4-18.2, and when the tumor volume was approximately 120 mm 3On the sixth day after inoculation, 64 animals with good tumor growth were selected. According to the tumor volume, the animals were divided into 8 groups of 8 each (day 0), and 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), SYJS001-ADC at 0.5, 1, 2, 4 mg / kg (single dose), IMAB362-ADC at 0.5, 1, 2, 4 mg / kg (single dose), and SYJS001-mAb at 4 mg / kg (single dose) were intravenously administered respectively. The tumor diameter was measured twice a week, the body weight of the mice was measured, the data were recorded, and the growth changes of the tumor were dynamically observed by measuring the tumor diameter at different times after administration. The experiment was terminated on the 20th day. After the animals were asphyxiated with carbon dioxide, the tumors were removed and weighed.

[0096] Results: In this experiment, for SYJS001-ADC at 0.5, 1, 2, 4 mg / kg (single dose), IMAB362-ADC at 0.5, 1, 2, 4 mg / kg (single dose), and SYJS001-mAb at 4 mg / kg (single dose), the tumor volumes were 59.1%, 27.3%, 1.8%, 0%, 68.2%, 30%, 1.8%, 0%, and 83.6% of the solvent control group respectively. Compared with the solvent control group, each group except the 4 mg / kg group of SYJS001-mAb could significantly inhibit tumor growth (p<0.001). The in vivo tumor inhibitory effect of SYJS001 ADC on NUGC4-CLDN18.2 was almost the same as that of IMAB362-ADC at high doses, but was significantly superior to IMAB362-ADC at low doses (effective doses) (see Figure 21).

[0097] Similar to the situation of the previous comparative experiment, the low dose in this experiment was a more promising dose for clinical application, and the clinical future and drug formability of SYJS001-ADC were significantly superior to those of IMAB362-ADC.

[0098] Example 10: Experiments on MMAE release and bystander effect After co - culturing the positive group BxPC - 3 - CLDN18.2 cells, HEK293 - Luc cells (cells that do not express CLDN18.2), and SYJS001 ADC for 4 days, the number of HEK293 - Luc cells in the mixed cells was shown by chemiluminescence color development. Compared with the negative group BxPC - 3 and the HEK293 - Luc co - culture group, all three concentrations of 5 μg / mL, 1 μg / mL, and 200 ng / mL could cause growth inhibition of HEK293 - Luc cells to varying degrees. This indicates that BxPC - 3 - Human CLDN18.2 in the positive group binds to SYJS001 ADC, then enters the cell by endocytosis, and the MMAE released intracellularly realizes the apoptosis of BxPC - 3 - human CLDN18.2. The MMAE released after cell apoptosis decomposition causes growth inhibition of bystander cells. The specific results are shown in detail in Figure 22. From this, it was suggested that MMAE is efficiently released from SYJS001 ADC and exerts cytotoxic activity on adjacent cells due to its membrane permeability, resulting in the bystander effect.

[0099] Summarizing the results of the above various examples, the anti - CLDN18.2 monoclonal antibody obtained in the present invention can specifically bind to CLDN18.2 and internalize with high efficiency in CLDN18.2 - positive cells. It was found that the ADC drug obtained in the present invention has extremely strong killing effects on tumor cells including pancreatic cancer, gastric cancer, and lung cancer.

[0100] Example 11: Investigation of the stability of ADC 11.1 Plasma and Serum Stability The purpose of this experiment is to investigate the in vitro metabolic stability of SYJS001-ADC in plasma and serum of different species (human, cynomolgus monkey, and Sprague-Dawley rat). With the incubation concentration of SYJS001-ADC set at 100 μg / mL, it was incubated for 0 to 168 h (7 days) under sterile conditions at 37°C. The LC-MS / MS method was used to detect the concentration of MMAE, and the concentration results of MMAE in 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma, and Sprague-Dawley rat plasma are shown in Tables 5 to 8. With the increase in incubation time, the generation of free MMAE was observed in all substrates. After incubation of SYJS001-ADC for 168 h (7 days) at 37°C, the dropout rates in 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma, and Sprague-Dawley rat plasma were 0.672%, 0.327%, 0.209%, and 0.405% respectively. The experiment showed that the dropout rate of MMAE in SYJS001-ADC was less than 1.0% in all cases, indicating that SYJS001-ADC was stable in any of 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma, and Sprague-Dawley rat plasma.

[0101]

Table 5

[0102]

Table 6

[0103]

Table 7

[0104]

Table 8

[0105] 11.2 Accelerated stability Store SYJS001-ADC in buffer at 2°C to 8°C (the components include 5% w / v L-histidine and 25% L-histidine hydrochloride). After leaving it for different periods of time, the results of detecting its stability are shown in the following table. IMAB362-ADC (IMAB362-vcMMAE) reached a free toxin level of 0.3% on the 28th day (see Table 7 of CN107667118A), which is much higher than the 0.000026% of the drug SYJS001-ADC of the present invention over three months, indicating that the stability of SYJS001-ADC is much better than that of IMAB362-ADC.

[0106]

Table 9

[0107] Example 12: Comparative study of SYJS001 and IMAB362 12.1 Affinity Resuspend the CLDN18.2 high-expression cell line BxPC3-CLDN18.2 using PBS buffer, and adjust the cell concentration to 1×10 6Adjusted to cells / mL and added to a 96-well V-bottom culture plate at 100 μL per well. Centrifuged at 2500 rpm for 5 min and the supernatant was discarded. The sample was diluted using the experimental buffer, and the initial concentration of the antibody was set at 40 μg / mL and serially diluted 5-fold for a total of 10 gradients. The diluted samples were added to a 96-well V-bottom culture plate at 100 μL / well and incubated at 4°C for 60 - 90 min. After incubation, the V-bottom culture plate was placed in a centrifuge and centrifuged at 2500 rpm for 5 min. The centrifuged supernatant was carefully removed, 100 μL of PBS resuspended cells were added, and centrifuged at 2500 rpm for 5 min. This was repeated twice, the centrifuged supernatant was carefully removed, and based on the experimental sequence, a fluorescent antibody detection reagent (1:1000 dilution) was added and incubated at 4°C in the dark for 30 - 60 min. After incubation, the V-bottom culture plate was placed in a high-speed centrifuge and centrifuged at 2500 rpm for 5 min. The centrifuged supernatant was carefully removed, 100 μL of the experimental buffer was added to resuspend the cells, and then detection was performed using a flow cytometer, and the fluorescence signal value of the corresponding well of the experimental plate was read by plate reading. Experimental data were derived and the data were analyzed using GraphPad Prism 5 software. A regression model of the four-parameter equation was selected to create an "S" curve, and the median effective dose ED 50 (C value) was automatically generated by the software. As a result, as shown in the following figure (Figure 23, MFI: mean fluorescence intensity), the affinity of SYJS001 was more than twice that of IMAB362.

[0108] 12.2 Antigen Epitope Analysis To detect the antigen epitope competition between SYJS001-ADC and IMAB362-ADC, the Octet epitope pairing was used. 5 μg / mL of human CLDN18.2 protein was immobilized on the HIS1K capture sensor and loaded for 180 s. The two antibodies were each diluted to 100 nM. The immobilized sensor was first bound to one antibody for 180 s (Association), then to the other antibody for 180 s (Association), and the binding signal of the other antibody was detected to determine whether the two antibodies recognize the same epitope. The results were as follows: 60 - 100%: no competition at all. 20 - 60%: partial competition. <20%: completely competitive, and in this case, the two antibodies were determined to be in competition. As a result, as shown in the following table (Table 10), the epitopes of SYJS001-ADC and IMAB362-ADC were completely competitive.

[0109]

Table 10

[0110] 12.3 Endocytosis detection BxPC3-CLDN18.2 cells were seeded in a 6-well plate, and the number of cells per well was approximately 4 - 6×10 5Cells were used. They were transferred to an incubator and cultured overnight at 37°C and 5% CO2. The antibody was diluted to 5 μg / mL using complete medium (DMEM (Hyclone, product number: SH30243.01) + 10% FBS (Gibco, product number: 10091 - 148) + 1 μg / mL puromycin (Gibco, product number: A11138 - 02)). The existing medium in the 6 - well plate was discarded, and 2 mL of the complete medium containing the antibody was added to each well. After leaving it at 4°C for 2 h, the supernatant was discarded, washed twice with complete medium, and 2 mL of the complete medium without the antibody was added to each well. Except at this point, 2 - 3 duplicate wells were taken, trypsinized, and this point was taken as the total amount of antibody bound to the cells. The remaining cells were transferred to the incubator and subjected to endocytosis at 37°C and 5% CO2 incubator for 4 h, 21 h, 25 h, and 48 h. After washing the trypsinized cells at each time point, a fluorescent antibody detection reagent (diluted 1:1000) was added, and incubated at 4°C in the dark for 60 min. The centrifugation supernatant was carefully removed, washed twice, and the fluorescence signal value was detected with a flow cytometer. Endocytosis rate calculation formula: (Total antibody fluorescence signal - Fluorescence signal at different time points) / Total antibody fluorescence signal × 100% As a result of the following figure (Figure 24, MFI: Mean Fluorescence Intensity), the endocytosis rate of SYJS001 was much higher than that of IMAB362 - ADC in the initial stage (0 - 21 h), and was almost the same as that of IMAB362 - ADC from 21 - 48 h. Under the same other conditions, the faster the endocytosis rate, the stronger the ability of the drug to enter tumor cells, so the toxic molecule is released better and objectively the effect is accelerated. Therefore, it was expected that SYJS001 would have a faster effect compared to IMAB362 - ADC.

[0111] 12.4 In vitro cell growth inhibition experiment 1. Adenocarcinoma cell model BxPC-3-CLDN18.2 cells were collected and resuspended in complete medium (the same complete medium as used in the section of "12.3 Endocytosis Detection" above). The resuspended target cells were gently pipetted several times to a single-cell suspension, the cell viability was identified using the trypan blue staining method, and the cells were counted. The cell density was adjusted to 1×10 5 cells / mL. It was added to a 96-well black flat-bottom cell culture plate at 100 μL per well. With the starting concentration of the antibody being 5 μg / mL, it was serially diluted 2-fold to make a total of 11 gradients. The diluted test articles were added to the 96-well black flat-bottom cell culture plate seeded with cells at 20 μL each. The antibody and the cells were co-cultured and placed in a cell incubator (37 °C, 5% CO2) and incubated for 63 - 69 h. After incubation, resazurin sodium solution (w / v 0.03%) was added at 20 μL per well. It was allowed to act at 37 °C for 3 - 4 h, the fluorescence value was read at 550 nm / 610 nm using a microplate reader, and a figure was created using Magellan6 or similar figure-making software, and the half-inhibitory concentration IC 50 of the reference standard and the sample was fitted. The output parameter C is IC 50 (unit ng / mL). As a result, as shown in the following figure (Figure 25, RLU: relative luminescence unit), SYJS001-ADC had a significantly better in vitro inhibitory effect on BxPC-3-CLDN18.2 cells than IMAB362-ADC.

[0112] 2. Gastric cancer cell model NUGC4-CLDN18.2 cells were collected and resuspended in complete medium (the same complete medium as used in the section of 12.3 Endocytosis Detection). The resuspended target cells were gently pipetted several times to a single-cell suspension, the cell viability was identified using the trypan blue staining method, and the cells were counted. The cell density was adjusted to 1×10 5Adjusted to cells / mL. Added to a 96-well black flat-bottom cell culture plate at 100 μL per well. The initial concentration of the antibody was set at 10 mg / mL and serially diluted 5-fold to give a total of 11 gradients. 20 μL of the diluted test article was added to each well of the 96-well black flat-bottom cell culture plate seeded with cells. The antibody and cells were co-cultured and incubated in a cell incubator (37 °C, 5% CO2) for 63 - 69 h. After incubation, 20 μL of resazurin sodium solution (w / v 0.03%) was added per well. It was allowed to act at 37 °C for 3 - 4 h, the fluorescence value was read at 550 nm / 610 nm using a microplate reader, and a figure was created using Magellan6 or similar figure creation software. The half-inhibitory concentration IC 50 of the reference standard and the sample was fitted. Output parameter C is IC 50 (unit: ng / mL). As a result, as shown in the following figure (Figure 26, RLU: relative light unit), SYJS001-ADC had a superior in vitro inhibitory effect on NUGC4-CLDN18.2 cells compared to IMAB362-ADC, exceeding twice that of IMAB362-ADC.

[0113] Overall, the SYJS001-ADC obtained in the present invention was clearly superior to the control IMAB362-ADC in terms of stability, related antibody affinity, endocytosis efficiency, and in vitro and in vivo tumor cell inhibition.

[0114] The above is not intended to limit the combinations of features necessary for the implementation of the present invention, but is merely a preferred embodiment used as an example. The provided title is not intended to limit the various embodiments of the present invention. Terms such as "comprising", "containing", and "including" are not intended to be limiting. Further, unless otherwise stated, when there is no numerical modification, the plural form is included, and "or", "or", "or" means "and / or", "as well as / or". Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. All publications and patents referred to in this application are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the methods and combinations described in this specification will be apparent to those skilled in the art. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention for which protection is sought should not be unduly limited to these specific embodiments. In fact, various modifications of the forms described for carrying out the invention that will be apparent to those skilled in the art are intended to be included within the scope of the appended claims.

Claims

1. A conjugate comprising an antibody or an antigen-binding fragment thereof conjugated to one or more drug molecules, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising three CDR regions having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain comprising three CDR regions having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, the drug molecule is MMAE (Monomethyl auristatin E), the drug molecule is conjugated to the antibody or an antigen-binding fragment thereof via a linker, the linker being linked to the antibody or an antigen-binding fragment thereof via an amino group, and the linker is NH ２ -(CH ２ -CH ２ -O) ｍ -CH ２ -C(=O)-Val-Cit-pABC, wherein m is an integer from 1 to 8 and the average drug-to-antibody ratio is 2.

2. The conjugate according to claim 1, wherein the antibody is a monoclonal antibody, a bispecific antibody, a humanized monoclonal antibody, or a fully human monoclonal antibody.

3. The conjugate according to claim 1, wherein the heavy chain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7, and the light chain comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO:

8.

4. The conjugate according to claim 1, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

10.

5. The conjugate according to claim 1, wherein m is 3.

6. A pharmaceutical composition comprising the conjugate according to claim 1 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, which is for use in the treatment or prevention of cancer.

8. The pharmaceutical composition according to claim 7, wherein the cancer is a CLDN18.2 positive cancer.

9. The pharmaceutical composition according to claim 7, wherein the cancer is gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, or gallbladder cancer.

10. The pharmaceutical composition according to claim 7, wherein the cancer is an adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung, or ovary.

11. The pharmaceutical composition according to claim 7, wherein the cancer is gastric cancer or pancreatic cancer.

12. A kit comprising the conjugate according to claim 1 and a container for containing the conjugate.

13. A pharmaceutical composition comprising the conjugate according to claim 1 and an anti-proliferative agent.

14. The pharmaceutical composition according to claim 13, wherein the anti-proliferative agent is selected from the group consisting of paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, and iproplatin.

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