Claudin18.2-targeting antibody drug conjugates and uses thereof

Abstract

The invention relates to 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; r is 1 to 5; a heavy chain variable region of the anti-Claudin18.2 antibody or an antigen binding fragment of the anti-Claudin18.2 antibody comprises an HCDR1 as shown in SEQ ID NO: 1, an HCDR2 as shown in SEQ ID NO: 2, and an HCDR3 as shown in SEQ ID NO: 3; the light chain variable region comprises an LCDR1 as shown in SEQ ID NO: 4, an LCDR2 as shown in SEQ ID NO: 5 and an LCDR3 as shown in SEQ ID NO: 6; toxin medicine with following structure: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an antibody-drug conjugate targeting Claudin18.2 and an application thereof. Background Art

[0002] The number of newly diagnosed gastric cancer cases worldwide increased from 980,000 in 2016 to 1,089,000 in 2020. It is estimated that by 2025, the total number of new gastric cancer patients worldwide will reach 1.256 million. The incidence and mortality of pancreatic cancer rank among the top 10 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, which is very close to the incidence rate, the annual death toll is about 196,000.

[0003] Chemotherapy is widely used in the treatment of advanced gastric cancer. The treatment of gastric cancer is mainly divided into two categories according to whether the HER2 target expression is positive: 1. For HER2-positive patients, the first-line treatment mainly uses trastuzumab combined with platinum or fluorouracil; for patients who fail targeted therapy, the second-line treatment mainly uses chemotherapy drugs such as paclitaxel, docetaxel, and irinotecan; the third-line treatment is mainly apatinib and nivolumab; 2. For HER2-negative patients, the treatment method is mainly chemotherapy, and the first-line treatment mainly includes platinum, taxanes, 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 in gastric cancer has positive HER2 target expression, and 80% of gastric cancer patients have negative HER2 target expression. At present, both chemotherapy drugs and PD-1 targeted drugs have very limited efficacy in gastric cancer. Therefore, there is an urgent need to develop new targeted therapeutic drugs targeting new targets.

[0004] Claudin 18.2 is a four-transmembrane protein with highly restricted expression in normal tissues, but highly specific expression in patients with gastric cancer, pancreatic cancer, etc. Claudin18 has two splice variants, Claudin18.1 and claudin18.2, which differ by only 8 amino acids; Claudin 18.2 is involved in tumorigenesis and development and is located in the outer cell membrane. Its drug forms include monoclonal antibodies, bispecific antibodies, antibody-drug conjugates (ADCs), CAR-T, etc.

[0005] There are many forms of Claudin18.2-ADCs at present, most of which are obtained by coupling antibodies targeting Claudin18.2 with cleavable linkers-toxins. Most of the toxins are tubulin inhibitors, and a small number of toxins are camptothecins. The ADCs currently under development for the Claudin18.2 target have the defect of poor stability of the linker-toxin, and the efficacy of Claudin18.2-ADC products is limited.

[0006] The contents of the background technology section are merely the technologies known to the inventors and do not necessarily represent the prior art in the field. Summary of the invention

[0007] In view of the above problems, the present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein 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, such as 1-9, 2-8, 3-7, 4-6, 2-6, such as 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 present invention combines a novel topoisomerase inhibitor toxin drug with a high affinity and high endocytosis antibody of Claudin18.2 and a novel linker, and uses the N297 position of the antibody IgG-Fc to obtain a Claudin18.2-ADC product with better in vitro plasma stability than the ADC product obtained by opening the disulfide bond coupling with an antibody. In addition, the ADC for the Claudin18.2 target obtained by the present invention has a better anti-tumor effect in multiple tumor models in animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

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

[0014] Figure 2 The results of the endocytosis of naked antibody P1026 and IMA-362 in NUGC4 cells

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

[0016] Figure 4 These are the in vitro plasma stability results of P1026-Dxd and P1026-ADC of the present invention;

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

[0018] Figure 6 The efficacy results of P1026-ADC of the present invention and control ADC in CDX model in animals;

[0019] Figure 7 The efficacy results of P1026-ADC of the present invention and control ADC in PDX model in animals;

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

[0021] In the following description, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0022] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context otherwise clearly indicates, the singular forms "one", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include all values ​​between the endpoints and the endpoints. The preferred embodiments of the present invention are described below, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0023] definition

[0024] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0025] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having 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 any one of the alternatives or two or more of the alternatives.

[0027] As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the elements, integers or steps described. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0028] The term "Claudin" or "CLDN" used herein is the most important skeletal protein that determines the tight junction structure between cells. It participates in adhesion junctions and plays an important role in the metastasis and invasion of tumor cells. Claudin protein is widely present in mammalian epithelial and endothelial cells, and its distribution is mainly on the side of epithelial cells and on the plasma membrane of basal cells. Different Claudin proteins have their own specific expression in different tissues. Among them, the Claudin18 (CLDN18) gene is located at 3q22.3, with a molecular weight of 24kDa and 261 amino acid residues. It is a member of the Claudins superfamily, and its protein structure includes 2 extracellular loops and 4 transmembrane regions. The two subtypes of human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1), and Claudin18.2 or CLDN18.2 (UniProtID: P56856-2). In the primary structure sequence of the two proteins, only the amino acid residues at certain positions of the N-terminal signal peptide to the extracellular loop 1 (Loop1) structure are different, 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 is also very high. Among them, the extracellular loop 1 of CLDN18.2 is completely consistent in sequence in different species such as humans, mice, and macaques, and the homology of CLDN18.2 proteins in humans and mice reaches 84%, indicating that the CLDN18.2 protein sequence is extremely conservative (O. Tureci et al., Gene 481: 83-92, 2011). CLDN18.2 or any variants and isoforms thereof can be isolated from cells or tissues that naturally express 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 terms "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 refer 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 without binding to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (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 and refer to antibody molecules having the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, full-length antibodies comprise two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, full-length antibodies comprise two heavy chains (H) interconnected by disulfide bonds.

[0031] For conventional four-chain IgG antibodies, the full-length antibody heavy chain is usually composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated as VL herein) 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 are composed of three CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites / parts, respectively. The distribution of amino acids in various regions or domains follows the numbering definition in Kabat.

[0032] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3.

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

[0034] "Antigen-binding fragment" refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody and binds to 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 antibody; single-chain antibody (e.g., scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.

[0035] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. The skilled person will understand that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0036] An "IgG-type antibody" refers to an IgG-type antibody to which the heavy chain constant region belongs. The heavy chain constant region of all antibodies of the same type is the same, and the heavy chain constant region of antibodies of different types is different. For example, an IgG4-type antibody means that its heavy chain constant region is from IgG4, or an IgG1-type antibody means that its heavy chain constant region is from IgG1.

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

[0038] As used herein, "antibody drug conjugate (ADC)" refers to a structure obtained by linking an antibody to a toxin drug.

[0039] The general term "sugar" used herein indicates monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" used herein means 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 represented herein as E(A)x, wherein E is a sugar or a sugar derivative, and wherein E includes x functional groups A.

[0040] A core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as a GlcNAc bonded to the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. The core-GlcNAc substituent may be present at the native glycosylation site of the antibody, but it may also be introduced at a different site of the antibody. Herein, a core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fucα1-6)GlcNAc (fucosyl-α-1,6-N-acetylglucosamine) substituent - also referred to as GlcNAc(Fuc).

[0041] "Glycosylation modification" refers to the process of changing the sugar chains of antibodies by glycosylation engineering. The glycosylation of antibodies can be further modified for various purposes to obtain antibodies with new glycosylation, for example, to remove glycosylation in order to eliminate FcγR affinity and complement binding / effector function, to reduce fucose and sialic acid groups and increase bisecting N-acetylglucosamine, galactose and mannose in order to enhance Fc-mediated ADCC and CDC effects. Glycosylation modification methods are known in the art, for example, by changing the glycosylation sites of antibodies to increase or decrease the sugar chains on the surface of antibodies, or by modifying sugar chains in vitro by chemical or enzymatic methods, or by changing the glycosylation pathway of the expression system (for example, composed of enzymes such as glycosidases and glycosyltransferases) to catalyze the glycosylation of antibodies, and the glycosylation of antibodies can also be changed by the influence of cell culture conditions. In some embodiments, the "glycosylation modification" of the present invention is carried out by modifying sugar chains by enzymatic methods in vitro. Preferably, the glycosylation modification of the present invention is performed by modifying the sugar chain using a glycosidase (eg, endoglycosidase or glycosyltransferase).

[0042] The antibody with modified glycosylation of the present invention refers to an antibody whose glycosylation pattern is modified compared to an antibody with a natural glycosylation pattern. Preferably, the antibody with modified glycosylation refers to an antibody obtained after the antibody is expressed in an expression system (e.g., a mammalian cell) and the sugar chain is modified by an enzymatic method in vitro (e.g., by a glycosidase (e.g., an endoglycosidase or a glycosyltransferase)). More preferably, the antibody with modified glycosylation of the present invention refers to an antibody comprising a core-GlcNAc and a sugar derivative E(A)x connected thereto, wherein the GlcNAc is bonded to the antibody via C1, preferably via an N-glycosidic bond to an amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, the fucose is usually connected to C6 of the -GlcNAc substituent via α-1,6. The fucosylated-GlcNAc substituent refers to core-GlcNAc(Fuc) and the fucosylated GlcNAc-E(A)x substituent refers to GlcNAc(Fuc)-E(A)x.

[0043] The term "site-specific conjugation" as described herein refers to conjugation in which a toxin drug / active substance is specifically linked to a specific site of an antibody via a linker.

[0044] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the antibody drug conjugate of the present invention, and the salt is not biologically or otherwise undesirable. The antibody drug conjugate of the present invention may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the antibody drug conjugate of the present invention and an organic or inorganic acid, and the organic or inorganic acid includes but is 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, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the antibody drug conjugates of the present invention and organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as ammonium salts formed by organic bases containing N groups.

[0045] The term "solvate" refers to an association formed by one or more solvent molecules and the antibody drug conjugate of the present invention. 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] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0047] The term "drug:antibody ratio" or "DAR" refers to the ratio of the small molecule toxin drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety. In some embodiments described herein, the DAR can be represented by the formula Ab-[L-(D) r ] p The DAR can be determined by p and r in, for example, DAR can be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. DAR can also be calculated as the average DAR of the population of molecules in the product, that is, the overall ratio of the small molecule toxin drug portion (D) coupled to the Ab portion described herein to the Ab portion in the product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), and this DAR is referred to as the average DAR herein. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 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, 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, and ranges with two of these values ​​as endpoints.

[0048] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

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

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

[0051] The term "small molecule toxin drug" refers to a low molecular weight organic compound that can regulate biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10kD, usually less than 2kD and preferably less than 10kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

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

[0053] The term "effective amount" refers to an amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses administered to a patient, produces the desired effect in a patient in need of treatment or prevention.

[0054] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious 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 into which exogenous nucleic acid is introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as in the initially transformed cell are included herein.

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

[0057] "Individuals" or "subjects" include mammals. Mammals include, but are not limited to, domestic 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 an antibody or molecule that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody or ADC molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[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 decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease 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 invention include bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer, and gallbladder 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 precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0063] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0064] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0065] The term "drug 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) the ADC molecules of the present invention, and (ii) other therapeutic agents) are administered to the patient in sequence as separate entities, without specific time restrictions or at the same or different time intervals, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient. In some embodiments, the ADC molecules of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those when they are used alone. The term "fixed combination" means that two or more active agents are administered to the patient simultaneously in the form of a single entity. It is preferred that the dosage and / or time interval of the two or more active agents be selected so that the combined use of each part can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0066] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to a desired dose before administration. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating a disorder or condition described herein.

[0067] As used herein, "treat," ...

[0068] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, 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 vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably 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 solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0071] Antibody-drug conjugates

[0072] The present invention discloses an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein 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, such as 1-9, 2-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r is 1-5, preferably 1 or 2;

[0073] 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;

[0074] Among them, 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; SEQ ID NO: 6 is QQANSFPLT.

[0075] The toxin drug has the following structure

[0076]

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

[0078] Among them, SEQ ID NO.7 is QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMNWVRQ APGKGLEWVAYITGSGRTIYYADSVKGRFTISRDDNAKKSLYLQMNSLRSEDTAVYYCAIY DYGDFDFWGQGTLVTVSS; SEQ ID NO.8 is DIQMTQSPSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPLTFGGGTKVEIK.

[0079] Furthermore, the antibody is selected from the following group: whole antibody, bispecific antibody, monoclonal antibody, chimeric antibody, humanized antibody and fully human antibody.

[0080] Furthermore, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein: the antibody heavy chain constant region is selected from the IgG series of antibodies; and the light chain constant region is selected from κ or λ chain.

[0081] Furthermore, the IgG series antibodies are selected from one or more of IgG1, IgG2 and IgG4, preferably IgG1.

[0082] Furthermore, the antigen-binding fragment is selected from the following group: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment and ScFv.

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

[0084] In a preferred embodiment, the linker of the present invention is a linker connected to an antibody oligosaccharide. The "linker connected to an antibody oligosaccharide" defined herein refers to any linker that is connected to a reactive group on a sugar chain at an antibody glycosylation site to couple an antibody to a drug. The sugar chain on the antibody glycosylation site is generally an N-sugar chain, and is usually modified to transform the non-uniform structure N sugar chain into a single structure N sugar chain with a reactive group, and then the reactive group carried by the sugar chain is connected to the "linker" to achieve site-specific coupling of the drug to the antibody to obtain an antibody-drug conjugate. In a preferred embodiment, the N-glycosylation site of the antibody is an antibody Fc domain, preferably a conserved N-glycosylation site on the CH2 domain, such as Asn297. Therefore, in one embodiment, the "linker attached to an antibody oligosaccharide" of the present invention is particularly any linker that can achieve site-specific coupling with a reactive group on an N-sugar chain at a conserved N-glycosylation site (e.g., Asn297) on the Fc domain of an antibody, such as a linker described in PCT / NL2013 / 050744 or a linker of PCT / EP2021 / 075401, which are incorporated herein in their entirety. In one embodiment, the reactive group of the present invention is an azido, keto, or alkynyl group. In one embodiment, the linker of the present invention is a linker comprising an alkynyl group. In one embodiment, the reactive group of the present invention is an azido, keto, or alkynyl group, preferably an azido, and the linker of the present invention is a linker comprising an alkynyl group. When the present invention refers to such a linker, a new group is formed after the reactive group reacts with the group of the linker, so the group formed after the reactive group in the antibody-drug conjugate reacts can also be defined as part of the "linker".

[0085] Linkers suitable for use in the present invention 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, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, and ultraviolet-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.). The linker applicable to the present invention can also be a combination of one or more linkers, for example, a linker degraded by cathepsin can be combined with other types of linkers to form a new linker. Therefore, the "linker" described in the present invention covers a single type of linker, or a combination of different types of linkers, as long as it can couple the antibody of the present invention to the drug. Therefore, in one embodiment, the linker applicable to the present invention is MC-VC-PAB, vc-PAB, SMCC or MC-GGFG.

[0086] D in the present invention can be any antitumor compound, as long as it has an antitumor effect and has a substituent or a partial structure that can be connected to the linker structure, and there is no particular limitation. For example, the antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For antitumor compounds, part or all of the linker can preferably be cut off in tumor cells to release the antitumor compound part, thereby showing an antitumor effect. When the linker is cut off from the connecting part of the drug, the antitumor compound is released in an unmodified structure, and its original antitumor effect can be exerted.

[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 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 (topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), an auristatin compound such as monomethyl auristatin E (MMAE) or a maytansine compound such as a small molecule microtubule 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 invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

[0089] Furthermore, the Claudin18.2 is selected from the following group: human Claudin18.2, mouse Claudin18.2 and monkey Claudin18.2.

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

[0091] Further, the antibody is an antibody with engineered glycosylation, for example, an antibody obtained after enzymatic modification of sugar chains in vitro (for example, modification of sugar chains by glycosidase (for example, endoglycosidase or glycosyltransferase)). Preferably, the antibody with engineered glycosylation refers to an antibody in which the sugar chains at the glycosylation sites of the antibody are transformed from non-uniformly structured N sugar chains to single-structured N sugar chains with reactive groups (for example, any reactive groups that can react with linker moieties, such as azide, keto and alkyne). More preferably, the N-glycosylation site is a conserved N-glycosylation site on the Fc domain of the antibody, such as Asn297.

[0092] Methods for modifying antibody glycosylation suitable for the present invention 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 of the invention with engineered glycosylation 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 azido, keto and halogen and X is 1, 2, 3 or 4; wherein the GlcNAc-E(A)x substituent is bonded to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent, wherein the N-acetylglucosamine is optionally fucosylated. In the case where the N-acetylglucosamine is fucosylated, it is bonded to fucose (Fuc) via C6.

[0094] Further, the linker is a linker capable of achieving site-specific coupling, preferably a linker connected to an antibody oligosaccharide, preferably, the oligosaccharide linker is a linker comprising an alkynyl group, and most preferably, the oligosaccharide linker is a linker capable of achieving specific site coupling with a reactive group (e.g., an azide group) on the N-sugar chain at a conserved N-glycosylation site (e.g., Asn297) on the antibody Fc domain.

[0095] In some embodiments of the present invention, a composition is disclosed, which comprises the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof and an optional pharmaceutically acceptable excipient.

[0096] In some specific embodiments of the present invention, disclosed is the use of the antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for preventing and / or treating cancer or tumors.

[0097] Furthermore, the drug is a drug for cell therapy.

[0098] Furthermore, the cancer or tumor is a cancer or tumor that is positive for CLDN18.2 expression.

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

[0100] Example

[0101] The invention 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 to limit the invention. In addition, the experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0102] Example 1: Immunoscreening of Preferred Antibodies

[0103] By immunizing human Claudin18.2 transgenic mice, a preferred antibody against the Claudin18.2 target was screened and obtained, and the preferred antibody codename is P1026. The heavy chain variable region of the antibody 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 shown in SEQ ID NO: 7; and the light chain variable region is shown in SEQ ID NO: 8. For the specific sequence and preparation method of the antibody, please refer to Chinese patent CN 115947851 B.

[0104] Example 2: SPR determination of affinity between preferred antibodies and antigens for Claudin18.2

[0105] The affinity of the obtained preferred antibody (P1026-IgG) against the Claudin18.2 target and the human Claudin18.2 recombinant protein was determined by SPR (Surface plasmon resonance) technology. The affinity of the antibody was determined by SPR according to the general method (e.g., the operating method described in the instrument manual). In brief, a CM5 chip (Cytiva, CAT#50-105-5509) coupled with an Anti-His antibody (Cytiva, CAT#27-4710-01) was used to capture the recombinant human Claudin18.2 protein (GenScript, CAT#Z03504) with a His tag as a ligand, and a Claudin18.2 antibody with a starting concentration of 200 nM was used for 2-fold gradient dilution. A total of 6 samples at different concentrations were used as analytes, and a multi-concentration kinetic affinity test was performed with the captured recombinant Claudin18.2 protein. The binding curve was Kinetics fitted using Biacore software 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-house 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: Binding activity of preferred antibodies to MC38-Claudin18.2 cells

[0109] The human Claudin18.2 sequence (NCBI, NP_001002026) was constructed into the pcDNA3.4 vector, and then transfected into MC38 cells (Nanjing Kebai) using Lipo3000 liposome reagent to construct a stable expression of human Claudin18.2 overexpression cell line MC38-Claudin18.2. 1×10^5 MC38-Claudin18.2 overexpression cell lines were plated in a 96-well plate, and the preferred antibody P1026 and the control antibody IMAB-362 of 100ug / ml mother solution were diluted 5 times, and a total of 8 concentration gradients were set. The gradient diluted antibodies were incubated with the cells for 1h, centrifuged and discarded the supernatant, washed three times with PBS, and then Goat-anti human IgG, PE cojugated fluorescent secondary antibody (Thermo, CAT#12-4998-82) was added for incubation for 1h. After the incubation, the supernatant was centrifuged and discarded, and washed three times with PBS. Flow cytometry was used to detect the binding activity of different concentrations of the preferred antibody and control antibody to MC38-Claudin18.2 cells. The results showed that the cell binding activity of the preferred antibody P1026 was increased by about 16.8 times compared with the control antibody IMAB-362.

[0110]

[0111] Example 4: Endocytic activity of preferred antibodies in NUGC4 cell line

[0112] Gastric cancer cell line NUGC4 has a low level of endogenous expression of Claudin18.2. In order to explore the endocytic activity of the preferred antibody in NUGC4 cells, 10ug / ml of P1026 antibody and 25ul of IMAB362 control antibody were placed in a 96-well plate. TM 25ul of iFL secondary antibody (Thermo, CAT#Z25611) was added to a 96-well plate and mixed well. Then 50ul of NUGC4 overexpressing target cells (purchased from Nanjing Kebai Biotechnology) were added at a density of 2x10 6 The cells and antibodies were mixed evenly and incubated at 37°C for 1h, 2h, 4h, 6h, and 19h. The endocytic activity of the preferred antibody P1026 and the control antibody IMAB362 in NUGC4 cells was detected by flow cytometry. The results are shown in Figure 2As shown, it shows that the preferred antibody P1026 is significantly better than the control antibody IMAB-362 in Claudin18.2 target-mediated internalization of the P1026 antibody in NUGC4 cells.

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

[0114] Suzhou Qide Pharmaceutical Technology Co., Ltd. was commissioned to perform site-specific glycosidic bond coupling of P1026 naked antibody and toxin-linker through the N297 site of the Fc region of the antibody (the coupling method refers to PCT / CN2023 / 098081) to prepare the preferred ADC, named P1026-ADC, whose structure is as follows Figure 1 As shown. Where 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), antibody P1026 removes its 297-position N-sugar chain to obtain an antibody with an Fc region N-glycosylation site modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine, and then an appropriate amount of toxin-linker compound (chemical structure as shown in Figure 8 The preferred ADC antibody drug (denoted as P1026-ADC) after coupling was analyzed by hydrophobic interaction chromatography (HIC). The results are as follows: Figure 3 As shown, the DAR (Drug-antibody ratio) value of P1026-ADC of 4 accounts for more than 95%, indicating that the uniformity of the product obtained 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 ADCs

[0117] Yantai MyPlex International Biopharmaceuticals Co., Ltd. was commissioned to prepare an ADC product (P1026-Dxd) whose linker-toxin is GGFG-Dxd by reducing the four pairs of disulfide bonds of the antibody. The prepared P1026-Dxd and P1026-ADC were divided into five equal parts, each with 200ug, and then 500ul of human plasma was added and mixed, and then incubated at 37°C for 0h, 24h, 48h, 72h, and 96h. The samples incubated for the specified time were purified by CNBr activated Sepharose 4B (Cytiva). Finally, the average DAR value of the purified ADC was detected by mass spectrometry to characterize the stability of the ADC in human plasma. The experimental results are shown in the following table and Figure 4As shown in the figure, as the incubation time of P1026-Dxd with human plasma increases, the toxin shedding ratio gradually increases. After incubation with human plasma for 48 hours, more than 40% of the toxin is shed. After incubation for 72 hours, the mass spectrometer can hardly detect the complete antibody-drug conjugate; while after incubation of P1026-ADC with human plasma for different time periods, the toxin-linker of its ADC is almost not shed. This experiment fully shows that the stability of P1026-ADC is significantly better than that of P1026-Dxd.

[0118]

[0119]

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

[0121] In order to explore the in vitro activity of the P1026-ADC molecule, a cell killing experiment was performed using the NUGC4 cell line of tumor cells that stably overexpressed the Claudin18.2 target (cells were purchased from Nanjing Kebai Biotechnology Co., Ltd., Cat. Number: CBP74135). The experimental process is briefly described as follows: when the NUGC4-Claudin18.2 cell line grows to the logarithmic growth phase, the cells are plated in a 96-well plate (Corning, CAT#: 3599) and cultured overnight at 37°C in a carbon dioxide incubator. The next day, the 1ug / ml P1026-ADC mother solution sample was diluted 3 times in a gradient, with a total of 8 concentration gradient points. The diluted P1026-ADC molecule to be tested was added to a 96-well plate containing the NUGC4-Claudin18.2 cell line and placed 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 reacting for 2 hours, the SpectraMAX i3x multifunctional microplate reader from Molecular Device was used to detect the wavelength OD450nm in ABS mode, and the reference wavelength OD630nm was set. The cell killing percentage was calculated, and the four-parameter fitting was performed using Graphpad software to obtain the IC50 value of the ADC molecule. The results showed that the killing activity IC50 of P1026-ADC against the NUGC4-Claudin18.2 cell line was 0.742nM. The cell killing results are shown in Figure 5 shown.

[0122] Experimental Example 8: Optimization of the efficacy of ADC in a cell line-derived xenograft tumor (CDX) model in animals

[0123] In order to explore the in vivo efficacy of P1026-ADC molecules, the NUGC4 cell line stably overexpressing Claudin18.2 target (cells purchased from Nanjing Kebai Biotechnology Co., Ltd., Cat. Number: CBP74135) was used to establish an in vivo NUGC4-Claudin18.2 CDX model in animals to evaluate the efficacy of the preferred ADC. Specifically, each male severely immunodeficient (NDG) mouse was subcutaneously inoculated with 5x10 6 NUGC4-CLDN18.2 cells were inoculated, and tumors were selected to grow to about 120 mm 5 days after the cells were inoculated. 3 The mice were randomly divided into groups, with 5 mice in each group. The drugs were injected into the tail vein on the 6th day. The specific drug information is shown in the table below. Figure 6 As shown, a single administration of P1026-ADC can significantly inhibit the growth of tumors in animals, and the tumors were basically regressed after 45 days of observation. Although the clinical products CMG-901 and SHR1904-ADC can inhibit tumor growth in the early stage, the tumors gradually resumed growth after 30 days.

[0124]

[0125] Experimental Example 9: Optimizing the efficacy of ADC in PDX models

[0126] We commissioned Sino-US Crown Biotech Co., Ltd. to use GA0006 tumor tissue from gastric cancer patients to heterotopically transplant Balb / c Nude mice to construct a humanized tissue xenograft (PDX) model. Specifically, after the mice were implanted with tumor pieces, the tumors in the mice grew to an average volume of 150mm. 3 The subjects were randomly divided into two groups: the control group (Ctl. group) was given PBS; the experimental group was given CMG901-ADC at a dose of 2 mpk, once a week for a total of 3 times; the experimental group was given P1026-ADC at a single dose of 10 mpk. The entire experiment lasted for 25 days from administration to the end of the experiment. The experimental results are shown in Figure 7 As shown, a single administration of P1026-ADC can significantly inhibit the growth of tumors in animals.

[0127] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein 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, such as 1-9, 2-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r is 1-5, preferably 1 or 2; 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; The toxin drug has the following structure:

2. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises: the heavy chain variable region set forth in SEQ ID NO:7; and the light chain variable region set forth in SEQ ID NO:

8.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 2, wherein the anti-Claudin18.2 antibody is selected from the following group: a whole antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and a fully human antibody; and / or, 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; And / or, the antibody heavy chain constant region is selected from IgG series antibodies; preferably, the IgG series antibodies are selected from one or more of IgG1, IgG2 and IgG4, and IgG1 is further preferred; And / or, the light chain constant region is selected from a kappa or lambda chain.

4. The antibody drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the linker is one or more of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide; wherein the GGFG tetrapeptide Linker is preferred.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the antibody-drug conjugate has the following structure: in, p is 1-10, such as 1-9, 2-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, of which 2 is preferred.

6. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein the antibody drug conjugate has an average DAR of 1-15, such as 2-10, 2-8 or 3-5.

7. The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody is an antibody with engineered glycosylation, such as an antibody obtained after enzymatic modification of sugar chains in vitro (e.g., modification of sugar chains by glycosidases (e.g., endoglycosidases or glycosyltransferases)), preferably, the antibody with engineered glycosylation refers to an antibody in which the sugar chains at the glycosylation sites of the antibody are transformed from non-uniformly structured N sugar chains to single-structured N sugar chains with reactive groups (e.g., any reactive groups that can react with linker moieties, such as azide, keto and alkyne), more preferably, the N-glycosylation site is a conserved N-glycosylation site on the Fc domain of the antibody, such as Asn297.

8. The antibody drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the linker is a linker capable of achieving site-specific coupling, preferably a linker connected to an antibody oligosaccharide, preferably, the oligosaccharide linker is a linker comprising an alkynyl group, most preferably, the oligosaccharide linker is a linker capable of achieving specific site coupling with a reactive group (e.g., an azide group) on the N-sugar chain at a conserved N-glycosylation site (e.g., Asn297) on the antibody Fc domain.

9. A composition comprising the antibody drug conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or solvate thereof and optionally a pharmaceutically acceptable excipient.

10. Use of the antibody drug conjugate according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for preventing and / or treating cancer or tumor; in, The cancer or tumor is a cancer or tumor that is positive for CLDN18.2 expression, wherein the cancer or tumor is selected from bladder cancer, ovarian cancer, lung cancer, adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, kidney cancer, colon cancer, small intestine cancer, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, esophageal cancer and gallbladder cancer; And / or, wherein the drug is a drug for cell therapy.

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