Antibody drug conjugates targeting claudin 18.2 and uses thereof

Abstract

An antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the antibody drug conjugate having a molecular formula of: Ab-[L-(D) r ] p wherein Ab represents an anti-Claudin18.2 antibody or an antigen binding fragment thereof, L represents a linker, D represents a toxin drug, p is 1-10; r is 1-5; the heavy chain variable region of the anti-Claudin18.2 antibody or the antigen binding fragment thereof comprises a HCDR1 as shown in SEQ ID NO: 1, a HCDR2 as shown in SEQ ID NO: 2, and a HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises a LCDR1 as shown in SEQ ID NO: 4, a LCDR2 as shown in SEQ ID NO: 5, and a LCDR3 as shown in SEQ ID NO: 6; the toxin drug has a structure as follows:

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an antibody drug conjugate targeting Claudin 18.2 and application thereof. BACKGROUND

[0002] The number of new cases of gastric cancer worldwide increased from 98.0 million in 2016 to 108.9 million in 2020, and it is estimated that the total number of new gastric cancer patients worldwide will reach 125.6 million by 2025. The incidence and mortality of pancreatic cancer rank among the top 10 of malignant tumors worldwide. In recent years, the incidence of pancreatic cancer has gradually increased. There are about 200,000 new cases of pancreatic cancer worldwide each year, accounting for 2% of all malignant tumors. At the same time, due to the high mortality rate of pancreatic cancer, it is very close to the incidence, and the annual death toll is about 196,000 people.

[0003] Chemotherapy is widely used in the treatment of advanced gastric cancer. The treatment of gastric cancer is mainly based on the expression of HER2 target points. It is divided into two categories: 1. For HER2 positive patients, the first-line treatment mainly uses trastuzumab combined with platinum or fluorouracil; for patients who fail to target therapy, the second-line treatment mainly uses chemotherapy drugs such as paclitaxel, docetaxel, irinotecan, etc.; the third-line treatment is mainly apatinib and nivolumab; 2. For HER2 negative patients, the main treatment method is chemotherapy, and the first-line treatment mainly includes platinum, taxane, fluorouracil, etc.; the second-line treatment mainly includes irinotecan, fluorouracil, capecitabine, tegafur, paclitaxel, docetaxel, albumin paclitaxel; the third-line treatment is mainly apatinib and nivolumab. However, only 20% of the population has positive expression of HER2 target points in gastric cancer, and 80% of gastric cancer patients have negative expression of HER2 target points. The current chemotherapy drugs and PD-1 targeted drugs have very limited efficacy in gastric cancer. Therefore, it is urgent to develop new targeted therapeutic drugs for new targets.

[0004] Claudin 18.2 is a four-transmembrane protein, and its expression in normal tissues is highly restricted, but it is highly specifically expressed in patients with gastric cancer, pancreatic cancer, etc. Claudin 18 has two splice variants, Claudin 18.1 and claudin 18.2, which differ by only 8 amino acids; Claudin 18.2 is involved in tumor occurrence and development, and is located on the outer cell membrane. The drug forms include monoclonal antibodies, bispecific antibodies, antibody drug conjugates (ADC), CAR-T, etc.

[0005] Currently, there are many forms of Claudin18.2-ADC, most of which are obtained by coupling antibodies of Claudin18.2 target with cleavable linker-toxins to obtain ADC. Most of the toxins are microtubule inhibitors, and a small part of the toxins are camptothecins. The currently researched ADCs developed for the Claudin18.2 target have the defect of poor stability of the linker-toxin, and the Claudin18.2-ADC products have limited efficacy.

[0006] The contents of the background section only represent the technology known to the inventors, and do not necessarily represent the prior art in the field. SUMMARY

[0007] In view of the above problems, the present application provides an antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the molecular formula of the antibody drug conjugate is: Ab-[L-(D) r ] p , wherein Ab represents an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, L represents a linker, D represents a toxin drug, p is 1-10, for example 1-9, 2-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r is 1-5, preferably 1 or 2;

[0008] The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;

[0009] The toxin drug has the following structure:

[0010]

[0011] The Claudin18.2-ADC product of the present application is obtained by coupling a new topoisomerase inhibitor toxin drug with high affinity and high endocytosis of Claudin18.2 antibody, and a new linker, using the N297 site of antibody IgG-Fc for site-specific coupling. Compared with the ADC product obtained by coupling with the opening of the disulfide bond of the antibody, the in vitro plasma stability is better. Moreover, the ADC obtained by the present application for the Claudin18.2 target has better anti-tumor effect in multiple tumor models in animals. BRIEF DESCRIPTION OF DRAWINGS

[0012] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and are meant to explain the present application and are not intended to limit the application. In the drawings:

[0013] Figure 1 is the chemical structure of an exemplary antibody drug conjugate of the present application;

[0014] Figure 2 is the endocytosis result of P1026 naked and IMA-362 in NUGC4 cells

[0015] Figure 3 is the HIC analysis result of P1026-ADC and P1026 naked;

[0016] Figure 4 is the in vitro plasma stability result of P1026-Dxd and P1026-ADC;

[0017] Figure 5 is the cell killing activity result of P1026-ADC;

[0018] Figure 6 is the efficacy result of P1026-ADC and control ADC in CDX model in animals;

[0019] Figure 7 is the efficacy result of P1026-ADC and control ADC in PDX model in animals;

[0020] Figure 8 is the chemical structure of an exemplary toxin-linker compound of the present application. DETAILED DESCRIPTION

[0021] In the following description, certain specific embodiments will be described in simple terms. As would be apparent to one of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit or scope of the application. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0022] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the term "or" means "and / or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms such as "include", "includes," and "included" is not limiting. Also, the terms "comprise", "comprising", "include", "including" and "comprises" and / or "comprising" when used in this specification, specify the presence of stated features, integers, steps, operations, objects, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, objects, and / or components. Furthermore, to the extent that any definition or usage provided herein conflicts with any definition or usage provided in a document incorporated herein by reference, the definition or usage provided herein prevails.

[0023] Definitions

[0024] To better understand the present application, definitions and explanations of relevant terms are provided as follows.

[0025] The term "about" when used in connection with a numerical value means a range of numerical values that includes a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0026] As used herein, the term "and / or" means either or both of the items it connects.

[0027] As used herein, the term "comprising" or "including," means including, but not limited to, whatever follows the term. In this application, when used in the specification and / or claims, the terms "comprise", "comprising", "include", "including" and the like are used in the sense of "including but not limited to", except when the context requires otherwise. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass an antibody variable region consisting of the particular sequence.

[0028] The term "Claudin" or "CLDN" as used herein is the most important backbone protein that determines the structure of intercellular tight junctions, which is involved in adherent junctions and plays an important role in the metastasis and invasion of tumor cells. Claudin proteins are widely present in mammalian epithelial and endothelial cells, and their distribution is mainly on the lateral cell membrane of epithelial cells and the basal cell cytoplasm. Different Claudin proteins have their own specific expression in different tissues, among which Claudin 18 (CLDN18) gene is located at 3q22.3, with a molecular weight of 24 kDa, containing 261 amino acid residues, belonging to the Claudins superfamily member, and its protein structure contains 2 extracellular loops and 4 transmembrane regions. Two subtypes of human CLDN18 or Claudin 18 protein are Claudin 18.1 or CLDN18.1 (UniProt ID: P56856-1) and Claudin 18.2 or CLDN18.2 (UniProt ID: P56856-2), and the only difference between the two protein primary structure sequences is the amino acid residues from the N-terminal signal peptide to the extracellular loop 1 (Loop 1) structure of some positions, especially in the extracellular loop 1, CLDN18.1 and CLDN18.2 are only 8 amino acids different. The inter-species sequence homology of the two subtypes of CLDN18 protein is also very high. Among them, the extracellular loop 1 of CLDN18.2 is completely consistent in different species such as human, mouse, and rhesus monkey, and the homology of human and mouse CLDN18.2 proteins reaches 84%, indicating that the sequence of CLDN18.2 protein is extremely conservative (O. Tureci. et al., Gene 481:83-92, 2011). CLDN18.2 or any variant and isoform thereof can be isolated from cells or tissues naturally expressing them or recombinantly produced using techniques well known in the art and / or those described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0029] The term "anti-CLDN18.2 antibody", "anti-CLDN18.2", "CLDN18.2 antibody" or "antibody that binds to CLDN18.2" or "antibody that specifically binds to CLDN18.2" as used herein refers to an antibody that is capable of binding to (human) CLDN18.2 with sufficient affinity so that the antibody can be used as a therapeutic agent targeting (human) CLDN18.2. In one embodiment, the (human) CLDN18.2 antibody binds to (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, the (human) CLDN18.2 antibody does not bind to CLDN18.1. In one embodiment, the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1. In some embodiments, the binding is measured, e.g., by radioimmunoassay (RIA), Bio-Layer Interferometry assay (BLI), MSD assay or surface plasmon resonance (SPR) or flow cytometry.

[0030] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein to refer to an antibody molecule having a native immunoglobulin molecule structure. In the case of a conventional four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) which are interconnected by disulfide bonds. In the case of a heavy chain antibody having only heavy chains but lacking light chains, a full-length antibody comprises two heavy chains (H) which are interconnected by disulfide bonds.

[0031] For a conventional four-chain IgG antibody, a full-length antibody heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. A full-length antibody light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region VL consists of three CDRs and four FRs arranged in the following order from the amino-terminal to the carboxy-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light pair (VH and VL) form an antigen binding site / portion, respectively. The distribution of amino acids in the various regions or domains follows the numbering defined in Kabat.

[0032] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an epitope. The CDRs of a heavy chain and light chain are typically referred to as CDR1, CDR2, and CDR3, numbered in order from the N-terminus. CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0033] The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen binding fragment.

[0034] An "antigen binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; diabodies or fragments thereof; or a camelid antibody.

[0035] The term "antigen" refers to a molecule that elicits an immune response. This immune response can involve either antibody production or the activation of specific immune cells, or both. The skilled artisan will understand that any macromolecule, including essentially any protein or peptide, can serve as an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA. The term "epitope" as used herein refers to the portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0036] An "antibody of IgG format" refers to an antibody whose heavy chain constant region is of IgG format. The heavy chain constant region of all antibodies of the same format is identical, and differs between antibodies of different formats. For example, an antibody of IgG4 format refers to an antibody whose heavy chain constant region is from IgG4, or an antibody of IgG1 format refers to an antibody whose heavy chain constant region is from IgG1.

[0037] The terms "binds" or "binds specifically" as used herein mean that the binding is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or bio-layer interferometry assay or MSD assay or surface plasmon resonance (SPR).

[0038] As used herein, "antibody drug conjugate (ADC)" refers to a structure resulting from the linkage of an antibody to a toxin drug.

[0039] The general term "sugar" as used herein indicates monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" as used herein denotes a derivative of a monosaccharide, i.e. a monosaccharide comprising a substituent and / or a functional group. Examples of sugar derivatives include amino sugars and sugar acids, such as glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). Examples of sugar derivatives also include compounds denoted herein as E(A)x, wherein E is a sugar or sugar derivative, and wherein E comprises x functional groups A.

[0040] A core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as a GlcNAc which is bound via C1 to the antibody, preferably via an N-glycosidic bond to the amide nitrogen atom of the side chain of an asparagine amino acid of the antibody. Said core-GlcNAc substituent can be present at a natural glycosylation site of the antibody, but it can also be introduced onto a different site of the antibody. In the present context, a core-N-acetylglucosamine substituent is a monosaccharide substituent, or, if the core-GlcNAc substituent is fucosylated, a disaccharide core-(Fucαl-6)GlcNAc (fucosyl-alpha-1,6-N-acetylglucosamine) substituent, also referred to as GlcNAc(Fuc).

[0041] "glycosylation engineering" refers to the process of altering the glycosylation of an antibody. The glycosylation of an antibody can be further engineered for various purposes to obtain an antibody with new glycosylation, for example, to remove the glycosylation for the elimination of Fc gamma R affinity and complement binding / effect functions, to reduce fucose and sialic acid groups and to increase bisecting N-acetylglucosamine, galactose and mannose for the enhancement of Fc-mediated ADCC and CDC effects. Means of glycosylation engineering are known in the art, for example, by altering the glycosylation sites of an antibody to increase or decrease the glycosylation of the antibody surface, or by modifying the sugar chains in vitro by chemical or enzymatic means, or by catalyzing the glycosylation of the antibody by altering the glycosylation pathway of the expression system, e.g. by enzymes such as glycosidases and glycosyltransferases, or by influencing the glycosylation of the antibody by cell culture conditions. In some embodiments, the "glycosylation engineering" of the present application is performed by modifying the sugar chains in vitro by enzymatic means. Preferably, the glycosylation engineering of the present application is performed by modifying the sugar chains by glycosidases, such as endoglycosidases or glycosyltransferases.

[0042] The antibody with engineered glycosylation according to the present application means an antibody whose glycosylation pattern is engineered compared to an antibody with a native glycosylation pattern. Preferably, the antibody with engineered glycosylation means an antibody obtained after modification of the sugar chain in vitro by enzymatic method (e.g., modification of the sugar chain by glycosidase (e.g., endoglycosidase or glycosyltransferase)) after expression in an expression system (e.g., mammalian cell). More preferably, the antibody with engineered glycosylation according to the present application means an antibody comprising a core-GlcNAc and a sugar derivative E(A)x linked thereto, wherein GlcNAc is bonded to the antibody through C1, preferably through N-glycosidic bond to the amide nitrogen atom of the side chain of asparagine amino acid of the antibody. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, typically fucose is linked to C6 of the -GlcNAc substituent through a-1,6. The fucosylated -GlcNAc substituent means core-GlcNAc(Fuc), and the fucosylated GlcNAc-E(A)x substituent means GlcNAc(Fuc)-E(A)x.

[0043] The term "site-specific conjugation" as used herein refers to conjugation of a toxin drug / active agent specifically to a particular site of an antibody through a linker.

[0044] The term "pharmaceutically acceptable salt" means a salt that retains the biological effectiveness and properties of the antibody drug conjugate of the present application and is not biologically or otherwise undesirable. The antibody drug conjugate of the present application can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present application, the pharmaceutically acceptable non-toxic acid addition salt means a salt of the antibody drug conjugate of the present application formed with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. The pharmaceutically acceptable non-toxic base addition salt means a salt of the antibody drug conjugate of the present application formed with an organic or inorganic base, including but not limited to alkali metal salts such as lithium, sodium or potassium salt; alkaline earth metal salts such as calcium or magnesium salt; organic base salts such as ammonium salt formed with an N-containing organic base.

[0045] The term "solvate" means an association or complex of one or more solvent molecules and the antibody drug conjugate of the present application. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0046] "pharmaceutically acceptable" and "pharmaceutical" are used interchangeably herein.

[0047] The term "drug: antibody ratio" or "DAR" refers to the ratio of small molecule toxin drug moieties (D) conjugated to the Ab moiety described herein to the Ab moiety. In some embodiments described herein, the DAR can be determined by the formula Ab-[L-(D) r ] p The DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the overall ratio of small molecule toxin drug moieties (D) conjugated to the Ab moiety described herein to the Ab moiety in the product as measured by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and / or HPLC), which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the application is from 1 to 20, e.g., 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, ranges with two of these values as endpoints.

[0048] The term "therapeutic agent" described herein encompasses any agent effective in the prevention or treatment of a tumor, e.g., a cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressive agents).

[0049] The term "cytotoxic agent" as used herein refers to an agent which inhibits or prevents the function of cells and / or causes death or destruction of cells.

[0050] "Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer or immune system disorders.

[0051] The term "small molecule toxin drug" refers to an organic compound of low molecular weight capable of modulating a biological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more able to penetrate cells, less susceptible to degradation, and less likely to elicit an immune response than macromolecules.

[0052] The term "immunomodulatory agent" as used herein refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immune modulators include immunosuppressants. In some embodiments, the immunomodulatory agents of the present application include immune checkpoint inhibitors or immune checkpoint agonists.

[0053] The term "effective amount" refers to the amount or dose of an antibody or fragment or composition or combination of the present application, which elicits the intended effect in a patient in need of treatment or prevention after administration at a single or multiple doses.

[0054] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0055] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells in which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny can not be completely identical to the parent cell both in nucleic acid content and in physical identity, but, can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein.

[0056] The term "label" as used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or antibody, and facilitates detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope label or fluorescent label) or, in the case of an enzymatic label, can catalyze chemical changes in a substrate compound or composition that changes can be detected. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling by conjugating the probe or antibody to a separate, directly labeled, second reagent.

[0057] An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0058] An "isolated" antibody or other molecule (e.g., ADC molecule) is one which has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, an antibody or ADC molecule is purified to greater than 95% or 99% purity as determined by, e.g., electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods.

[0059] The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0060] The terms "tumor" and "cancer" are used interchangeably herein and encompass both solid tumors and hematological tumors.

[0061] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the application include bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cancer, colon cancer, small bowel cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer, and gall bladder cancer, including metastatic forms of those cancers.

[0062] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive as referred to herein.

[0063] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc. that is useful in

[0064] The term "pharmaceutical composition" refers to a composition that is in a form suitable for its intended use, and that contains at least one active ingredient in an effective amount to achieve the desired effect.

[0065] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an ADC molecule of the present application, and (ii) another therapeutic agent) are presented in physically separated form, such as in separate units of a kit, for administration to a patient simultaneously, without specific time limitations relative to one another, or sequentially, in any order, wherein such administration provides therapeutically effective levels of the two or more active agents in the body of the patient. In some embodiments, the ADC molecule of the present application and the other therapeutic agent used in the pharmaceutical combination are administered at levels that do not exceed the levels at which they are used individually. The term "fixed combination" means that the two or more active agents are administered to a patient as a single entity. Preferably, the dosage of each active agent and / or the time interval between the administration of the active agents is / are selected so that the combined use of the active agents results in an effect that is greater than the effect achievable with the use of any of the active agents alone. The active agents can each be present in a separate formulation, which formulations can be the same or different.

[0066] The term "combination therapy" refers to the administration of two or more therapeutic agents, or treatment modalities (such as radiotherapy or surgery), to treat a disease described herein. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration

[0067] As used herein, "treatment" refers to slowing, interrupting, arresting, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0068] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0069] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0070] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, such as from a fresh, frozen and / or preserved organ or tissue sample or biopsy or puncture sample; blood or any blood component; a body fluid, such as cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; a cell from a subject at any time of gestation or development. The tissue sample can contain compounds not naturally admixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0071] Antibody-drug conjugate

[0072] The present disclosure discloses an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the molecular formula of the antibody-drug conjugate is: Ab-[L-(D) r ] p , wherein Ab represents an anti-Claudin 18.2 antibody or an antigen-binding fragment thereof, L represents a linker, D represents a toxin drug, p is 1-10, for example 1-9, 2-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r is 1-5, preferably 1 or 2;

[0073] The anti-Claudin 18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6;

[0074] SEQ ID NO: 1 is NYEMN; SEQ ID NO: 2 is YITGSGRTIYYADSVKG; SEQ ID NO: 3 is YDYGDFDF; SEQ ID NO: 4 is RASQGISSWLA; SEQ ID NO: 5 is AASSLQS; and SEQ ID NO: 6 is QQANSFPLT.

[0075] The toxin drug has the following structure

[0076]

[0077] Further, the antibody or antigen-binding fragment thereof further comprises: a heavy chain variable region as shown in SEQ ID NO: 7; and a light chain variable region as shown in SEQ ID NO: 8.

[0078] SEQ ID NO. 7 is QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMNWVRQ APGKGLEWVAYITGSGRTIYYADSVKGRFTISRDNAKKSLYLQMNSLRSEDTAVYYCAIY DYGDFDFWGQGTLVTVSS; and SEQ ID NO. 8 is DIQMTQSPSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPLTFGGGTKVEIK.

[0079] Further, the antibody is selected from the group consisting of a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0080] Further, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from an IgG series antibody; and the light chain constant region is selected from a kappa or lambda chain.

[0081] Further, the IgG series antibody is selected from one or more of IgG1, IgG2, and IgG4, preferably IgG1.

[0082] Further, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab)2 fragment, a Fv fragment, and a ScFv.

[0083] In some embodiments, the linker suitable for use in the present application can be any linker capable of effecting conjugation of an antibody to a drug. In some embodiments, the linker can be a linker used in techniques capable of effecting site-specific conjugation.

[0084] In a preferred embodiment, the linker of the present application is a linker that is attached to an antibody oligosaccharide. As defined herein, "a linker that is attached to an antibody oligosaccharide" refers to any linker that is attached to a reactive group on a sugar chain at an antibody glycosylation site to effect conjugation of an antibody to a drug. The sugar chain at an antibody glycosylation site is typically an N-glycan, and is usually engineered to convert a non-uniform structure N-glycan to a uniform structure N-glycan bearing a reactive group, which is then used to attach the linker to effect site-specific conjugation of a drug to an antibody, resulting in an antibody-drug conjugate. In a preferred embodiment, the N-glycosylation site of the antibody is a conserved N-glycosylation site on the Fc domain, preferably the CH2 domain, of the antibody, such as Asn297. Thus, in one embodiment, the "linker that is attached to an antibody oligosaccharide" of the present application is particularly any linker that is capable of effecting site-specific conjugation to a reactive group on an N-glycan at a conserved N-glycosylation site (such as Asn297) on the Fc domain of an antibody, such as the linker described in PCT / NL2013 / 050744 or the linker of PCT / EP2021 / 075401, which are incorporated herein in their entirety. In one embodiment, the reactive group of the present application is an azido group, a keto group or an alkyne group. In one embodiment, the linker of the present application is a linker comprising an alkyne group. In one embodiment, the reactive group of the present application is an azido group, a keto group or an alkyne group, preferably an azido group, and the linker of the present application is a linker comprising an alkyne group. When such linkers are mentioned in the present application, the group formed after the reactive group of the linker reacts with the group of the antibody can also be defined as part of the "linker" since the new group is formed after the reactive group of the linker reacts with the group of the antibody.

[0085] Linkers suitable for use in the present application also include, for example, cathepsin-degradable linkers, such as Val-Cit linkers (e.g., vc-PAB), cBu-Cit linkers, and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers, silicon aliphatic structure linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared sensitive linkers, UV-sensitive linkers such as PC4AP (Antibody-drug conjugates: Recent advances in linker chemistry, Su, Z., Xiao, D., Xie, F., Liu, L., Wang, Y., Fan, S.,…Li, S. (2021). Antibody-drug conjugates: Recent advances in linker chemistry. Acta Pharmaceutica Sinica B.). Linkers suitable for use in the present application can also be a combination of one or more linkers, for example, a cathepsin-degradable linker can be combined with another type of linker to form a new linker. Thus, “linker” as described herein encompasses a single type of linker, or a combination of different types of linkers, as long as it is capable of coupling an antibody of the present application to a drug. Thus, in one embodiment, a linker suitable for use in the present application is MC-VC-PAB, vc-PAB, SMCC, or MC-GGFG.

[0086] The D in the present application can be any antitumor compound, as long as it is a compound having an antitumor effect and having a substituent or a partial structure that can be linked to the linker structure, and is not particularly limited. For example, the antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For the antitumor compound, a part or all of the linker is preferably cleaved in a tumor cell to release the antitumor compound part, thereby exhibiting an antitumor effect. Upon cleavage of the linker at the linking part to the drug, the antitumor compound is released as an unmodified structure, and can exert its original antitumor effect.

[0087] In some embodiments, the antibody drug conjugate has an average DAR of 1-15, such as 2-10, 2-8, or 3-5, such as an average DAR of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

[0088] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent or a chemotherapeutic agent, such as a camptothecin compound exatecan (a topoisomerase I inhibitor), Dxd (a novel topoisomerase I inhibitor exatecan derivative), an auristatin compound such as monomethyl auristatin E (MMAE) or a maytansinoid compound such as the small molecule microtubulin inhibitor DM1. The structures in the Examples of the present application show the structures of representative compounds of these anti-tumor compounds. In one embodiment, the present application provides an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof.

[0089] Further, wherein the Claudin 18.2 is selected from the group consisting of human Claudin 18.2, mouse Claudin 18.2 and monkey Claudin 18.2.

[0090] Further, the linker is a cleavable linker such as valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide, etc. Coupling to a cysteine, lysine, N297 site glycosidic bond on the antibody sequence.

[0091] Further, the antibody is an antibody with engineered glycosylation, such as an antibody obtained after in vitro enzymatic modification of the sugar chain (such as modification of the sugar chain by a glycosidase (such as an endoglycosidase or a glycosyltransferase)), preferably, the antibody with engineered glycosylation refers to an antibody in which the sugar chain at the glycosylation site of the antibody is modified from a non-uniform structure N-glycan to a single structure N-glycan with a reactive group (such as any reactive group capable of reacting with the linker moiety, such as an azido group, a ketone group and an alkyne group), more preferably, the N-glycosylation site is a conserved N-glycosylation site on the Fc domain of the antibody, such as Asn297.

[0092] Methods suitable for engineering the glycosylation of the antibody of the present application are described, for example, in PCT / NL2013 / 050744, PCT / EP2016 / 059194 or PCT / EP2017 / 052792, which are incorporated herein in their entirety.

[0093] In a preferred embodiment, the antibody with engineered glycosylation of the present application is an antibody comprising a GlcNAc-E(A)x substituent, wherein GlcNAc is N-acetylglucosamine, wherein E(A)x is a sugar derivative comprising X functional groups A, wherein A is independently selected from the group consisting of an azido group, a keto group and an aldehyde group and X is 1, 2, 3 or 4; wherein said GlcNAc-E(A)x substituent is bound to said antibody via the CI of the N-acetylglucosamine of said GlcNAc-E(A)x substituent, wherein said N-acetylglucosamine is optionally fucosylated. In case the N-acetylglucosamine is fucosylated, it is bound via C6 to a fucose (Fuc).

[0094] Further, said linker is a linker enabling site-specific conjugation, preferably a linker to an oligosaccharide of an antibody, preferably said oligosaccharide linker is a linker comprising an alkyne group, most preferably said oligosaccharide linker is a linker enabling specific site conjugation to a reactive group (e.g. an azido group) on an N-glycan at a conserved N-glycosylation site (e.g. Asn297) on an Fc domain of an antibody.

[0095] In some embodiments of the present application, a composition comprising said antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof and optionally a pharmaceutically acceptable excipient is disclosed.

[0096] In some embodiments of the present application, the use of said antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof for the preparation of a medicament for the prevention and / or treatment of a cancer or a tumor is disclosed.

[0097] Further, said medicament is a medicament for cell therapy.

[0098] Further, said cancer or tumor is a CLDN18.2 positive cancer or tumor.

[0099] Further, said cancer or tumor is selected from the group consisting of bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer.

[0100] Examples

[0101] The present application generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting. Additionally, the experimental methods described in the following examples are routine methods unless otherwise specified. The starting materials, reagents, materials, etc. used in the following examples are commercially available unless otherwise specified.

[0102] Example 1: Immunological screening of preferred antibodies

[0103] The preferred antibody against Claudin18.2 target was screened by immunizing human Claudin18.2 transgenic mice, and the preferred antibody was named P1026. The antibody heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; the heavy chain variable region is as shown in SEQ ID NO: 7; and the light chain variable region is as shown in SEQ ID NO: 8. For specific sequence and preparation method information of the antibody, see Chinese Patent CN 115947851 B.

[0104] Example 2: SPR assay of Claudin18.2 preferred antibody and antigen affinity

[0105] The affinity of the preferred antibody (P1026-IgG) obtained against the Claudin18.2 target was determined using the SPR (Surface plasmon resonance) technique. The affinity of the antibody was determined according to the general method (for example, the method described in the instrument manual). Briefly, a CM5 chip (Cytiva, CAT#50-105-5509) coupled with Anti-His antibody (Cytiva, CAT#27-4710-01) was used to capture the His-tagged recombinant human Claudin18.2 protein (GenScript, CAT#Z03504) as the ligand. The Claudin18.2 antibody was diluted 2-fold in gradient, with a starting concentration of 200nM, and 6 different concentration points were used as analytes for kinetic affinity detection with the captured recombinant Claudin18.2 protein. The Biacore software was used to perform Kinetics fitting on the binding curve to obtain the affinity. The results showed that compared with the control antibody IMAB-362 (sequence source reference Chinese Patent CN103509114A, 175D10 clone sequence, prepared in the laboratory), the affinity KD of the preferred antibody P1026-IgG to the human Claudin18.2 target was increased by about 17.1 times.

[0106]

[0107]

[0108] Example 3: Preferred antibody binding activity to MC38-Claudin18.2 cells

[0109] MC38-Claudin18.2 cell line was constructed by constructing human Claudin18.2 sequence (NCBI, NP_001002026) into pcDNA3.4 vector, and then transfecting into MC38 cells (Nanjing Kebai) using Lipo3000 liposome reagent. 1 x 10^5 MC38-Claudin18.2 overexpression cell lines were plated in a 96-well plate, and 100 ug / ml of preferred antibody P1026 and control antibody IMAB-362 were diluted by 5 times to set 8 concentration gradients. The gradient-diluted antibodies were incubated with the cells for 1 h, centrifuged to discard the supernatant, washed with PBS three times, then Goat-anti human IgG, PE conjugated secondary antibody (Thermo, CAT#12-4998-82) was added and incubated for 1 h. After incubation, the supernatant was discarded by centrifugation, and the cells were washed with PBS three times. The binding activity of preferred antibodies and control antibodies to MC38-Claudin18.2 cells was detected by flow cytometry. The results showed that the cell binding activity of preferred antibody P1026 was about 16.8 times higher than that of control antibody IMAB-362.

[0110]

[0111] Example 4: Endocytosis activity of preferred antibody in NUGC4 cell line

[0112] Gastric cancer cell line NUGC4 has low level of Claudin18.2 endogenous expression. To explore the endocytosis activity of preferred antibodies in NUGC4 cells, 10 ug / ml of P1026 antibody and IMAB362 control antibody 25ul were taken in a 96-well plate, and pHrodo TM iFL secondary antibody (Thermo, CAT#Z25611) 25ul was added to the 96-well plate and mixed, and 50ul NUGC4 overexpression target cells (purchased from Nanjing Kebai) were added at a density of 2x10 6 The cells were mixed with the antibodies and incubated at 37°C for 1h, 2h, 4h, 6h, and 19h at different time points. The endocytosis activity of preferred antibody P1026 and control antibody IMAB362 in NUGC4 cells was detected by flow cytometry. The results showed that the endocytosis activity of preferred antibody P1026 was about 2.5 times higher than that of control antibody IMAB-362. Figure 2As shown, it indicates that the P1026 antibody is obviously superior to the control antibody IMAB-362 in mediating P1026 antibody endocytosis in NUGC4 cells compared with the control antibody IMAB-362.

[0113] Example 5: Preparation of preferred ADC and determination of DAR value

[0114] The Suzhou Kaidi Pharmaceutical Technology Co., Ltd. was commissioned to site-couple the P1026 naked antibody and the toxin-linker through the N297 site of the Fc region of the antibody by a glycosidic bond (the coupling method is referred to PCT / CN2023 / 098081). The preferred ADC was prepared by coupling, named P1026-ADC, and its structure is as shown in Figure 1 . Among them, p is the number of toxin-linkers connected to each antibody. Specifically, first, under the catalysis of glycosidase Endo S2 (MCE, CAT#37278-88-9), the antibody P1026 is cut off at its 297th N-glycan chain to obtain an antibody with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine modified N-glycosylation site in the Fc region, and then an appropriate amount of toxin-linker compound (chemical structure as shown in Figure 8 ) is added to the system to couple with the obtained modified antibody to obtain a conjugate. By using hydrophobic interaction chromatography (HIC) analysis on the preferred ADC antibody drug after coupling (denoted as P1026-ADC), the results are as shown in Figure 3 , which shows that the proportion of P1026-ADC with a DAR (Drug-antibody ratio) value of 4 is more than 95%, indicating that the uniformity of the obtained product after coupling is high.

[0115] Sample DAR0 DAR2 DAR4 P1026-ADC 0.00％ 5.44％ 94.56％

[0116] Example 6: In vitro plasma stability of preferred ADC

[0117] The Yantai Maiborui International Biomedical Co., Ltd. was commissioned to prepare the ADC product (P1026-Dxd) with the linker-toxin GGFG-Dxd by reducing the four pairs of disulfide bonds of the antibody. The prepared P1026-Dxd and P1026-ADC were each divided into five parts, 200 ug each, and then 500 ul of human plasma was added to each part and mixed, and then incubated at 37°C for 0h, 24h, 48h, 72h, and 96h, respectively. The samples incubated for a specified time were purified by using CNBr activated Sepharose 4B (Cytiva). Finally, the average DAR value change of the purified ADC was detected by mass spectrometry analysis to characterize the stability of the ADC in human plasma. The experimental results are as shown in the following table and Figure 4As shown, the proportion of toxin shedding of P1026-Dxd gradually increased with the extension of incubation time with human plasma. When incubated with human plasma for 48 hours, more than 40% of the toxin was shed. When incubated for 72 hours, the intact antibody-drug conjugate was hardly detected by mass spectrometry. After incubation of P1026-ADC with human plasma for different time, the toxin-linker of ADC was hardly shed. This experiment fully demonstrated that the stability of P1026-ADC was significantly better than that of P1026-Dxd.

[0118]

[0119]

[0120] Example 7: Preferred ADC-mediated cell killing activity

[0121] To explore the in vitro activity of P1026-ADC molecules, a tumor cell line NUGC4 stably overexpressing Claudin 18.2 target was used for cell killing experiments. The experimental process is briefly described as follows: when the NUGC4-Claudin 18.2 cell line grew to the logarithmic growth phase, the cells were plated in a 96-well plate (Corning, CAT#: 3599) and cultured overnight in a carbon dioxide incubator at 37°C. The next day, 1 ug / ml of P1026-ADC stock sample was diluted by 3-fold gradient, with a total of 8 concentration gradient points. The diluted P1026-ADC molecules were added to the 96-well plate containing NUGC4-Claudin 18.2 cell line and incubated in a carbon dioxide incubator at 37°C for about 72 hours. Cell Counting Kit-8 cell proliferation toxicity detection reagent (Tongren Chemical, Cat#: CK04) was added, and after 2 hours of reaction, the SpectraMAX i3x multifunctional enzyme labeler of Molecular Device Company was used for wavelength OD450nm detection in ABS mode, with OD630nm reference wavelength set. The cell killing percentage was calculated, and the Graphpad software was used for four-parameter fitting to obtain the IC50 value of the ADC molecule. The results showed that the killing activity IC50 of P1026-ADC on NUGC4-Claudin 18.2 cell line was 0.742 nM. The cell killing results are shown in Figure 5

[0122] Experimental Example 8: Preferred ADC animal in vivo cell line-derived xenograft tumor (CDX) model efficacy

[0123] ​To explore the in vivo efficacy of P1026-ADC molecule, NUGC4 cell line stably overexpressing Claudin18.2 target (cells purchased from Nanjing Kebai Biotechnology Co., Ltd., Cat. Number: CBP74135) was used to establish animal in vivo NUGC4-Claudin18.2 CDX model to evaluate the preferred ADC efficacy. Specifically, each male severe immunodeficiency (NDG) mouse was subcutaneously injected with 5x10 6 NUGC4-CLDN18.2 cells, and 5 days after inoculation, mice with tumors growing to about 120mm 3 were randomly divided into groups, 5 mice per group; on the 6th day, tail vein injection was performed for drug administration, and the specific drug information is shown in the table below. The experimental results are shown in Figure 6 P1026-ADC single administration can significantly inhibit the growth of tumors in animals, and the tumor is basically regressed by 45 days of observation, while the clinically researched products CMG-901 and SHR1904-ADC can inhibit tumor growth in the early stage, but the tumor gradually resumes growth after 30 days.

[0124]

[0125] Experimental Example 9: Preferred ADC animal in vivo PDX model efficacy

[0126] The human-derived tissue xenotransplantation (PDX) model was constructed by using gastric cancer patient-derived GA0006 tumor tissue to ectopically transplant Balb / c Nude mice by the China-US Crown Biotechnology Co., Ltd. Specifically, after the mice were inoculated with tumor blocks, when the average tumor volume of the mice reached 150mm 3 , they were randomly divided into groups, the control group (Ctl. group) was given PBS; the experimental group CMG901-ADC was given a 2mpk dose, once a week for a total of 3 times; the experimental group P1026-ADC was given a single dose of 10mpk. The entire experiment lasted for 25 days from drug administration to the end of the experiment. The experimental results are shown in Figure 7 P1026-ADC single administration can significantly inhibit the growth of tumors in animals.

[0127] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, said antibody-drug conjugate having the molecular formula: Ab-[L-(D)] r ] p Where Ab represents anti-Claudin18.2 antibody, L represents linker, D represents toxin drug; r is 2; The anti-Claudin18.2 antibody includes a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and the light chain variable region includes LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; The toxin drug has the following structure: ； The antibody-drug conjugate has the following structure: Where p is 1-10; The antibody is a modified glycosylated antibody, obtained by modifying the glycan chain in vitro using the glycosidase Endo S2.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The antibody further comprises: the heavy chain variable region shown in SEQ ID NO: 7; and the light chain variable region shown in SEQ ID NO:

8.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The anti-Claudin18.2 antibody mentioned therein is selected from the group consisting of bispecific antibodies, monoclonal antibodies, chimeric antibodies, and fully human antibodies.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The anti-Claudin18.2 antibody is selected from whole antibodies.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The anti-Claudin18.2 antibody is selected from humanized antibodies.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, The antibody further includes a heavy chain constant region and a light chain constant region.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that, The constant region of the antibody heavy chain is selected from IgG series antibodies; the constant region of the light chain is selected from κ or λ chains.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 7, characterized in that, The IgG series antibodies are selected from one or more of IgG1, IgG2 and IgG4.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, characterized in that, The IgG series antibodies are selected from IgG1.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein the antibody-drug conjugate has an average DAR of 1-15.

11. The antibody-drug conjugate according to claim 10, or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate has an average DAR of 2-10.

12. The antibody-drug conjugate according to claim 10, or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate has an average DAR of 2-8.

13. The antibody-drug conjugate according to claim 10, or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate has an average DAR of 3-5.

14. A composition comprising an antibody-drug conjugate according to any one of claims 1-13 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

15. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-13 in the preparation of a medicament for treating cancer; in, The cancer is a cancer that expresses CLDN18.2 positively, wherein the cancer is selected from ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, head and neck cancer, bile duct cancer, colon cancer, small bowel cancer, cervical cancer, esophageal cancer, and gallbladder cancer. The drug in question is a cell therapy drug.

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