Anti-CLDN18.2 Antibody, Drug Conjugate, Preparation Method and Use Thereof

Abstract

The present application provides an anti-CLDN18.2 antibody and an antibody-drug conjugate prepared from the antibody, and the antibody or antibody-drug conjugate can effectively treat CLDN18.2-positive tumors, including but not limited to cancers represented by gastric cancer and pancreatic cancer.

Description

[0001] Related Applications

[0002] This application claims the priority of Chinese Patent Application No. 202011385844.4, titled "Anti-CLDN18.2 Antibody, Drug Conjugate and Their Preparation Methods and Uses", filed on November 30, 2020. The entire text of this patent application is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This application generally relates to the field of biomedicine, and particularly to anti-CLDN18.2 antibodies, related conjugates (such as antibody-drug conjugates), and their uses in the treatment or prevention of tumors. Background Art

[0004] Tight junctions (TJs) are important functional components of the adhesion between normal epithelial cells. They mechanically connect cells to form an epithelial barrier, prevent the transport of macromolecules between cells, and maintain epithelial cell polarity. The proteins that make up tight junctions mainly include Claudin (also known as occludin, sealing protein, tight junction protein, etc. To avoid any ambiguity, this specification uses the English name Claudin or the abbreviation CLDN), Occludin, ZO-1, ZO-2, ZO-3, cingulin, Pals1, MUPP1, among which Claudin protein and Occludin protein are the most critical proteins.

[0005] Claudin protein is the scaffold protein that makes up tight junctions. Its abnormal expression can lead to the destruction of the structure and function of epithelial cells and endothelial cells, and may play an important role in the pathogenesis of various diseases. So far, it has been found that the Claudin gene family includes 24 members, and the members are highly conserved in function during evolution. The molecular mass of Claudin is 22-27 kD. Each Claudin molecule has the same structure. Claudin is widely distributed in normal tissues and different tumor tissues, and there are differential expressions. Abnormal expressions of several Claudin proteins have also been found in precancerous lesions and gastric cancer of the stomach, and are related to prognosis.

[0006] The humanized Claudin18 gene has two different first exons, so it can produce two splice variants, Claudin18.1 (hereinafter referred to as CLDN18.1) and Claudin18.2 (hereinafter referred to as CLDN18.2). The amino acid sequences of human CLDN18.1 and 18.2 are both 261 amino acid residues in length, and 21 of them are different among the 0-70 amino acid residues; the two subtypes of CLDN18 are transcribed and amplified in different tissues respectively. Among them, CLDN18.1 is selectively expressed in normal lung cells, while the expression of CLDN18.2 in the normal stomach is limited to the short-lived cells of the differentiated gastric epithelium.

[0007] Scientific research shows that although CLDN18.1 and CLDN18.2 are extremely similar in structure, their expressions in tumors are very different. For example, in normal tissues, CLDN18.1 is only expressed in the lung, while CLDN18.2 is expressed in a limited way in the stomach; in tumor tissues, there is no obvious high expression of CLDN18.1 in the lung, while the expression of CLDN18.2 is up-regulated in cancer types such as gastric cancer, esophageal cancer, and pancreatic cancer. For example, when the gastric epithelial tissue undergoes malignant transformation, the disorder of cell polarity will lead to the exposure of the epitope of the CLDN18.2 protein on the cell surface. At the same time, the CLDN18.2 gene will also be abnormally activated, highly selectively and stably expressed in specific tumor tissues, and participate in the proliferation, differentiation and migration of tumor cells, which makes it a potential effective molecular target for anti-tumor drugs.

[0008] The annual incidence rate of gastric cancer worldwide is 13.86 / 100,000. A large proportion of patients have reached the middle and advanced stages at the time of diagnosis, and the recovery after surgery is extremely unsatisfactory. In addition, the incidence rate is high in the elderly, the overall survival time is on average less than one year, and the 5-year survival rate is less than 20%.

[0009] Pancreatic cancer is also one of the tumors with the highest malignancy at present, with a median survival time of less than 6 months and an overall 5-year survival rate of less than 6%.

[0010] The research and development of anti-tumor drugs targeting CLDN18.2 has been in full swing globally. Currently, there are numerous projects targeting CLDN18.2 (about 28), and the project types include monoclonal antibodies, bispecific antibodies, and CAR-T targeting CLDN18.2. Among all the projects, the monoclonal antibody Claudiximab (renamed Zolbetuximab after acquisition) of Ganymed company (acquired by Astellas) has the fastest R & D progress. Its gastric cancer indication trial has reached the phase III clinical stage. A total of 9 projects have entered the clinical stage, and most of them are in the preclinical stage, with great uncertainty in the treatment effect. Based on the current data, no reports on clinical studies related to antibody-drug conjugates (ADCs) targeting CLDN18.2 have been found.

[0011] Although CLDN18.2 is an excellent target for digestive tract cancer and pancreatic cancer, due to the difference of 7 amino acid residues in the extracellular domain ECD1 of about 50 amino acids between CLDN18.1 and CLDN18.2, how to design an antibody that specifically recognizes CLDN18.2 but not CLDN18.1 has become a difficult problem in the development of monoclonal antibody drugs for this target.

[0012] In addition, generally speaking, although monoclonal antibody therapy has the characteristics of high target specificity and low side effects, its efficacy when used alone is relatively limited. Therefore, most monoclonal antibody drugs are used in combination with chemotherapy drugs. Currently, the main way to improve the efficacy of monoclonal antibodies is antibody-drug conjugates. Antibody-drug conjugates (also known as antibody-drug conjugates) belong to a new type of anti-cancer biological missile drug, which mainly consists of three parts: an antibody, a drug molecule, and a linker connecting the two. After chemically conjugating a monoclonal antibody with a drug, the antibody-drug conjugate uses the targeting property of the monoclonal antibody to specifically recognize the receptor targeted by the antibody on the surface of cancer cells, bind to the receptor, and then enter the cell interior. It releases the drug using intracellular proteases to prevent cancer cells from multiplying and kill cancer cells. The antibody-drug conjugation technology combines small molecule drugs with biological proteins, taking advantage of the strengths of both, enhancing the drug efficacy and reducing the toxic side effects, becoming a new generation of therapeutic products.

[0013] As of September 2020, the FDA has approved a total of 9 ADC drugs for marketing, including Adcetris from Seattle, Kadcyla, Polivy from Genentech, Besponsa, Mylotarg from Wyeth, Lumoxiti, Enhertu from AstraZeneca, and Trodelvy from immunomedics. Currently, no ADC drugs developed in China have been approved for marketing.

[0014] Both single antibody drugs and antibody-drug conjugates are drug types with very good prospects, and more in-depth research and development are urgently needed in the pharmaceutical field. Summary of the Invention

[0015] In a first aspect, the present application provides a conjugate, which comprises an anti-CLDN18.2 antibody of the present application or an antigen-binding fragment of the antibody conjugated with one or more drug molecules.

[0016] In a second aspect, the present application provides a pharmaceutical composition, which comprises the conjugate described in the first aspect and a pharmaceutically acceptable carrier.

[0017] In a third aspect, the present application provides the use of the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a drug for treating or preventing cancer.

[0018] In a fourth aspect, the present application provides a method for treating cancer in an individual, which comprises administering a therapeutically effective amount of the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect to an individual suffering from the cancer.

[0019] In a fifth aspect, the present application provides a medical product (such as a medicine box), which comprises the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect.

[0020] In a sixth aspect, the present application provides the use of the conjugate described in the first aspect and an anti-proliferative agent in the preparation of a drug for treating tumors.

[0021] In a seventh aspect, the present application provides a pharmaceutical composition, which comprises the conjugate described in the first aspect and an anti-proliferative agent.

[0022] In an eighth aspect, the present application provides a method for treating a tumor in an individual, which comprises administering a therapeutically effective amount of the conjugate described in the first aspect or the pharmaceutical composition described in the second aspect and an anti-proliferative agent to an individual suffering from the tumor.

[0023] In a ninth aspect, the present application provides an anti-CLDN18.2 antibody or an antigen-binding fragment of the antibody, a pharmaceutical composition comprising the antibody or antigen-binding fragment, the pharmaceutical use of the antibody or antigen-binding fragment, and a method for treating tumors / cancers using the antibody or antigen-binding fragment. Brief Description of the Drawings

[0024] Figure 1 Shows a schematic diagram of the Claudin protein structure.

[0025] Figure 2Shows the schematic diagram of the SYJS001 ADC structure, where L&D represents the part of the linker (Linker, "L") + drug molecule (Drug, "D"). The L&D on the right shows the complete structure, and the circled part indicates the amide bond connection between the linker in L&D and the antibody. The SYJS001 ADC shown in this application is a site-specific antibody-drug conjugate. Each molecule consists of 1 fully human monoclonal antibody against CLDN18.2 (SYJS001 mAb) conjugated with 1 molecule of MMAE derivative at the amino acid position Q298 (i.e., Kabat number Q295) on each heavy chain through a linker (NH2-PEG3-Val-Cit). The connection mode between the antibody and the linker is a stable amide bond (isopeptide bond). The average ratio of drug molecule to antibody (DAR) is 2.0, and the relative molecular weight is 150KD.

[0026] Figure 3 Shows the plasmid map of pGenHT1.0-DGV.

[0027] Figure 4 Shows the double digestion identification map of the SYJS001 in pGenHT1.0-DGV plasmid, where lane M: KBLadder; lane 1: supercoiled state; lane 2: linearized plasmid formed by digestion with PvuI enzyme; lane 3: heavy chain, light chain fragments and remaining fragments formed by digestion with AscI / PmLI enzymes.

[0028] Figure 5 Shows the schematic diagram of the SYJS001 in pGenHT1.0-DGV plasmid.

[0029] Figure 6 Shows the modification rate identification map of SYJS001 ADC, where the peaks at 4.89 and 95.11 represent the anti-CLDN18.2 monoclonal antibody and the anti-CLDN18.2 monoclonal antibody-drug conjugate, respectively.

[0030] Figure 7 Shows the DAR distribution identification map of SYJS001 ADC.

[0031] Figure 8 Shows the binding curves of SYJS001 ADC to cells expressing human, murine, and simian CLDN18.2.

[0032] Figure 9 Shows the results of the cross-reactivity experiment of the SYJS001 antibody.

[0033] Figure 10 Shows the results of the specific binding experiment of SYJS001 ADC to CLDN18.2 protein.

[0034] Figure 11 Shows the endocytosis observation of SYJS001 ADC in HEK293-CLDN18.2 cells.

[0035] Figure 12 Shows the in vitro growth inhibitory effects of SYJS001 naked antibody and SYJS001 ADC on NCI-N87-CLDN18.2, where Group 1: SYJS001 ADC (square dots), Group 2: SYJS001 naked antibody (round dots).

[0036] Figure 13 Shows the in vitro growth inhibitory effects of SYJS001 naked antibody and SYJS001 ADC on KATOⅢ, where Group 1: SYJS001 ADC (square dots), Group 2: SYJS001 naked antibody (round dots).

[0037] Figure 14 Shows the in vitro growth inhibitory effects of SYJS001 naked antibody and SYJS001 ADC on NCI-H460-CLDN18.2, where Group 1: SYJS001 ADC (square dots); Group 2: SYJS001 naked antibody (round dots).

[0038] Figure 15 Shows the in vitro growth inhibitory effects of SYJS001 naked antibody and SYJS001 ADC on NUGC4-CLDN18.2, where Group 1: SYJS001 ADC (square dots); Group 2: SYJS001 naked antibody (round dots).

[0039] Figure 16 Shows the in vitro growth inhibitory effects of SYJS001 naked antibody and SYJS001 ADC on PATU8988S, where Group 1: SYJS001 ADC (square dots); Group 2: SYJS001 naked antibody (round dots).

[0040] Figure 17 Shows the in vitro growth inhibitory effects of SYJS001 naked antibody and SYJS001 ADC on BxPC-3-CLDN18.2, where Group 1: SYJS001 ADC (square dots); Group 2: SYJS001 naked antibody (round dots).

[0041] Figure 18 Shows the in vivo tumor inhibitory effect of SYJS001 ADC on BxPC-3-CLDN18.2 (compared with gemcitabine).

[0042] Figure 19 Shows the in vivo tumor inhibitory effect of SYJS001 ADC on NUGC4-CLDN18.2 (compared with cisplatin).

[0043] Figure 20 Shows the in vivo antitumor effect of SYJS001 ADC on BxPC-3-CLDN18.2 (compared with IMAB362-ADC).

[0044] Figure 21 Shows the in vivo antitumor effect of SYJS001 ADC on NUGC4-CLDN18.2 (compared with IMAB362-ADC).

[0045] Figure 22 Shows the experimental results of MMAE release and bystander effect.

[0046] Figure 23 Shows the experimental results of the affinity comparison between SYJS001 and IMAB362.

[0047] Figure 24 Shows the experimental results of the endocytosis rate comparison between SYJS001 and IMAB362.

[0048] Figure 25 Shows the in vitro cell inhibition experimental results of SYJS001 ADC and IMAB362 ADC (adenocarcinoma cell model).

[0049] Figure 26 Shows the in vitro cell inhibition experimental results of SYJS001 ADC and IMAB362 ADC (gastric cancer cell model).

[0050] Detailed Description of the Invention

[0051] Definitions

[0052] Unless otherwise defined, all technical terms used herein have the same meaning as understood by those of ordinary skill in the art. For definitions and terms in this field, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0053] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. Additionally, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting from the minimum value 1 or greater, such as 1 to 6.1, and all sub-ranges ending with the maximum value 10 or less, such as 5.5 to 10. Additionally, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0054] As used herein, the terms "pharmaceutical composition", "combination drug", and "drug combination" are used interchangeably and denote a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient combined together to achieve a particular purpose. In certain embodiments, the pharmaceutical composition includes combinations that are separated in time and / or space, provided that they can act together to achieve the purposes of this application. For example, the components contained in the pharmaceutical composition (such as antibodies, nucleic acid molecules, combinations of nucleic acid molecules, and / or conjugates according to this application) can be administered to a subject as a whole, or separately to the subject. When the components contained in the pharmaceutical composition are administered to the subject separately, the components can 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, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends in particular on whether the composition according to this application is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition according to this application can contain wetting agents, emulsifying agents, or buffering substances as additives.

[0055] The pharmaceutical composition, vaccine, or pharmaceutical preparation according to this application can be administered by any suitable route, such as orally, nasally, intradermally, subcutaneously, intramuscularly, or intravenously.

[0056] As used herein, "therapeutically effective amount" or "effective amount" refers to a dose sufficient to demonstrate its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the individual circumstances and severity of the subject being treated. The prescription of treatment (such as the determination of dosage, etc.) is ultimately the responsibility of the general practitioner and other physicians and depends on their decision-making, usually considering the disease being treated, the individual circumstances of the patient, the site of delivery, the method of administration, and other factors known to the physician.

[0057] As used herein, the term "subject" refers to a mammal, such as a human, but may also be other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.) or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0058] The term "antibody" broadly encompasses intact antibodies and any antigen-binding fragments ("antigen-binding portions") thereof or single-chain forms. A "full-length / intact antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by 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, and the light-chain constant region comprises one domain, CL. The VH and VL regions can also be further subdivided into multiple regions with high variability, called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to tissues or factors of the host, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (Clq). Chimeric or humanized antibodies are also encompassed within the antibodies according to the present application. The full-length / intact antibody can be any type of antibody, such as IgD, IgE, IgG, IgA or IgM (or subclasses thereof), but the antibody does not need to belong to any specific class. Immunoglobulins can be designated as different classes according to the amino acid sequence of the constant domain of the heavy chain. Generally, immunoglobulins have five main classes: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further differentiated into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains 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 well known.

[0059] Complementarity-determining regions (CDRs, usually CDR1, CDR2 and CDR3) are the regions in the variable region that have the greatest influence on the affinity and specificity of the antibody. There are several common definitions for the CDR sequences of VH or VL, including IMGT, Chothia definition and Kabat definition. For the variable region sequence of a given antibody, the CDR sequences in the VH and VL sequences can be determined according to the IMGT, Chothia definition or Kabat definition.

[0060] The term "humanized antibody" refers to an antibody that may contain CDR regions derived from a human antibody, and other parts of the antibody molecule are derived from one (or several) human antibodies. Moreover, in order to retain the binding affinity, some residues in the framework (referred to as FR) regions may be modified; humanized antibodies or fragments thereof according to the present application can be prepared by techniques known to those skilled in the art;

[0061] The term "semi-humanized antibody" refers to an antibody in which one antibody chain contains a murine variable region (as in a chimeric antibody) and the other antibody chain contains a humanized variable region, as compared to a humanized antibody or a fully humanized antibody.

[0062] The term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, for example, an antibody in which the variable region sequence is from a murine antibody and the constant region sequence is from a human antibody. Chimeric antibodies or fragments thereof according to the present application can be prepared by using genetic recombination techniques. For example, the chimeric antibody can be produced by cloning recombinant DNA that contains a promoter and a sequence encoding the variable region of a non-human, especially murine, monoclonal antibody according to the present application, as well as a sequence encoding the constant region of a human antibody. The chimeric antibody according to the present application encoded by such a recombinant gene will be, for example, a murine-human chimera, the specificity of the antibody being determined by the variable region derived from murine DNA, and its isotype being determined by the constant region derived from human DNA. For methods of preparing chimeric antibodies, reference can be made, for example, to the document Verhoeyn et al. (BioEssays, 8:74, 1988).

[0063] The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0064] The term "bispecific antibody" refers to an antibody that has the ability to bind two antigen epitopes simultaneously. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two binding portions respectively fused thereto (similar to a natural antibody, except that the two arms bind different antigen targets or epitopes), and the antigen-binding portion can be a single-chain antibody (scfv) or a Fab fragment.

[0065] As used herein, the term "antigen-binding fragment" particularly refers to antibody fragments such as Fv, scFv (sc refers to single-chain), Fab, F(ab')2, Fab', scFv-Fc fragments or diabodies, or any fragment that should be able to increase the half-life through chemical modification or by incorporation into liposomes. The chemical modification such as adding poly(alkylene) glycol such as polyethylene glycol ("PEGylation"), and the PEGylated fragments are called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG ("PEG" is polyethylene glycol). The antigen-binding fragment of the anti-CLDN18.2 antibody of the present application has CLDN18.2 binding activity. For example, the antigen-binding fragment is composed of or contains partial sequences of the heavy or light chain variable chains of its source antibody, and the partial sequences are sufficient to retain the same binding specificity and sufficient affinity as its source antibody. Such an antigen-binding fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence of its source.

[0066] Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which can be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which can be divalent fragments having two Fab' fragments, and the two Fab' fragments are connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) Fv fragments having the VL and VH domains of a single arm of an antibody; (4) single-chain Fv (scFv), which can be a single polypeptide chain composed of a VH domain and a VL domain via a peptide linker; and (5) (scFv)2, which can contain two VH domains and two VL domains connected by a peptide linker, and the two VL domains are combined with the two VH domains via disulfide bridges.

[0067] The terms "Fc fragment", "Fc domain", "Fc portion" or similar terms refer to a part of the constant region of the antibody heavy chain, including the hinge region, the CH2 fragment and the CH3 fragment of the constant region.

[0068] Generally, to prepare monoclonal antibodies or antigen-binding fragments thereof, especially murine monoclonal antibodies or antigen-binding fragments thereof, reference can be made to the techniques described especially in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the techniques for preparation from hybridoma cells described by Kohler and Milstein (Nature, 256: 495-497, 1975).

[0069] According to the structural information of the anti-CLDN18.2 monoclonal antibody given in the present application, it can be prepared in CHO-K1 (ATCC Number: CCL-61, Lot No.: 59965043) cells by methods known in the art.

[0070] The term "homology / identity / consistency" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of homology. Methods and computer programs for alignment are well known in the art.

[0071] The term "specifically binds" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0072] The inventors of the present application first obtained a monoclonal antibody against CLDN18.2, which can bind to CLDN18.2 on CLDN18.2-positive cells and be efficiently internalized, and is very suitable for the development of ADC. In subsequent ADC research and development, the inventors of the present application found that when NH2-PEG3-Val-Cit was used as the linker, good stability of the conjugation of the linker and the drug molecule was achieved. Thus, the humanized antibody was conjugated with a small molecule drug (e.g., MMAE) through the linker, and the obtained ADC drug has extremely strong killing effects on CLDN18.2 highly expressed cancer cells, especially pancreatic cancer, gastric cancer and lung cancer cells, and has good stability. Specifically, in vivo experiments showed that intravenous administration of the antibody-drug conjugate in nude mice with CLDN18.2-positive gastric or pancreatic xenograft tumors resulted in dose-dependent tumor growth inhibition, and significant therapeutic effects were observed at a single-dose intravenous administration of about 1 to 8 mg / kg; the best therapeutic effect was obtained at 8 mg / kg, and the individuals showed good tolerance; the overall therapeutic effect was significant, and the ADC drug obtained in the present application can produce a bystander effect, further enhancing the therapeutic effect.

[0073] In one aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to CLDN18.2. Specifically, the antibody comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions, and the amino acid sequence of at least one of the CDR regions has the 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 thereto; and / or (ii) the light chain comprises three CDR regions, and the amino acid sequence of at least one of the CDR regions has the 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 thereto.

[0074] In some specific embodiments, the antibody comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NO: 1, 2 and 3, respectively; and / or (ii) the light chain comprises three CDR regions, and the CDR regions have the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, respectively.

[0075] In certain specific embodiments, the antibody or the antigen-binding fragment thereof of the present application is isolated.

[0076] In certain specific embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8.

[0077] In certain specific embodiments, the heavy chain comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 9, and / or the light chain comprises a light chain having the amino acid sequence shown in SEQ ID NO: 10.

[0078] In certain specific embodiments, the antibody of the present application is a monoclonal antibody.

[0079] In certain specific embodiments, the antibody of the present application is a bispecific antibody. For example, one arm of the bispecific antibody can be an antigen-binding fragment (such as Fab or scfv) of the anti-CLDN18.2 antibody described in the present application, and the other arm can be an antigen-binding fragment (such as Fab or scfv) targeting other antigens (for example, other antigen targets that can be used for ADC construction) or targeting another CLDN18.2 epitope (different from the CLDN18.2 binding epitope of the anti-CLDN18.2 antibody described in the present application).

[0080] In certain specific embodiments, the antibodies of the present application are humanized antibodies, including semi-humanized antibodies and fully human antibodies.

[0081] In certain specific embodiments, the antibody or its antigen-binding fragment of the present application has ADCC activity.

[0082] In certain specific embodiments, the antibody or its antigen-binding fragment of the present application has CDC activity.

[0083] In certain specific embodiments, the antibody or its antigen-binding fragment of the present application specifically binds to CLDN18.2 and substantially does not bind to CLDN18.1.

[0084] In certain specific embodiments, the antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype or IgG4 subtype.

[0085] In certain specific embodiments, the heavy chain constant region of the antibody can be human IgG1 subtype, human IgG2 subtype, human IgG4 subtype, murine IgG1 subtype or murine IgG2a subtype.

[0086] In certain specific embodiments, the heavy chain constant region is of the IgG1 subtype, that is, the antibody is an IgG1-type antibody.

[0087] In certain specific embodiments, the antibody comprises a light chain constant region selected from the κ subtype or λ subtype.

[0088] In certain specific embodiments, the light chain constant region of the antibody can be human κ subtype, human λ subtype, murine κ subtype or murine λ subtype.

[0089] In certain specific embodiments, the antibody of the present application is an IgG1κ antibody.

[0090] In certain specific embodiments, the antibody or its antigen-binding fragment of the present application can be used for treating or preventing cancer, wherein the cancer overexpresses CLDN18.2.

[0091] 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.

[0092] In another aspect, the present application provides an isolated polynucleotide encoding the antibody of the present application.

[0093] In yet another aspect, the present application provides a combination of isolated polynucleotides, said combination comprising a polynucleotide encoding the light chain of an antibody or an antigen-binding fragment thereof of the present application and a polynucleotide encoding the heavy chain of an antibody or an antigen-binding fragment thereof of the present application.

[0094] In another aspect, the present application provides an expression vector comprising the polynucleotide or the combination of polynucleotides of the present application, said polynucleotide being operably linked to regulatory sequences that permit the expression of the polypeptide encoded thereby in a host cell or a cell-free expression system.

[0095] In some embodiments of the present application, the host cell can be a prokaryotic host cell, a eukaryotic host cell or a phage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic host cell can be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., an insect cell such as Spodoptera frugiperda, a plant cell such as Nicotiana tabacum, or a mammalian cell such as BHK cell, CHO cell, COS cell, myeloma cell, etc. In some embodiments, the host cell of the present application is preferably a mammalian cell, more preferably a BHK cell, a CHO cell, an NSO cell or a COS cell.

[0096] In another aspect, the present application provides an antibody-drug conjugate comprising the anti-CLDN18.2 antibody or an antigen-binding fragment thereof of the present application conjugated to one or more drug molecules. For the description of the embodiments and technical features of the anti-CLDN18.2 antibody or an antigen-binding fragment thereof of the present application, see above.

[0097] In view of the fact that 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 should understand that when CLDN18.2 is a target for other diseases other than cancer / tumor cells, the drug molecule can be selected according to the target disease.

[0098] The types of anti-cancer drugs include but are not limited to cytotoxic drugs, immunopotentiators or radioisotopes.

[0099] In some embodiments, the types of cytotoxic drugs include tubulin inhibitors (such as alkaloids), DNA topoisomerase inhibitors, DNA damaging agents, antimetabolites or antitumor antibiotics.

[0100] In some embodiments, the tubulin inhibitors include, but are not limited to, auristatin derivatives (such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (such as DM1, DM4, ansamitocin, mertansine or dolastatin and their derivatives).

[0101] In some embodiments, the DNA topoisomerase inhibitors are 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-hydroxyellipticine, topotecan, lurtotecan, belotecan, 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-propenamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxy-3-phenylpropyl)-(E)-2-propenamide, 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-acridinylcarboxamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acridinylcarboxamide.

[0102] In some embodiments, the DNA damaging agents include, but are not limited to, calicheamicin, duocarmycin, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0103] In some embodiments, the immunopotentiators include, but are not limited to, levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic:polycytidylic acid or polyinosinic:polyuidylic acid.

[0104] In some embodiments, the antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine or 5-fluorouracil.

[0105] In some embodiments, the anti-tumor antibiotics include, but are not limited to, polypeptide antibiotics (such as actinomycin D or bleomycin) or anthraquinone drugs (such as doxorubicin or mitoxantrone hydrochloride).

[0106] In some embodiments, the radioisotopes include, but are not limited to, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 Co or 177 Lu.

[0107] In some embodiments, an antibody having the ability to bind to CLDN18.2 is covalently linked to a drug moiety via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is cleavable under intracellular conditions. In one embodiment, the linker is hydrolyzable at a pH less than 5.5. In some embodiments, the linker is cleavable by intracellular proteases. 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 through a cysteine thiol of the antibody. In one embodiment, the antibody is linked to the linker through an amino group of the antibody (especially the amino group of a glutamine residue).

[0108] Non-limiting examples of the linker 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.

[0109] In certain specific embodiments, the antibody-drug conjugate described in the present application has the following general formula Ab-(L-U)n, where Ab represents the antibody targeting CLDN18.2 of the present application, 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 can 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), and the DAR value can be an average value and can be any value from 1 to 8 (not limited to integers and can also be decimals), preferably an integer from 1 to 8, preferably 2, 4, 6, 8, and more preferably 2.

[0110] In another aspect, the present application provides a pharmaceutical preparation (such as a pharmaceutical composition), which comprises the antibody-drug conjugate of the present application, and a pharmaceutically acceptable diluent, carrier or excipient.

[0111] In another aspect, the present application provides a medical preparation, which comprises the antibody-drug conjugate of the present application. In some embodiments, the medical preparation exists in the form of a kit, and the kit comprises a container for accommodating the antibody-drug conjugate of the present application.

[0112] In one embodiment, the medical preparation further comprises printed instructions for using the preparation in a method for treating or preventing cancer (especially cancer expressing CLDN18.2).

[0113] The antibody-drug conjugate provided by the present application can effectively treat and / or prevent cancers associated with cells expressing CLDN18.2 (CLDN18.2 positive). As non-limiting examples, the cancer can be gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer, and metastases of the above cancers, especially gastric cancer metastases, peritoneal metastases and lymph node metastases. The cancers suitable for treatment with the antibody-drug conjugate provided by the present application can be adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung and ovary, and the antibody-drug conjugate provided by the present application is particularly suitable for the treatment of gastric cancer and pancreatic cancer.

[0114] Therefore, the present application also provides a number of inventions related to the above-mentioned therapeutic uses.

[0115] In one aspect, the present application provides the use of the above-described antibody-conjugate in the preparation of a drug for treating or preventing cancer.

[0116] In another aspect, the present application provides a method for treating cancer in an individual, comprising administering a therapeutically effective amount of the antibody-conjugate described above or a pharmaceutical preparation or composition comprising said conjugate to an individual suffering from said cancer.

[0117] In another aspect, the present application provides the use of the antibody-conjugate described above and an anti-proliferative agent in the preparation of a medicament for treating a tumor (such as the cancer described above).

[0118] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-conjugate described above and an anti-proliferative agent.

[0119] In another aspect, the present application provides a method for treating a tumor in an individual, comprising administering a therapeutically effective amount of the antibody-conjugate described above or a pharmaceutical preparation or composition comprising said antibody-conjugate and an anti-proliferative agent to an individual suffering from said tumor.

[0120] In specific embodiments, the anti-proliferative agent includes, but is not limited to, paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin, iproplatin. In certain specific embodiments, the anti-proliferative agent may also be other antibodies, antibody-drug conjugates or fusion proteins. Examples

[0121] The following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

[0122] Experimental equipment and materials

[0123]

[0124]

[0125] Cell lines used in the experiments

[0126]

[0127] Example 1: Preparation of a Fully Human Anti-CLDN18.2 Monoclonal Antibody

[0128] The anti-CLDN18.2 antibody used in the present application was 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 detecting the titer of anti-CLDN18.2 in the mouse plasma. Mice with sufficient anti-CLDN18.2 titer in the plasma were used for hybridoma fusion. Before finally harvesting the spleens and lymph nodes of the mice, immune boosting was also carried out by injecting the immunogen intraperitoneally, into the footpad or into the tail vein of the mouse.

[0129] Screen the sera of immunized mice by fluorescence-activated cell sorting (FACS) to select mice that produce antibodies binding to CLDN18.2. First, incubate cell lines expressing CLDN18.2 (CHO or 3T3) with serially diluted sera from immunized mice, and then detect specific antibody binding with PE-fluorescently labeled anti-mouse IgG Ab on a fluorescence-activated cell sorter (iQue plus, Sartorius). In addition, the mouse sera are also confirmed by imaging tests. CHO (or 3T3 cells) expressing CLDN18.2 are incubated with diluted sera from immunized mice, the cells are washed, fixed with formaldehyde, washed again, and then specific antibody binding is detected with Alexa488-fluorescently labeled goat anti-mouse antibody, and scanned and analyzed on a cell imager (Cytation 5, Biotek). After confirming the mice that produce antibodies binding to CLDN18.2, spleens and lymph nodes are taken from the immunized mice, lymphocytes are isolated, and by electrofusion, fused to mouse myeloma cells Sp2 / 0 (ATCC, CRL 1581), and the resulting hybridomas are used to screen for anti-CLDN18.2 specific antibodies. The cells are plated in flat-bottom 96-well tissue culture plates and then incubated in selection medium (HAT medium) for 2 weeks, and then switched to hybridoma medium for culture. Approximately 10 - 14 days after cell plating, anti-CLDN18.2 specific binding imaging screening is performed on the hybridoma supernatants from individual wells by the aforementioned cell imaging method. The relevant hybridomas are screened from the hybridomas generated from an immunized group of three mice. The hybridoma cells generated by electrofusion after immunizing mice are plated in flat-bottom 96-well tissue culture plates, and each well contains one or several hybridoma cells. The hybridoma supernatants from individual wells are detected by the cell imaging method, and the clone supernatants secreting positive antibodies specifically bind to CHO cells transfected with CLDN18.2, but do not bind to CHO cells transfected with CLDN18.1 and CHO cells. The hybridomas secreting positive antibodies are transferred to 24-well plates and screened and confirmed again. The confirmed positive antibody hybridomas are sorted into monoclonal by using a single-cell sorter. 96 subclones are sorted from each positive hybridoma and screened and confirmed again. The positive subclones generating preliminary candidate molecules are amplified and cultured in vitro for sequencing, and a small amount of antibodies produced by the positive subclones are used for purification and antibody characterization verification. The obtained antibody SYJS001 has the following related sequences:

[0130] Table 1

[0131]

[0132] SEQ ID NO:7 (heavy chain variable region)

[0133]

[0134] SEQ ID NO:8 (Light chain variable region)

[0135]

[0136] SEQ ID NO:9 (Heavy chain)

[0137]

[0138]

[0139] SEQ ID NO:10 (Light chain)

[0140]

[0141] Example 2: Vector Construction and Antibody Expression

[0142] 2.1 Design of the Vector

[0143] The plasmid vector pGenHT1.0-DGV used for the expression of SYJS001 monoclonal antibody was provided by Nanjing Genscript Biotech Co., Ltd. (referred to as "Genscript"). The vector map is shown in Figure 3 , and the information of each key element is shown in Table 2:

[0144] Table 2: Information of key elements of pGenHT1.0-DGV vector

[0145]

[0146] 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.

[0147] Design and synthesis of the heavy chain:

[0148] The artificially synthesized heavy chain was named SYJS001-HC. An NruI restriction endonuclease site was introduced at the 5' end, a PmLI restriction endonuclease site was introduced at the 3' end. Meanwhile, a Kozak sequence and a signal peptide sequence (19 amino acids): MGWSCIILFLVATATGVHS (SEQ ID NO:19) were introduced behind the NruI restriction endonuclease site at the 5' end. The expression cassette of the heavy chain was designed as:

[0149] NruI-Kozak sequence-signal peptide-SYJS001-HC-stop codon-PmLI

[0150] Design and synthesis of the light chain:

[0151] The synthetic light chain is named SYJS001-LC. During the synthesis process, an AscI restriction enzyme site is introduced at the 5' end of the light chain, and an FseI restriction enzyme site is introduced at the 3' end. Meanwhile, a Kozak sequence and a signal peptide sequence (19 amino acids): MGWSCIILFLVATATGVHS (SEQ ID NO:19) are introduced after the AscI restriction enzyme site at the 5' end. The expression cassette of the light chain is designed as:

[0152] AscI-Kozak sequence-signal peptide-SYJS001-LC-stop codon-FseI

[0153] 2.2 Construction of the Recombinant Vector

[0154] The insertion site of SYJS001-HC is the downstream multiple cloning NruI / PmLI site of the pGenHT1.0-DGV vector, and the insertion site of SYJS001-LC is the upstream multiple cloning AscI / FseI site. The promoter of both multiple cloning sites is CMV, and the heavy chain and light chain are constructed on the same empty vector. The PCR amplification product SYJS001-HC and the plasmid vector pGenHT1.0-DGV are digested with NruI / PmLI and ligated and transformed. Positive clones are screened by the Kan+ resistance marker to obtain a correctly constructed recombinant heavy chain expression vector, named: SYJS001-HC in pGenHT1.0-DGV. Next, SYJS001-LC and SYJS001-HC in pGenHT1.0-DGV are digested with AscI / FseI, ligated and transformed, and cloned and screened. The positive clones are digested with AscI / PmLI for identification (see Figure 4 ) and sequenced to obtain a correctly constructed recombinant heavy and light chain expression vector, named: SYJS001 in pGenHT1.0-DGV. The structural schematic diagram is shown in Figure 5 . The SYJS001 in pGenHT1.0-DGV is digested and identified, and the size of the target region sequence is 4527bp. The obtained recombinant plasmid is electrotransformed into the host cell CHO K1 to obtain a stable cell line highly expressing the SYJS001 antibody protein.

[0155] 2.3 Expression and purification of antibodies

[0156] The stable cell line highly expressing SYJS001 antibody was cultured in a shaker flask with serum-free CD FortICHO. After a certain period of time, the culture supernatant was collected. A HiTrap MabSelect SuRe 1 ml column (product of GE Healthcare Life Sciences, catalog number: 11-0034-93) was equilibrated with PBS solution at pH = 7.4 for 10 column volumes at a flow rate of 0.5 ml / min; the culture supernatant was filtered through a 0.45 μm filter membrane and loaded onto the column at a flow rate of 0.5 ml / min. It was then washed with PBS solution at pH 7.4 for another 5 - 10 column volumes at a flow rate of 0.5 ml / min; eluted with 100 mM citrate buffer (pH 3.6) at a flow rate of 0.5 ml / min, and the elution peak was collected to obtain SYJS001 antibody with a purity > 95%.

[0157] Example 3: Preparation of SYJS001 ADC

[0158] Certain volumes of L&D (whose structure is as Figure 2 shown), reaction buffer, SYJS001 antibody, mTGase (transglutaminase), and H2O were transferred into an elastic ethylene-vinyl acetate disposable reaction bag in an appropriate order using a peristaltic pump. After the reaction bag was sealed and mixed evenly, it was placed at 30 °C for 24 - 144 h. Samples were taken every 24 h during the reaction process for analysis and detection of the coupling rate by C4-HPLC. When the coupling rate >= 95%, the reaction was terminated and purified immediately.

[0159] The amino acid sequence of the actually used transglutaminase is as follows:

[0160]

[0161] Example 4: Physicochemical Property Analysis and Identification of SYJS001 ADC

[0162] 1. Identification of the modification rate of SYJS001 ADC

[0163] Experimental steps:

[0164] 1) Loading: Take 10 μl of the supernatant liquid of the reduced ADC reaction solution sample and load it onto the chromatographic column (waters xbridge C4, 3.5 μm, 4.6 mm * 250 mm)

[0165] 2) Elution: Mobile phase A is 0.1% aqueous TFA solution, and mobile phase B is 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 for elution at 0, 5, 8, 15, 20, 22, 25, 30 min respectively. Control the flow rate at 0.8 ml / min and the detection wavelength at 280 nm.

[0166] The experimental results are as Figure 6 shown, showing that the modification rate of SYJS001 ADC is 95.11%.

[0167] 2. Detection of the DAR value of SYJS001 ADC

[0168] Experimental steps:

[0169] 1. Loading: Take 10 μg of SYJS001 ADC sample and load it onto the chromatographic column (Agilient PLRP-S, 5 μm, 2.1 mm * 50 mm)

[0170] 3. Elution: Mobile phase A is 0.1% TFA aqueous solution, and mobile phase B is 0.1% acetonitrile solution. Adjust the ratio of mobile phase A: B to 7.3:2.1, 6.5:3.5, 5.7:4.3, 0.5:9.5, 7.3:2.7 at the 0th, 8th, 25th, 26th, and 31.5th minutes respectively for elution. Control the flow rate at 0.25 ml / min and the detection wavelength at 280 nm; The results are as Figure 7 shown, and the average DAR is 2.

[0171] Example 5: Flow Cytometry Detection of the Binding Ability of SYJS001 ADC to CLDN18.2 Protein

[0172] In this experiment, flow cytometry was used to detect the binding ability of SYJS001 ADC to CLDN18.2 proteins of different species.

[0173] After incubating overexpressing cell lines (HEK293 - human CLDN18.2, HEK293 mouse CLDN18.2, cynomolgus monkey GLDN18.2, CHO - K1) with SYJS001 ADC samples at different concentrations, incubate with a secondary antibody conjugated to IgG (goat anti - human IgG (H + L) cross - adsorbed secondary antibody), and analyze the binding ability of the sample to CLDN18.2 of different species by detecting the fluorescence signal values at different concentrations through a flow cytometer.

[0174] Specific experimental steps:

[0175] The initial concentration of the protein sample SYJS001 ADC is 45 μg / ml, and it is diluted in a 3 - fold gradient for a total of 11 gradients. Take 100 μl of antibodies at different concentrations and incubate them with 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, and the cells were washed to remove unbound samples. Goat anti-human IgG (H+L) cross-adsorbed secondary antibody (diluted 1:1000) 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 the four-parameter equation was selected to make an "S"-shaped curve, and the software automatically generated the median effective dose ED 50 (C value); the experimental results are as Figure 8 shown in

[0176] Table 3, indicating that SYJS001 ADC has good affinity for human, mouse, and cynomolgus monkey CLDN18.2. 50 value

[0177] Sample Name <![CDATA[ED 50 (ng / ml)]]> HEK293 - Human CLDN18.2 439.1 HEK293 - Mouse CLDN18.2 717.0 Cynomolgus Monkey CLDN18.2 CHO-K1 647.9

[0178] Example 6: Specific Binding of SYJS001 ADC to CLDN18.2

[0179] 6.1 Specificity and cross-reactivity of SYJS001 monoclonal antibody with CLDN18 family members

[0180] CHOK1-CLDN18.2 and HEK293-CLDN18.1 cells were cultured in the corresponding complete medium and passaged every 2 - 3 days; when the cell confluence reached 90%, the cell density was adjusted to 2 - 3×10 6 cells / mL using a multi-channel pipette, and the cell suspension was added to a flow cytometry 96-well detection plate, 100 μL per well; centrifuged at 2500 rpm for 5 min, and the supernatant was discarded; washed twice with 2% FBS / PBS; SYJS001 monoclonal antibody was diluted with 2% FBS / PBS buffer, the starting concentration of the naked antibody was 6 μg / ml, diluted in 3-fold gradients, a total of 8 gradients, and 2 replicates were set for each drug concentration, and corresponding blank controls were set. 100 μL per well, incubated at 4 °C for 2 h; washed three times with 2% FBS / PBS, added 488-labeled goat anti-human IgG (diluted 1:5000), incubated at 4 °C for 1 h; washed three times with 2% FBS / PBS, and then the corresponding fluorescence channel was selected for reading.

[0181] 6.2 Specific binding of SYJS001 ADC to CLDN18.2

[0182] HEK293-CLDN18.2 cells were cultured in the corresponding complete medium and passaged every 2 - 3 days; when the cell confluence reached 90%, the cell density was adjusted to 2 - 3×10 6Cells / mL. Use a multi-channel pipette to add the cell suspension to a flow cytometry 96-well detection plate, 100 μL per well; centrifuge at 2500 rpm for 5 min, discard the supernatant; wash twice with 2% FBS / PBS; dilute SYJS001 monoclonal antibody and SYJS001 ADC with 2% FBS / PBS buffer respectively. The initial concentration of ADC is 15 μg / ml, and it is diluted in a 3-fold gradient for a total of 11 gradients. Set 2 replicates for each drug concentration and set corresponding blank controls. Add 100 μL per well and incubate at 4°C for 2 h; wash three times with 2% FBS / PBS, add 488-labeled goat anti-human IgG (diluted 1:1000), and incubate at 4°C for 1 h; wash three times with 2% FBS / PBS, and then select the corresponding fluorescence channel for reading.

[0183] The results show (see Figure 9 and Figure 10 ), the SYJS001 monoclonal antibody obtained in this application can specifically bind to CLDN18.2 and has no obvious cross-reaction with CLDN18.1; compared with the naked antibody, SYJS001 ADC also has specific binding to CLDN18.2, and the affinity is not significantly affected after coupling with the toxin.

[0184] Example 7: Verification Experiment of the Endocytosis of SYJS001 ADC

[0185] Experimental procedure:

[0186] Collect HEK293-CLDN18.2 cells and resuspend the cells with DMEM complete medium; gently pipette the resuspended target cells several times to obtain a single-cell suspension, and use the trypan blue staining method to identify cell viability and cell count; adjust the cell density to 1×10 5 cells / ml; inoculate 100 μl per well into a confocal 96-well plate cell culture dish, and the number of cells inoculated per well is 1×10 4 , add SYJS001 labeled with Zenon TM pHrodo TM iFL to the 96-well plate, with a final concentration of 2 μg / ml, and then continuously culture in an incubator at 37°C and 5% CO2 for 24 hours. All images were observed and captured with a 20X objective lens of a laser confocal microscope.

[0187] The experimental results are as shown in Figure 11 , and the results show that SYJS001 ADC was internalized in HEK293-CLDN18.2 cells and localized in lysosomes in an acidic environment (Zenon TM pHrdo TMThe iFL IgG Labeling Reagents (Z25611) show fluorescence only in the acidic environment of lysosomes. For example: Figure 11 In the rightmost HEK293-CLDN18.2 figure, the parts circled by the three boxes show green fluorescent dots under the microscope), while endocytosis does not occur in the HEK293 and HEK293-CLDN18.1 cell figures, and no green fluorescent dots are observed.

[0188] Example 8: Inhibitory Effects of SYJS001 Naked Antibody and ADC on the Growth of Different Cells

[0189] Experimental procedure: Collect target cells and resuspend them as a single-cell suspension. Use trypan blue staining to identify cell viability and cell count; adjust the cell density to 1×10 5 cells / ml; add 100 μl per well to a 96-well black flat-bottom cell culture plate; add 20 μl of the diluted test article per well to the 96-well black flat-bottom cell culture plate already seeded with cells; incubate in a cell culture incubator (37 °C, 5% CO2) for 66 ± 3 hr; add resazurin sodium solution (w / v 0.03%), 20 μl per well; incubate at 37 °C for 3 - 4 h, read the fluorescence value with an ELISA reader at 550 nm / 610 nm, use Magellan6 or similar graphing software to plot the graph, and fit the half-inhibitory concentration IC 50 of the reference standard and the sample. The output parameter C is IC 50 , with the unit of ng / mL.

[0190] The experimental results are as Figures 12 - 17 and shown in Table 4, indicating that SYJS001 ADC can inhibit the growth of cancer cells 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) in vitro.

[0191] Table 4: In vitro proliferation inhibitory effects of SYJS001 naked antibody and SYJS001 ADC on different cells (Note: N / A = no inhibitory effect)

[0192]

[0193] It can be seen that SYJS001 ADC has significant inhibitory effects on gastric cancer cells, lung cancer cells, and human pancreatic cancer cells overexpressing CLDN18.2, and has weak inhibitory effects on human gastric cancer cells KATOⅢ and human pancreatic cancer cells PATU8988S that do not express or express low levels of CLDN18.2.

[0194] Example 9: In Vivo Efficacy Evaluation of SYJS001 ADC

[0195] 9.1 Pharmacodynamic comparison data with gemcitabine and cisplatin as controls

[0196] 1) In this experiment, a nude mouse xenograft model of human pancreatic cancer Bxpc3-18.2 was selected. When the tumor volume grew to about 100 mm 3 (on the 39th day after inoculation), 48 animals with good tumor growth were selected. The animals were evenly divided into 6 groups according to tumor volume (on the 0th day), with 8 animals in each group. They were intravenously administered 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), SYJS001-ADC 2, 4, and 8 mg / kg (single-dose), SYJS001-mAb (SYJS001 naked antibody) 8 mg / kg (single-dose), and gemcitabine (GEM) 50 mg / kg (biw×4, twice a week for 4 weeks). The tumor diameter was measured twice a week, and the body weight of the mice was weighed. The data were recorded. By measuring the tumor diameter at different times after administration, the growth of the tumor was dynamically observed. The experiment ended on the 28th day. After asphyxiating the mice with carbon dioxide, the tumors were removed and weighed.

[0197] The results showed that in this experiment, the tumor weight inhibition rates of the SYJS001-ADC 2, 4, and 8 mg / kg (single-dose) groups, the SYJS001-mAb 8 mg / kg (single-dose) group, and the Gemcitabine 50 mg / kg (biw×4) group were 56.6%, 94.8%, 97.8%, -36.2%, and 51.0% respectively. Compared with the solvent control group (0.9% sodium chloride injection), both the SYJS001-ADC 2, 4, and 8 mg / kg (single-dose) groups and the GEM 50 mg / kg (biw×4: administered twice a week for a total of four times) group could significantly inhibit the growth of the tumor (P < 0.01). Compared with the positive control GEM 50 mg / kg (biw×4) group, the inhibitory effects of the SYJS001-ADC 4 and 8 mg / kg (single-dose) groups on the tumor were more significant, especially the 8 mg / kg group, and the inhibitory effect was nearly twice as much (p < 0.001) (see Figure 18 ).

[0198] 2) In this experiment, a nude mouse xenograft model of human gastric cancer NUGC-4-18.2 was selected. When the tumor volume reached about 120 mm 3At (the 6th day after inoculation), 64 animals with good tumor growth were selected. The animals were evenly divided into 8 groups according to tumor volume (at the 0th day), with 8 animals in each group. They were intravenously administered 0.9% sodium chloride injection (0.9% INJ NS, solvent control group); SYJS001-ADC at 1, 2, 4 mg / kg (qw×3); 4, 8 mg / kg (single dose); SYJS001-mAb at 4 mg / kg (qw×3); cisplatin at 6 mg / kg (qw×3, once a week for 3 weeks). The tumor diameters were measured 2 times a week, and the body weights of the mice were weighed. The data were recorded. By measuring the tumor diameters at different times after administration, the growth changes of the tumors were dynamically observed. The experiment ended on the 20th day. After the animals were asphyxiated with carbon dioxide, the tumors were dissected and weighed.

[0199] The results showed that in this experiment, the tumor weight inhibition rates of SYJS001-ADC at 1, 2, 4 mg / kg (qw×3); 4, 8 mg / kg (single dose); 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%; 66.5% respectively. Compared with the solvent control group: all groups except the SYJS001-mAb 4 mg / kg (qw×3) group could significantly inhibit the growth of tumors (P<0.001); the SYJS001-ADC at 1, 2, 4 mg / kg (qw×3); 4, 8 mg / kg (single dose) groups were significantly superior to the positive control group cisplatin at 6 mg / kg (qw×3) in terms of efficacy and toxicity (P<0.05) (see Figure 19 ).

[0200] 9.2 Comparative efficacy data with IMAB362-ADC as the control

[0201] IMAB362 (also known as claudiximab, zolbetuximab) is a reported chimeric monoclonal antibody of the IgG1 subtype. It selectively targets the first extracellular domain of CLDN18.2 and has little activity against CLDN18.1. It was selected as the control antibody in this experiment.

[0202] 1) In this experiment, a human pancreatic cancer Bxpc3-18.2 nude mouse xenograft tumor model was used. When the tumor volume grew to about 100 mm 3At 39 days after inoculation, 64 animals with good tumor growth were selected. The animals were evenly divided into 8 groups according to tumor volume on day 0, with 8 animals in each group. They were intravenously administered 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), SYJS001-ADC at 2, 4, and 8 mg / kg (single-dose), IMAB362-ADC (for the IMAB362 sequence, refer to patent CN201680021997.6. Gene synthesis was completed by Nanjing Genscript Biotech Co., Ltd., and protein expression was transiently expressed using the KOP293 transient transfection protein expression system of Zhuhai Kairui Biotech Co., Ltd. For specific steps, refer to its official website. The preparation of IMAB362-ADC refers to the method for preparing SYJS001 ADC in Example 3) at 2, 4, and 8 mg / kg (single-dose), and SYJS001-mAb at 8 mg / kg (single-dose). The tumor diameter was measured twice a week, and the body weight of the mice was weighed. Data were recorded. By measuring the tumor diameter at different times after administration, the growth of the tumor was dynamically observed. The experiment ended on day 28. After the mice were asphyxiated with carbon dioxide, the tumors were dissected and weighed.

[0203] The results showed that in this experiment, the tumor volumes of the SYJS001-ADC 2, 4, and 8 mg / kg (single-dose) groups, the SYJS001-mAb 8 mg / kg (single-dose) group, and the IMAB362-ADC 2, 4, and 8 mg / kg (single-dose) groups were 35.9%, 6.0%, 3.4%, 116.2%, 47.0%, 6.8%, and 5.1% of the solvent control group, respectively. Compared with the solvent control group, both the SYJS001-ADC 2, 4, and 8 mg / kg (single-dose) groups and the IMAB362-ADC 2, 4, and 8 mg / kg (single-dose) groups could significantly inhibit tumor growth (p < 0.01). Compared with the positive control IMAB362-ADC group, the effects were not much different at high doses, but at low doses (effective doses), the in vivo tumor inhibitory effect of SYJS001 ADC was significantly better than that of IMAB362-ADC. For details, refer to Figure 20 (RTV (relative tumor volume), relative tumor volume).

[0204] 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 administration dose range of ADC based on VC-MMAE is generally 0.1 - 3.2 mg. Therefore, the low dose in this experiment is a dose with more promising clinical applications, and the clinical prospect and drug-likeness of SYJS001-ADC are superior to those of IMAB362-ADC.

[0205] 2) In this experiment, a nude mouse xenograft model was established 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. The animals were evenly divided into 8 groups according to tumor volume (on the 0th day), with 8 animals in each group. They were intravenously administered 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); SYJS001-mAb at 4 mg / kg (single dose); The tumor diameter was measured 2 times a week, and the body weight of the mice was weighed, and the data were recorded. By measuring the tumor diameter at different times after administration, the growth changes of the tumor were dynamically observed. The experiment ended on the 20th day. After the animals were asphyxiated with carbon dioxide, the tumors were dissected and weighed.

[0206] The results showed that in this experiment, the tumor volumes of SYJS001-ADC at 0.5, 1, 2, 4 mg / kg (single dose); IMAB362-ADC at 0.5, 1, 2, 4 mg / kg (single dose); SYJS001-mAb at 4 mg / kg (single dose) were 59.1%, 27.3%, 1.8%, 0%, 68.2%, 30%, 1.8%, 0%, 83.6% of the solvent control group, respectively. Compared with the solvent control group: all groups except the SYJS001-mAb 4 mg / kg group could significantly inhibit tumor growth (p < 0.001); The in vivo antitumor effect of SYJS001 ADC on NUGC4-CLDN18.2 was not much different from that of IMAB362-ADC at high doses, but at low doses (effective doses), the in vivo antitumor effect of SYJS001 ADC was significantly better than that of IMAB362-ADC (see Figure 21 ).

[0207] Similar to the previous comparative experiment, the low dose in this experiment is a dose with more promising clinical applications. The clinical prospect and drug-likeness of SYJS001-ADC are significantly better than those of IMAB362-ADC.

[0208] Example 10: MMAE Release and Bystander Effect Experiment

[0209] After the positive group of BxPC-3-CLDN18.2 and HEK293-Luc cells (cells that do not express CLDN18.2) were co-cultured with SYJS001 ADC for 4 days, chemiluminescence color development was used to indicate the number of HEK293-Luc in the mixed cells. Compared with the negative group of co-cultured BxPC-3 and HEK293-Luc, 3 concentration points including 5 μg / ml, 1 μg / ml, and 200 ng / ml could all cause proliferation inhibition of HEK293-Luc cells to varying degrees; this indicates that the positive group of BxPC-3-Human CLDN18.2 can bind to SYJS001 ADC, and then enter the cells through endocytosis. The MMAE released in the cells can cause apoptosis of BxPC-3-human CLDN18.2, and the MMAE released after cell apoptosis lysis can cause growth inhibition of bystander cells. For the specific results, see Figure 22 . Thus, it can be seen that MMAE can be efficiently released from SYJS001 ADC and exert cytotoxic activity on neighboring cells due to its membrane permeability, producing a bystander effect.

[0210] Based on the results of the above multiple examples, it can be known that the monoclonal antibody against CLDN18.2 obtained in this application can specifically bind to CLDN18.2 on CLDN18.2-positive cells and be efficiently internalized, and the ADC drug obtained in this application has extremely strong killing effects on tumor cells represented by pancreatic cancer, gastric cancer, and lung cancer.

[0211] Example 11: ADC Stability Study

[0212] 11.1 Plasma and Serum Stability

[0213] The purpose of this experimental study is the in vitro metabolic stability of SYJS001-ADC in plasma and serum of different species (human, cynomolgus monkey, and SD rat). The incubation concentration of SYJS001-ADC is 100 μg / mL, and it is incubated at 37°C under sterile conditions for 0 - 168 h

[0214] (7 days). The concentration of MMAE was detected by LC-MS / MS method. The concentration results of MMAE in 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma and SD rat plasma are shown in Table 5 - Table 8. With the increase of incubation time, free MMAE was generated in all matrices; After incubation at 37 °C for 168 h (7 days), the percentage of dissociation rate of SYJS001-ADC in 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma and SD rat plasma was 0.672%, 0.327%, 0.209% and 0.405% respectively. The experiment showed that the dissociation rate of MMAE on SYJS001-ADC was less than 1.0%, indicating that SYJS001-ADC was relatively stable in 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma and SD rat plasma.

[0215] Table 5. Free concentration of MMAE in 0.5% BSA-PBS solution and dissociation rate on ADC

[0216]

[0217] Table 6. Free concentration of MMAE in human plasma and dissociation rate on ADC

[0218]

[0219] Table 7. Free concentration of MMAE in monkey plasma and dissociation rate on ADC

[0220]

[0221]

[0222] Table 8. Free concentration of MMAE in rat plasma and dissociation rate on ADC

[0223]

[0224] 11.2 Accelerated stability

[0225] SYJS001-ADC was stored in buffer solution (its components include: 5% w / v L-histidine, 25% L-histidine hydrochloride) at 2 °C to 8 °C for different times, and its stability was detected. The results are shown in the following table. IMAB362-ADC

[0226] (IMAB362-vcMMAE) reached 0.3% of free toxin in 28 days (see Table 7 of CN 107667118A), which was much higher than 0.000026% of the drug SYJS001-ADC in this application for 3 months, indicating that the stability of SYJS001-ADC was much better than that of IMAB362-ADC.

[0227] Table 9 Experimental results of SYJS001 - ADC accelerated stability (6℃ ± 2℃)

[0228]

[0229] Example 12: Comparative study of SYJS001 and IMAB362

[0230] 12.1 Affinity

[0231] Resuspend the CLDN18.2 - overexpressing cell line BxPC3 - CLDN18.2 with PBS buffer, and adjust the cells to 1×10 6 cells / ml. Add 100 μl per well to a 96 - well V - bottom culture plate; centrifuge at 2500 rpm for 5 min and discard the supernatant; dilute the samples with the experimental buffer. The initial concentration of the antibody is 40 μg / ml, and perform 5 - fold serial dilutions for a total of 10 gradients; add 100 μl of the diluted samples per well to the 96 - well V - bottom culture plate and incubate at 4℃ for 60 - 90 minutes; after incubation, place the V - bottom culture plate in a centrifuge and centrifuge at 2500 rpm for 5 min; carefully aspirate the centrifuged supernatant, add 100 μl of PBS to resuspend the cells, and centrifuge at 2500 rpm for 5 min; repeat the operation twice; carefully aspirate the centrifuged supernatant, add the fluorescent antibody detection reagent (diluted 1:1000) according to the experimental layout, and incubate at 4℃ in the dark for 30 - 60 minutes; after incubation, place the V - bottom culture plate in a high - speed centrifuge and centrifuge at 2500 rpm for 5 min; carefully aspirate the centrifuged supernatant, add 100 μl of the experimental buffer to resuspend the cells, and then detect using a flow cytometer. Read the fluorescence signal values of the corresponding wells in the experimental plate using a plate reader; export the experimental data and analyze the data using GraphPad Prism 5 software; select the regression model of the four - parameter equation to make an "S" - shaped curve, and the software automatically generates the half - maximal effective concentration ED50 (C value). The results are shown in the following figure ( Figure 23 , MFI: mean fluorescence intensity), and the affinity of SYJS001 is more than 2 times higher than that of IMAB362.

[0232] 12.2 Epitope analysis

[0233] Use Octet epitope pairing to detect the epitope competition between SYJS001-ADC and IMAB362-ADC. Immobilize 5 μg / ml human CLDN18.2 protein on the HIS1K capture sensor for 180 s of loading. Dilute the two antibodies to 100 nM respectively. First, bind the immobilized sensor to one of the antibodies for 180 s of association, and then bind it to the second antibody for 180 s of association. Detect the binding signal of the second antibody to determine whether the two antibodies recognize the same epitope. Results: 60%-100%: complete non-competition; 20%-60%: partial competition; <20%: complete competition, then the two antibodies are considered to have competition. The results are shown in the following table (Table 10), and SYJS001-ADC and IMAB362-ADC are completely competitive in epitopes.

[0234] Table 10

[0235] SYJS001 IMAB362 SYJS001 9.02％ 7.98％ IMAB362 7.88％ 6.6％

[0236] 12.3 Endocytosis detection

[0237] Seed BxPC3-CLDN18.2 cells in a 6-well plate, with approximately 4-6×10 5 cells per well; transfer to an incubator and culture overnight at 37°C with 5% CO2; dilute the antibody to 5 μg / ml with complete medium (DMEM (Hyclone, catalog number: SH30243.01) + 10% FBS (Gibco, catalog number: 10091-148) + 1 μg / ml puromycin (Gibco, catalog number: A11138-02)), discard the original medium in the 6-well plate, and add 2 ml of complete medium containing the antibody to each well; place at 4°C for 2 hr, then discard the supernatant, wash twice with complete medium, and add 2 ml of complete medium without the antibody to each well; at this time, take 2-3 duplicate wells for trypsin digestion, and this time point is used as the total amount of antibody bound to the cells; transfer the remaining cells to an incubator and culture at 37°C with 5% CO2 for 4 hr, 21 hr, 25 hr, and 48 hr for endocytosis; after washing the cells digested with trypsin at each time point, add a fluorescent antibody detection reagent (diluted 1:1000), and incubate at 4°C in the dark for 60 minutes; carefully discard the centrifuged supernatant, wash twice, and detect the fluorescence signal value using a flow cytometer;

[0238] Endocytosis rate calculation formula: (total antibody fluorescence signal - fluorescence signal at different time points) / total antibody fluorescence signal × 100%. The results are shown in the following figure ( Figure 24, MFI (Mean Fluorescence Intensity), the endocytosis rate of SYJS001 was significantly higher than that of IMAB362-ADC in the initial stage (0 - 21 hr), and was basically the same as that of IMAB362-ADC from 21 - 48 hr. Under the same other conditions, a faster endocytosis rate means a stronger ability of the drug to enter tumor cells, which can better release toxic molecules and objectively play a role in taking effect faster. Therefore, it is expected that SYJS001 will take effect faster than IMAB362-ADC.

[0239] 12.4 In vitro cell growth inhibition experiment

[0240] 1. Adenocarcinoma cell model

[0241] Collect BxPC-3-CLDN18.2 cells and resuspend the cells using complete medium (the same complete medium used in the endocytosis detection part of 12.3); gently pipette the resuspended target cells several times to obtain a single-cell suspension, and use the trypan blue staining method to identify cell viability and cell count; adjust the cell density to 1×10 5 cells / ml; add 100 μl per well to a 96-well black flat-bottom cell culture plate; the initial concentration of the antibody is 5 μg / ml, and it is diluted in a 2-fold gradient, with a total of 11 gradients; add 20 μl of the diluted test article per well to the 96-well black flat-bottom cell culture plate already seeded with cells; co-culture the antibody and cells, and incubate in a cell culture incubator (37 °C, 5% CO2) for 63 - 69 hr; after the incubation is completed, add resazurin sodium solution (w / v 0.03%), 20 μl per well; act at 37 °C for 3 - 4 h, read the fluorescence value with an ELISA reader at 550 nm / 610 nm, use Magellan6 or similar graphing software to plot the graph, and fit out the half-inhibitory concentration IC 50 of the reference standard and the sample. The output parameter C is IC 50 , with the unit of ng / mL. The results are shown in the following figure ( Figure 25 , RLU: Relative Light Unit), and the in vitro inhibitory effect of SYJS001-ADC on BxPC-3-CLDN18.2 cells is significantly better than that of IMAB362-ADC.

[0242] 2. Gastric cancer cell model

[0243] Collect NUGC4-CLDN18.2 cells and resuspend the cells using complete medium (the same complete medium used in the endocytosis detection part of 12.3); gently pipette the resuspended target cells several times to obtain a single-cell suspension, and use the trypan blue staining method to identify cell viability and cell count; adjust the cell density to 1×10 5cells / ml; Add 100 μl per well to a 96-well black flat-bottom cell culture plate; The initial concentration of the antibody is 10 mg / ml, and it is diluted in a 5-fold gradient for a total of 11 gradients; Add 20 μl of the diluted test sample per well to the 96-well black flat-bottom cell culture plate seeded with cells; Co-culture the antibody and cells, and incubate in a cell culture incubator (37 °C, 5% CO2) for 63 - 69 hr; After the incubation is completed, add resazurin sodium solution (w / v 0.03%), 20 μl per well; Act at 37 °C for 3 - 4 h, read the fluorescence value with an ELISA reader at 550 nm / 610 nm, use Magellan6 or similar graphing software to plot the graph, and fit out the half-inhibitory concentration IC 50 . The output parameter C is IC 50 , with the unit of ng / mL. The results are shown in the following figure ( Figure 26 , RLU: relative light unit), indicating that the in vitro inhibitory effect of SYJS001-ADC on NUGC4-CLDN18.2 cells is better than that of IMAB362-ADC, exceeding it by more than 2 times.

[0244] Based on the above experiments, SYJS001-ADC obtained in this application is significantly superior to the control IMAB362-ADC in terms of stability, related antibody affinity, endocytosis efficiency, and in vitro and in vivo tumor cell inhibition.

[0245] The above description only presents preferred implementation schemes, which are only examples and do not limit the combination of essential features required to implement this application. The provided titles are not intended to limit the various implementation schemes of this application. Terms such as "comprising", "containing", and "including" are not intended to be limiting. In addition, unless otherwise stated, when not modified by a numeral, it includes the plural form, and "or" or "either...or..." means "and / or". Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the methods and compositions described in this application are obvious to those skilled in the art without departing from the scope and spirit of this application. Although this application is described through specific preferred implementation schemes, it should be understood that the claimed application should not be unduly limited to these specific implementation schemes. In fact, various variations of the described modes for implementing this application that are obvious to those skilled in the relevant art are intended to be included within the scope of the appended claims.

Claims

1. A conjugate comprising an antibody or antigen-binding fragment thereof coupled to one or more drug molecules, wherein the antibody comprises a heavy chain and a light chain, wherein the amino acid sequences of the three CDR regions of the heavy chain are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the three CDR regions of the light chain are shown in SEQ ID NOs: 4, 5, and 6, respectively, wherein the drug molecule is MMAE (Monomethyl auristatin E), and the drug molecule is coupled to the antibody or antigen-binding fragment thereof via a linker, wherein the linker is connected to the antibody or antigen-binding fragment thereof via an amino group, and the linker is NH2-(CH2-CH2-O) m -CH2-C(=O)-Val-Cit-pABC, wherein m is an integer from 1 to 8, and the average drug-antibody ratio (DAR) is 2.

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

3. The conjugate according to claim 1, wherein the antibody is a humanized antibody.

4. The conjugate according to claim 1, wherein the antibody is a fully human antibody.

5. The conjugate according to claim 1, wherein the antibody is an IgG-type antibody.

6. The conjugate according to claim 5, wherein the antibody is an IgG1-type antibody.

7. The conjugate according to claim 1, wherein the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment or a single-chain Fv fragment (scFv).

8. The conjugate according to claim 1, wherein the amino acid sequence of the heavy-chain variable region of the heavy chain is as shown in SEQ ID NO:7, and the amino acid sequence of the light-chain variable region of the light chain is as shown in SEQ ID NO:

8.

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

10.

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

11. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is for treating or preventing cancer.

13. The pharmaceutical composition according to claim 12, wherein the cancer is a CLDN18.2-positive cancer.

14. The pharmaceutical composition according to claim 12 or 13, 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.

15. The pharmaceutical composition according to claim 12 or 13, wherein the cancer is adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung or ovary.

16. The pharmaceutical composition according to claim 12 or 13, wherein the cancer is gastric cancer or pancreatic cancer.

17. Use of the conjugate according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 in the preparation of a drug for treating or preventing cancer, wherein the cancer is gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, or adenocarcinoma of the bile duct.

18. The use according to claim 17, wherein the cancer is a CLDN18.2-positive cancer.

19. The use according to claim 17 or 18, wherein the cancer is adenocarcinoma of the stomach, esophagus, pancreatic duct, lung or ovary.

20. The use according to claim 17 or 18, wherein the cancer is gastric cancer or pancreatic cancer.

21. A medical product comprising the conjugate according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11.

22. The medical product according to claim 21, which exists in the form of a kit, and the kit comprises a container containing the conjugate according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11.

23. Use of the conjugate according to any one of claims 1-10 and an anti-proliferative agent in the preparation of a medicament for treating tumors, wherein the tumors are gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, or adenocarcinoma of the bile duct.

24. The use according to claim 23, wherein the anti-proliferative agent is selected from paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin or iproplatin.

25. A pharmaceutical composition comprising the conjugate according to any one of claims 1-10 and an anti-proliferative agent.

26. The pharmaceutical composition according to claim 25, wherein the anti-proliferative agent is selected from paclitaxel, doxorubicin, docetaxel, cisplatin, carboplatin or iproplatin.

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