Antibody-drug conjugates targeting Claudin18.2

Abstract

The present invention relates to antibody-drug conjugates (ADCs) targeting Claudin18.2 and compositions comprising said molecules. The present invention further relates to therapeutic and diagnostic uses of these antibodies or antibody fragments.

Description

[Technical Field]

[0001] The present invention relates to antibody-drug conjugates (ADCs) that target Claudin18.2 (CLDN18.2) and compositions comprising said molecules. The invention further relates to therapeutic and diagnostic uses of these antibodies or antibody fragments. [Background technology]

[0002] Claudins are a family of proteins that are important components of tight junctions in cells. They establish an intercellular barrier that regulates the movement of molecules between cells. Claudins family proteins contain four transmembrane domains, and both their N- and C-termini are cytoplasmic. Different Claudins proteins are expressed in different tissues, and their functional changes are associated with carcinogenesis in each tissue. For example, Claudin-1 is expressed in colon cancer and has been shown to have prognostic value, Claudin-18 is highly expressed in gastric and pancreatic cancers, and Claudin-10 is highly expressed in hepatocellular carcinoma. As cell membrane surface proteins, Claudins are useful targets for various therapeutic strategies.

[0003] Claudin 18 isotype 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker whose expression in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa, but is absent in the gastric stem cell region. CLDN18.2 is expressed in a significant number of primary gastric cancers and maintains its expression level in gastric metastatic cancer tissues. CLDN18.2 expression is also observed in pancreatic cancers, making it an ideal target molecule for treating these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as a new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017). https: / / doi.org / 10.1186 / s13045-017-0473-4).

[0004] In 2014, there were approximately 410,000 new cases and 290,000 deaths nationwide, accounting for nearly half of the world's total cases and deaths, and the number is still increasing. However, there is a significant unmet need for clinical oncology treatment, and the development of drugs targeting Claudin 18.2 is extremely necessary.

[0005] Despite the clinical success of therapeutic antibodies, naked MAbs targeting cell surface tumor antigens alone rarely provide sufficient efficacy. To increase the low activity of MAbs, new strategies focus on conjugation to toxic molecules. Plant and bacterial toxins and small chemotherapeutic molecules are potentially good candidates because they are highly effective and active in very small doses.

[0006] Due to technological advances over the past few years, the field of antibody-drug conjugates (ADCs) for the treatment of cancer has recently seen an increasing amount of development activity by pharmaceutical companies, aiming to solve the problems that initially present with regard to immunogenicity, affinity, specificity, unwanted toxicity, productivity, half-life, etc.

[0007] Although some progress has been achieved, there remains a need for other therapeutic strategies for treating tumors, as well as components for use in such therapeutic strategies, particularly antibodies against Claudin18.2 that have high affinity, high specificity, and / or low immunogenicity risk, and ADC molecules that have relatively high activity, low toxicity, long half-life, high specificity, or high affinity, and / or favorable half-life or pharmacophore properties. Summary of the Invention

[0008] The present invention provides antibody-drug conjugates (ADCs) targeting Claudin18.2, which have the following advantages:

[0009] (1) It binds to target cells expressing human CLDN18.2 with high affinity. (2) They can enter cells by endocytosis and kill target cells, and in some embodiments, the ADCs of the present invention have high endocytosis efficiency. (3) It has a significant bystander killing effect. (4) It has high antitumor efficacy. (5) It has low toxicity. (6) Good stability. (7) It has good drug discovery potential.

[0010] In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface. In some embodiments, the target cells are CHO cells or 293 cells that express CLDN18.2, such as CHO-S cells or HEK293 cells. In some embodiments, the target cells are cancer cells that express CLDN18.2, such as cells that naturally express CLDN18.2, cells that express CLDN18.2 by artificial transfection, or cells that have increased CLDN18.2 expression levels by artificial transfection, such as gastric cancer cells, pancreatic cancer cell lines, or colon or colorectal cancer cell lines that express CLDN18.2. In some embodiments, the target cells are cell lines with intermediate hCLDN18.2 expression levels, such as NUGC-4 and SNU620. In some embodiments, the target cells are cell lines with high hCLDN18.2 expression levels, such as DAN-G cells that overexpress hCLDN18.2. [Brief explanation of the drawings]

[0011] [Figure 1] This shows that the HB37A6 antibody specifically binds to CLDN18.2 on the cell surface. [Figure 2] This shows that the HB37A6 antibody does not bind to CLDN18.1 on the cell surface. [Figure 3] 1 shows the binding of the HB37A6 antibody to the gastric cancer cell line NUGC-4, the gastric cancer cell line KATO III-hCLDN18.2, and the pancreatic cancer cell line DAN-G-hCLDN18.2. [Figure 4] 1 shows the antitumor effect of HB37A6 antibody in a mouse model of pancreatic cancer. [Figure 5] 1 shows the antitumor effect of HB37A6 antibody in a mouse model of gastric cancer. [Figure 6] 1 shows the cell binding activity of the IEX019 molecule. [Figure 7] 1 shows endocytosis of IEX019 molecules in DANG-hCLDN18.2 cells. [Figure 8A]The killing effect of IEX019 molecules on cell lines with low hCLDN18.2 expression (Figure 8A), the killing effect of IEX019 molecules on cell lines with moderate hCLDN18.2 expression (Figure 8B), and the killing effect of IEX019 molecules on cell lines with high hCLDN18.2 expression (Figure 8C) are shown. [Figure 8B] The killing effect of IEX019 molecules on cell lines with low hCLDN18.2 expression (Figure 8A), the killing effect of IEX019 molecules on cell lines with moderate hCLDN18.2 expression (Figure 8B), and the killing effect of IEX019 molecules on cell lines with high hCLDN18.2 expression (Figure 8C) are shown. [Figure 8C] The killing effect of IEX019 molecules on cell lines with low hCLDN18.2 expression (Figure 8A), the killing effect of IEX019 molecules on cell lines with moderate hCLDN18.2 expression (Figure 8B), and the killing effect of IEX019 molecules on cell lines with high hCLDN18.2 expression (Figure 8C) are shown. [Figure 9] 1 shows the bystander killing effect of the IEX019 molecule. [Figure 10A] The tumor suppression effect (FIG. 10A) and body weight change (FIG. 10B) of the IEX019 molecule in mice are shown. [Figure 10B] The tumor suppression effect (FIG. 10A) and body weight change (FIG. 10B) of the IEX019 molecule in mice are shown. [Figure 11A] The tumor suppression effect (FIG. 11A) and body weight change (FIG. 11B) of the IEX019 molecule in mice are shown. [Figure 11B] The tumor suppression effect (FIG. 11A) and body weight change (FIG. 11B) of the IEX019 molecule in mice are shown. [Figure 12A] The tumor suppression effect (FIG. 12A) and body weight change (FIG. 12B) of the IEX019 molecule in mice are shown. [Figure 12B] The tumor suppression effect (FIG. 12A) and body weight change (FIG. 12B) of the IEX019 molecule in mice are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] I. Definition

[0013] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, configuration, or reagents described herein, as these may vary. Furthermore, the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is limited only by the claims. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0014] The following definitions will be used to interpret this specification, and where appropriate, terms used in the singular may also include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0015] The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​that is 5% less than the number or numerical value specified as the lower limit and 5% greater than the number or numerical value specified as the upper limit.

[0016] As used herein, the term "and / or" refers to any one of possible options or two or more of possible options.

[0017] As used herein, the term "comprise" or "include" means including the above-mentioned elements, integers, or steps, but not excluding any other elements, integers, or steps. When the term "comprise" or "include" is used herein, unless otherwise specified, it also encompasses combinations of the above-mentioned other elements, integers, or steps. For example, when an antibody variable region "comprising" a specific sequence is referred to, it is also intended to encompass an antibody variable region consisting of this specific sequence.

[0018] As used herein, "Claudin" or "CLDN" refers to the most important scaffolding protein that determines the structure of intercellular tight junctions. It is involved in adherens junctions and plays an important role in tumor cell metastasis and invasion. Claudin proteins are widely distributed in mammalian epithelial and endothelial cells, primarily distributed on the lateral and basal plasma membranes of epithelial cells. Different claudin proteins have specific expression profiles in different tissues. Among them, the Claudin18 (CLDN18) gene, located on 3q22.3, has a molecular weight of 24 kDa and contains 261 amino acid residues. It is a member of the Claudins superfamily and consists of two extracellular loops and four transmembrane domains. 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 (UniProt ID: P56856-2), respectively. In the primary structural sequences of both proteins, only amino acid residues at certain positions from the N-terminal signal peptide to the extracellular loop 1 (Loop 1) structure differ, and in particular, CLDN18.1 and CLDN18.2 differ by only eight amino acids in extracellular loop 1. The interspecies sequence homology of the two CLDN18 subtype proteins is also very high. The extracellular loop 1 of CLDN18.2 has a completely identical sequence across different organisms, including humans, mice, and rhesus monkeys, and the homology between human and mouse CLDN18.2 proteins reaches 84%, revealing that the CLDN18.2 protein sequence is highly conserved (O. Tureci et al., Gene 481:83-92, 2011). CLDN18.2 or any of its variants and isotypes can be isolated from cells or tissues that naturally express them or recombinantly produced using techniques well known in the art and / or described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0019] As used herein, the terms "anti-CLDN18.2 antibody," "anti-CLDN18.2," "CLDN18.2 antibody," "antibody that binds to CLDN18.2," or "antibody that specifically binds to CLDN18.2" refer to an antibody that binds to (human) CLDN18.2 with sufficient affinity and can be used as a therapeutic agent that targets (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 by, for example, radioimmunoassay (RIA), biolayer interferometry (BLI), MSD assay or surface plasmon resonance (SPR) or flow cytometry.

[0020] The expression of CLDN18.2 in cells can be determined by various means, for example, anti-CLDN18.2 antibody. For example, the binding strength (e.g., measured by FACS) between "highly CLDN18.2-expressing" cells and anti-CLDN18.2 antibody may be 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or preferably 1000-fold or more, for example, 1100-fold, 1200-fold, 1300-fold, 1400-fold or more, than the binding strength between anti-CLDN18.2 antibody and cells that do not express CLDN18.2. For example, the binding strength (e.g., as measured by FACS) between cells that "moderately express CLDN18.2" and an anti-CLDN18.2 antibody may be 5 to 500 times greater than the binding strength between the anti-CLDN18.2 antibody and cells that do not express CLDN18.2, such as 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times greater, but not more than 500 times greater.

[0021] The terms "complete antibody," "whole antibody," or "full-length antibody" are used interchangeably herein and refer to an antibody molecule having a natural immunoglobulin molecular structure. In the case of a typical four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of a heavy-chain antibody having only heavy chains and lacking light chains, a full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds.

[0022] In a typical four-chain IgG antibody, the full-length antibody heavy chain generally consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, where the heavy chain constant region comprises at least three domains, CH1, CH2, and CH3. The full-length antibody light chain generally consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, where the light chain constant region comprises one domain, CL. Each heavy chain variable region, VH, and each light chain variable region comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0023] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0024] "Antigen-binding fragment" refers to a molecule that contains a portion of an intact antibody and binds to the same antigen as the intact antibody, but is different from the intact antibody. 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 (e.g., VHH), bivalent antibody or fragment thereof, or camelid antibody.

[0025] The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that almost any macromolecule, including proteins or peptides, can be used as an antigen. Antigens may also be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0026] "Complementarity determining regions," "CDR regions," or "CDRs" are regions in antibody variable domains that are hypervariable in sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. Heavy and light chain CDRs are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs in an antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs in an antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3.For a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined by any one or a combination of many known antibody CDR assignment systems, such as the Chothia system, which is based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), the Kabat system, which is based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USDapartment of Health and Human Services, National Institutes of Health (1987)), the AbM (University of Bath), the Contact (University College London), and the International ImMunoGeneTics (ImmunoGeneTics). These include the database (IMGT) (available on the World Wide Web at imgt.cines.fr / ), and the North CDR definition, which is based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)).

[0027] Below are the CDR region ranges defined in the Kabat, AbM, Chothia, Contact and IMGT methods.

[0028] [Table 1]

[0029] A CDR may be determined by having the same Kabat numbering position as the sequence of a reference CDR (eg, any one of the exemplary CDRs of the invention).

[0030] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined by any one of the above methods.

[0031] Unless otherwise specified, in the present invention, when a residue position in an antibody variable region (including heavy chain variable region residues and light chain variable region residues) is referred to, it refers to a numbered position based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0032] In one embodiment, the CDRs of the heavy chain variable region of the antibody according to the present invention are determined according to the following rules.

[0033] VH CDR1 is determined according to the AbM rules, and VH CDR2 and VH CDR3 are both determined according to the Kabat rules.

[0034] In one embodiment, the CDRs of the light chain variable region of the antibody of the present invention are determined according to the Kabat rules.

[0035] In one embodiment, the heavy chain variable region CDRs of the antibody of the present invention are determined according to the following rules: VH CDR1 is determined according to the AbM rules, VH CDR2 and VH CDR3 are both determined according to the Kabat rules, and the light chain variable region CDRs are determined according to the Kabat rules.

[0036] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained using different allocation systems may differ. That is, there will be differences in the CDR sequences of the variable regions of the same antibody defined using different allocation systems. Therefore, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but whose CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the use of a different method (e.g., the rules or combinations of different allocation systems).

[0037] Antibodies with different specificities (i.e., targeting different binding sites of different antigens) have different CDRs (in the same assignment system). However, despite the differences in CDRs between antibodies, the number of amino acid positions directly involved in antigen binding is limited in the CDRs. The minimum overlapping region can be determined by at least two of the Kabat, Chothia, AbM, Contact, and North methods to provide a "minimum binding unit" for antigen binding. The minimum binding unit may be a subportion of a CDR. As known to those skilled in the art, the residues of the remaining CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention contemplates variants of any of the CDRs provided herein. For example, in a variant of one CDR, the amino acid residues of the minimum binding unit are kept unchanged, but the remaining CDR residues defined by Kabat or Chothia may be substituted with conservative amino acid residues.

[0038] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of IgG, IgA, and IgD class antibodies, or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) derived from the two heavy chains of IgM and IgE class antibodies. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is based on the EU numbering system (also referred to as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, nor the heavy chain constant region CH1 and light chain constant region CL, but optionally includes the hinge region at the N-terminus of the heavy chain constant region.

[0039] "IgG-type antibody" refers to the IgG type to which the antibody's heavy chain constant region belongs. All antibodies of the same type have the same heavy chain constant region, while antibodies of different types have different heavy chain constant regions. For example, an IgG4-type antibody has a heavy chain constant region derived from IgG4, and an IgG1-type antibody has a heavy chain constant region derived from IgG1.

[0040] As used herein, the terms "binding" or "specifically binding" mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other conventional binding assays known in the art, such as radioimmunoassay (RIA), biolayer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0041] As used herein, "antibody-drug conjugate (ADC)" refers to a structure obtained by linking an antibody and a drug.

[0042] As used herein, the general term "sugar" refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). As used herein, the term "sugar derivative" refers to a derivative of a monosaccharide, i.e., a monosaccharide containing a substituent and / or 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), N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Examples of sugar derivatives further include compounds represented herein as E(A)x, where E is a sugar or sugar derivative and E contains x functional groups A.

[0043] A core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as a GlcNAc attached to an 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 can be present at a native glycosylation site of the antibody, but can also be introduced into a different site on the antibody. As used herein, a core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fucα1-6)GlcNAc substituent, also referred to as GlcNAc(Fuc).

[0044] "Glycosylation modification" refers to the process of altering the carbohydrate moieties of an antibody through the glycosylation process. The glycosylation of an antibody can be further modified for various purposes to obtain a newly glycosylated antibody. For example, glycosylation can be removed to eliminate FcγR affinity and complement binding / effector function, or fucose and sialic acid groups can be reduced and bispecific N-acetylglucosamine, galactose, and mannose can be increased to enhance Fc-mediated ADCC and CDC effects. Methods for glycosylation modification known in the art include, for example, altering the glycosylation site of the antibody to increase or decrease the carbohydrate moieties on the antibody surface, chemically or enzymatically modifying the carbohydrate moieties in vitro, or catalyzing antibody glycosylation by altering the glycosylation pathway (e.g., composed of enzymes such as glycosidases and glycosyltransferases) in the expression system, and affecting cell culture conditions to alter the glycosylation of the antibody. In some embodiments, the "glycosylation modification" of the present invention is performed by enzymatic modification of the carbohydrate moieties in vitro. Preferably, the glycosylation modification of the present invention is carried out by modifying sugar chains with a glycosidase (such as an endoglycosidase or a glycosyltransferase).

[0045] The term "antibody with altered glycosylation" as used herein refers to an antibody whose glycosylation mode is altered compared to an antibody with a native glycosylation mode. Preferably, the term "antibody with altered glycosylation" refers to an antibody obtained after expression in an expression system (e.g., mammalian cells) and enzymatic modification of the sugar chain in vitro (e.g., modification of the sugar chain with a glycosidase, such as an endoglycosidase or glycosyltransferase). More preferably, the term "antibody with altered glycosylation" refers to an antibody comprising a core-GlcNAc and a sugar derivative E(A)x linked thereto, wherein the GlcNAc is attached 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. When the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, the fucose is generally linked to C6 of the -GlcNAc substituent via α-1,6. The fucosylated -GlcNAc substituent is referred to as core-GlcNAc(Fuc), and the fucosylated GlcNAc-E(A)x substituent is referred to as GlcNAc(Fuc)-E(A)x.

[0046] The term "site-specific coupling" as used herein refers to coupling in which a drug / active substance is specifically linked to a specific site on an antibody via a linker.

[0047] The term "alkyl group," as used herein, refers to a fully saturated, branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

[0048] The term "aryl group" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 20, e.g., 6 to 12, carbon atoms in the ring portion. Preferably, an aryl group is (C6-C 10 ) aryl groups. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl groups, each of which is optionally substituted with 1 to 4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)—O—, aryl-O—, heteroaryl-O—, amino, mercapto, alkyl-S—, aryl-S—, nitro, cyano, carboxy, alkyl-OC(O)—, carbamoyl, alkyl-S(O)—, sulfonyl, sulfonamido, heterocyclyl, and the like, where R is independently hydrogen, alkyl, aryl, heteroaryl, aryl-alkyl, heteroaryl-alkyl, and the like.

[0049] The term "cycloalkyl group" refers to a cyclic alkyl group, i.e., a monovalent saturated or unsaturated hydrocarbon group having a cyclic structure. Cycloalkyl groups include all saturated or partially saturated (containing one or two double bonds) hydrocarbon groups having a cyclic structure. Cycloalkyl groups may contain three or more, e.g., 3 to 18, 3 to 10, or 3 to 8 carbon atoms in the ring, and generally, according to the present invention, contain 3 to 6 atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0050] As used herein, the term "heteroaryl group" refers to a 5- to 20-membered (e.g., 5- to 14-membered, 5- to 8-membered, 5- to 6-membered) monocyclic, bicyclic, or fused polycyclic ring system containing 1 to 8 heteroatoms selected from N, O, or S. Preferably, the heteroaryl group is a 5- to 10-membered ring system. Representative heteroaryl groups are 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2- or 4- or 5-imidazolyl, 3- or 4-pyrazolyl, 2- or 4- or 5-thiazolyl, 3- or 4- or 5-isothiazolyl, 2- or 4- or 5-oxazolyl groups. , 3-, 4- or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-, 2-, 4- or 5-pyridinyl.

[0051] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and performance of the ADC conjugates of the present invention, and which is not biologically or otherwise undesirable. The ADC conjugates of the present invention can 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 between the ADC conjugates of the present invention and 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, etc. A pharmaceutically acceptable non-toxic base addition salt refers to a salt formed between the ADC conjugate of the present invention and an organic or inorganic base, and includes, but is not limited to, alkali metal salts such as lithium salts, sodium salts, or potassium salts, alkaline earth metal salts such as calcium salts or magnesium salts, and organic base salts such as ammonium salts formed with an N-containing organic base.

[0052] The term "solvate" refers to a combination formed between one or more solvent molecules and an ADC conjugate of the invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0053] Unless contradicted by context, "pharmaceutically acceptable" and "medicinal" are used interchangeably herein.

[0054] The term "drug:antibody ratio" or "DAR" refers to the ratio of small molecule drug moieties (D) coupled to Ab moieties described herein to Ab moieties. In some embodiments described herein, the DAR can be determined by p and r in Formula I, e.g., the DAR can be 1 to 20, e.g., 2 to 18, 4 to 16, 5 to 12, 6 to 10, 2 to 8, 3 to 8, 2 to 6, 4 to 6, 6 to 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR can also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of small molecule drug moieties (D) coupled to Ab moieties described herein to Ab moieties in the product, as determined by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA measurement, electrophoresis, and / or HPLC), and this DAR is referred to herein as the average DAR. In some embodiments, the average DAR of the conjugates of the present invention is 1 to 20, e.g., 2 to 18, 4 to 16, 5 to 12, 6 to 10, 2 to 8, 3 to 8, 2 to 6, 4 to 6, 6 to 10, e.g., 1.0 to 8.0, 2.0 to 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.

[0055] As used herein, the term "therapeutic agent" includes any substance effective in the treatment or prevention of tumors, such as cancer, including chemotherapeutic agents, cytokines, antiangiogenic agents, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0056] 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.

[0057] A "chemotherapeutic agent" includes a chemical compound useful in the treatment of cancer or immune system disorders.

[0058] The term "small molecule drug" refers to a low-molecular-weight organic compound that can regulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, generally less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules are more cell-permeable, more susceptible to degradation, and less likely to induce an immune response than large molecules.

[0059] As used herein, the term "immunomodulatory agent" refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response may be a humoral or cellular response. Immunomodulatory agents include immunosuppressants. In some embodiments, immunomodulatory agents of the invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0060] The term "effective amount" refers to an amount or dosage that will produce the desired effect in a patient in need of treatment or prevention after administration of one or more doses of an antibody or fragment or composition or combination of the invention to the patient.

[0061] A "therapeutically effective amount" refers to an amount that effectively achieves a desired therapeutic result at the necessary dosage for the necessary period of time. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody fragment, or composition, or combination, do not outweigh the beneficial effects of the treatment. Compared to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, and even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0062] A "prophylactically effective amount" refers to an amount effective to achieve a desired prophylactic result at a necessary dosage for a necessary period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is administered prior to or at an earlier stage of disease in a subject.

[0063] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be exactly identical to the parent cell in nucleic acid content and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as screened or selected from the primary transformed cell are included.

[0064] As used herein, the term "label" refers to a compound or composition that is directly or indirectly attached to or fused to a reagent (e.g., a polynucleotide probe or antibody) and that facilitates detection by the reagent to which it is attached or fused. The label may be itself detectable (e.g., a radioisotope label or a fluorescent label) or, when labeled by enzyme catalysis, may catalyze the chemical alteration of a substrate compound or composition that is detectable. The term is intended to include 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 of a probe or antibody by reaction with another reagent that is directly labeled.

[0065] An "individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, goats, cats, dogs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0066] An "isolated" antibody or other molecule (e.g., an ADC molecule) is an antibody or molecule that has been separated from a component of its natural environment or its expression environment. In some embodiments, the antibody or ADC molecule is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0067] The term "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a decrease in tumor volume, a decrease in tumor cell number, a decrease in tumor cell proliferation, or a decrease in tumor cell viability.

[0068] The terms "tumor" and "cancer" are used interchangeably herein and include solid tumors and hematological tumors.

[0069] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the invention include gastric, pancreatic, or gastroesophageal junction cancer, including metastatic forms of these cancers.

[0070] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells or tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0071] The term "pharmaceutical adjuvant" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete or incomplete)), excipient, vector, stabilizer, etc., administered with an active substance.

[0072] The term "pharmaceutical composition" refers to a composition that contains an active ingredient in a form that is effective for the biological activity of the active ingredient, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0073] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including, but not limited to, a drug kit and a pharmaceutical composition. The term "non-fixed combination" refers to active ingredients (e.g., (i) an ADC molecule of the present invention and (ii) another therapeutic agent) administered to a patient simultaneously as separate entities, without specific time restrictions, or sequentially at the same or different time intervals, where such administration provides prophylactically or therapeutically effective levels of the two or more active agents in vivo in the patient. In some embodiments, the ADC molecule of the present invention and the other therapeutic agent used in the pharmaceutical combination are administered at levels that do not exceed the levels at which they are administered alone. The term "fixed combination" refers to two or more active agents administered to a patient simultaneously in the form of a single entity. Preferably, by selecting the doses and / or time intervals of the two or more active agents, the combination of each component can achieve a better effect in treating a disease or condition than the use of any one component alone. Each component may be in a separate formulation, which may be the same or different formulations.

[0074] The term "combination therapy" refers to the treatment of a disease described herein by administering two or more therapeutic agents or forms of treatment (e.g., radiation therapy or surgery). Such administration includes co-administration of these therapeutic agents at approximately the same time, e.g., in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, such administration includes sequential use of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen provides the beneficial effect of the pharmaceutical combination in treating the disease or condition described herein.

[0075] As used herein, "treating" refers to alleviating, interrupting, slowing, ameliorating, arresting, reducing, or reversing the progression or severity of an existing symptom, condition, pathology, or disease.

[0076] As used herein, "prevention" includes inhibiting the onset or progression of a disease, condition, or symptom associated with a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a prevention program. Generally, in the context of cancer, the term "prevention" refers to the administration of a medication before symptoms or symptoms of cancer arise, particularly before cancer occurs in subjects at risk of developing cancer.

[0077] 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 and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0078] A "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 may be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or aspirates; blood or any blood component; bodily fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from any stage of pregnancy or development in a subject. Tissue samples may contain compounds that are not naturally associated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0079] II. Antibody-Drug Conjugates

[0080] The present invention relates to a compound of formula (I): Ab-(L-(D) r ) p (I) or a pharmaceutically acceptable salt or solvate thereof, During the ceremony, Ab is an antibody or fragment thereof that binds to CLDN18.2 (e.g., human CLDN18.2); L is a linker, D is a drug containing a prodrug, preferably an antitumor compound; and p is 1 to 10, for example 1 to 9, 2 to 8, 3 to 7, 4 to 6, or 2 to 6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and r is 1 to 5, for example, 1, 2, 3, 4, or 5, and preferably 1 or 2.

[0081] In some embodiments, the Ab in formula (I) of the present invention is a human antibody or a humanized antibody, preferably a human antibody. In some embodiments, the Ab in formula (I) of the present invention is an antibody fragment, preferably an antigen-binding fragment such as Fv, Fab, Fab', Fab'-SH, or F(ab')2, a dAb (domain antibody), a linear antibody, a single-chain antibody (e.g., scFv), a single-domain antibody (e.g., VHH), a bivalent antibody or fragment thereof, or a camelid antibody.

[0082] In some embodiments, the Ab in formula (I) of the present invention is a bispecific or multispecific antibody.

[0083] In a preferred embodiment of the invention, the Ab comprises three complementarity determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2 and HCDR3.

[0084] In a preferred embodiment of the invention, the Ab comprises three complementarity determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2 and LCDR3.

[0085] In some embodiments, the Ab comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0086] In some embodiments, the Ab comprises a heavy chain variable region (VH). In some embodiments, the Ab comprises a light chain variable region (VL). In some embodiments, the Ab comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementarity determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0087] In some embodiments, the VH is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 4; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 4; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, and preferably, the amino acid changes do not occur in the CDR regions.

[0088] In some embodiments, the VL is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 9; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 9; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 9, and preferably, the amino acid changes do not occur in the CDR regions.

[0089] In some embodiments, the three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 from a VH of the invention are: (i) the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 4; or (ii) A sequence containing at least one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in total in the three HCDR regions relative to the sequence of (i).

[0090] In some embodiments, the three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 from a VL of the invention are: (i) the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 9; or (ii) A sequence containing at least one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in total in the three LCDR regions compared to the sequence of (i).

[0091] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; alternatively, HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1.

[0092] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:2, or alternatively, HCDR2 comprises an amino acid sequence that has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:2.

[0093] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; alternatively, the HCDR3 comprises an amino acid sequence that has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3.

[0094] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 6, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6.

[0095] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 7.

[0096] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 8, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 8.

[0097] In some embodiments, the Ab in formula (I) of the present invention comprises a heavy chain constant region. In some embodiments, the Ab in formula (I) of the present invention comprises a light chain constant region. In some embodiments, the Ab in formula (I) of the present invention further comprises a heavy chain constant region and a light chain constant region.

[0098] In some embodiments, the heavy chain constant region HC of the present invention is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, e.g., a wild-type IgG1 heavy chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a lambda or kappa light chain constant region.

[0099] In some preferred embodiments, the heavy chain constant region H of the present invention is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 5; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 5; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 5.

[0100] In some embodiments, the antibody light chain constant region LC of the invention is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 10; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 10; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 10.

[0101] In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain. In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a light chain. In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain and a light chain.

[0102] In some specific embodiments of the invention, heavy chains of the invention comprise or consist of a heavy chain variable region and a heavy chain constant region, and in some specific embodiments of the invention, light chains of the invention comprise or consist of a light chain variable region and a light chain constant region.

[0103] In some specific embodiments, the Ab in formula (I) of the present invention specifically binds to CLDN18.2 and comprises three complementarity-determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 4, and / or three complementarity-determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 9.

[0104] In some specific embodiments of the present invention, the Ab in formula (I) of the present invention comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and / or LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively.

[0105] In some specific embodiments of the present invention, Ab in formula (I) of the present invention is a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or It comprises a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 90% identity thereto.

[0106] In some embodiments of the present invention, the Ab in formula (I) of the present invention comprises a VH and a VL, wherein the amino acid sequence of the VH is shown in SEQ ID NO:4 and the amino acid sequence of the VL is shown in SEQ ID NO:9.

[0107] In some specific embodiments of the present invention, the Ab in formula (I) of the present invention is an IgG antibody, i.e., comprises a heavy chain and a light chain that binds to CLDN18.2. In some embodiments, the Ab in formula (I) of the present invention is a complete antibody.

[0108] In some embodiments, the heavy chain of the Ab in formula (I) is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 11; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 11; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 11.

[0109] In some embodiments, the light chain of the Ab in formula (I) is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 12; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 12.

[0110] In some embodiments of the present invention, the Ab in formula (I) of the present invention comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 11 and the amino acid sequence of the light chain is set forth in SEQ ID NO: 12.

[0111] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid changes described herein occur in the Fc region of the antibody heavy chain constant region.

[0112] In some embodiments, the substitution is conservative. A conservative substitution refers to the replacement of one amino acid with another amino acid of the same type, for example, the replacement of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. In some embodiments, the substitution occurs in the CDR region of the antibody. Generally, the resulting variant has a modification (e.g., improvement) in certain biological properties (e.g., improved affinity) relative to the parent antibody and / or substantially retains certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody.

[0113] The antibody Ab of formula (I) of the present invention may be an antibody with altered glycosylation. In some embodiments, the antibody is obtained after in vitro enzymatic modification of its sugar chain (e.g., modification of the sugar chain with a glycosidase (e.g., endoglycosidase or glycosyltransferase)). In some embodiments, the antibody with altered glycosylation refers to an antibody in which the sugar chain at the glycosylation site of the antibody has been altered from a heterogeneous N-sugar chain to a monosaccharide N-sugar chain bearing a reactive group (e.g., any reactive group capable of reacting with a linker moiety, such as an azide group, a ketone group, or an alkynyl group). In one preferred embodiment, the N-glycosylation site of the antibody is a conserved N-glycosylation site in the antibody Fc domain, e.g., Asn297.

[0114] For methods of modifying antibody glycosylation suitable for the present invention, see, for example, PCT / NL2013 / 050744, PCT / EP2016 / 059194 or PCT / EP2017 / 052792, which patent applications are incorporated herein in their entirety.

[0115] In one preferred embodiment, the antibody with altered glycosylation of the invention is GlcNAc-E(A). x an antibody comprising a substituent, wherein GlcNAc is N-acetylglucosamine, and wherein E(A)x is a sugar derivative containing x functional groups A, where A is independently selected from azide, ketone, and alkynyl, and x is 1, 2, 3, or 4, wherein the GlcNAc-E(A)x substituent is attached to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent, and wherein the N-acetylglucosamine is optionally fucosylated. If the N-acetylglucosamine is fucosylated, it is attached to fucose (Fuc) via C6.

[0116] In one embodiment, the antibody with altered glycosylation of the present invention is an antibody of formula (III), where Ab represents an antibody, GlcNAc represents N-acetylglucosamine, Fuc represents fucose, b represents 0 or 1, and y represents 1 to 20, and E(A)x represents a sugar derivative containing x functional groups A, where A represents an azide group, a ketone group, and an alkynyl group, and x represents 1, 2, 3, or 4. In a preferred embodiment, y represents 1 to 10, more preferably 1, 2, 3, 4, 5, 6, 7, or 8, even more preferably 1, 2, 3, or 4, and most preferably y represents 1 or 2. [ka]

[0117] The sugar derivative E(A) in the GlcNAc-E(A) substituent of an antibody with altered glycosylation can be, for example, linked to C4 of the GlcNAc via a β(1,4)-glycosidic bond or to C3 of the GlcNAc via an α(1,3)-glycosidic bond, preferably linked to C4 of the GlcNAc via a β(1,4)-glycosidic bond. The N-acetylglucosamine of the GlcNAc-E(A) substituent is linked to the antibody via C1, preferably to the amide nitrogen atom in the side chain of asparagine of the antibody (GlcNAcβ1-Asn) via an N-glycosidic bond. The GlcNAc in the GlcNAc-E(A) substituent is optionally fucosylated. Correspondingly, if present, GlcNAc-E in the antibody-drug conjugate can also be linked as described above.

[0118] In a preferred embodiment, the functional group A is an azide group. When A is an azide group, A is preferably attached to C2, C3, C4, or C6. As mentioned above, one or more azide substituents in E(A) can be attached to C2, C3, C4, or C6 of the sugar or sugar derivative E to replace a hydroxy group (OH). It should be understood that the attachment position of the functional group A corresponds to the position at which the sugar containing A is connected to the linker.

[0119] In one preferred embodiment, the sugar derivative E(A)x is derived from a sugar or sugar derivative E. In one preferred embodiment, E is a sugar or sugar derivative and is selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose and GalNAc, and most preferably GalNAc.

[0120] In another preferred embodiment, E(A)x is GalNAc-N3, and preferably E(A)x is 6-azido-6-deoxy-2-acetamidogalactose.

[0121] Unless inconsistent, it should be understood that the above descriptions and explanations regarding saccharides (including, but not limited to, the glycosidic linkage of GlcNAc-E(A)x) apply equally to the corresponding saccharides, e.g., saccharide linkages, of the conjugate of formula (II) below. In some embodiments, a reactive group (e.g., functional group A) is linked to the sugar chain of an antibody in the glycosylation modification of the antibody, and therefore an "antibody with modified glycosylation" is defined as an antibody comprising such a reactive group; however, it will be understood by those skilled in the art that, when an antibody-drug conjugate is formed, the reactive group reacts with a linker moiety to form a new group with the linker moiety, and thereby the new group is considered to be part of the linker in the antibody-drug conjugate.

[0122] Preferably, the antibody of the present invention is a monoclonal antibody, more preferably an IgG antibody (e.g., a four-chain IgG antibody), most preferably an IgG1 antibody. In one embodiment, the antibody is a whole antibody.

[0123] When the modified antibody is a whole antibody, the antibody preferably comprises two or more, more preferably two, GlcNAc-E(A)x substituents, which are optionally fucosylated. However, when the modified antibody is an antibody fragment, such as an Fab or Fc fragment, the antibody may have only one GlcNAc-E(A)x substituent, which is optionally fucosylated. The GlcNAc-E(A)x substituent can be located anywhere in the antibody, so long as it does not interfere with antigen binding to the antibody's antigen-binding site. In one embodiment, the GlcNAc-E(A)x substituent is located in the Fc domain of the antibody, more preferably in the CH2 domain.

[0124] As noted above, antibodies with altered glycosylation of the invention include one or more GlcNAc-E(A)x substituents, for example, two GlcNAc-E(A)x substituents.

[0125] In one preferred embodiment, the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (e.g., a native conserved N-glycosylation site) of the antibody, for example, a glycosylation site in the Fc region (more preferably, the CH2 domain). In a further preferred embodiment, the antibody is an IgG antibody, and the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (a native conserved N-glycosylation site) of an IgG antibody. In a further preferred embodiment, the native site is the Asn297-glycosylation site of an IgG antibody. The Asn297-glycosylation site is present in the Fc region of the heavy chain of an IgG antibody. In one preferred embodiment, the GlcNAc-E(A)x substituent is present at the Asn297-glycosylation site of both heavy chains of the antibody.

[0126] In some embodiments, L in formula (I) of the present invention is a linker. Any linker known in the art can be used to link to the anti-human CLDN18.2 of the present invention, and preferably, the linker can achieve site-specific coupling of the ADC.

[0127] In some embodiments, the linker applied in the present invention may be any linker that can couple an antibody to a drug, or may be a linker used in a technology that can achieve site-specific coupling.

[0128] In a preferred embodiment, the linker of the present invention is a linker that links to an antibody oligosaccharide. As defined herein, a "linker that links to an antibody oligosaccharide" refers to any linker that links to a reactive group on a sugar chain at a glycosylation site of an antibody to couple the antibody to a drug. The sugar chain at the glycosylation site of an antibody is generally an N-glycan, and is usually modified from a heterogeneous N-glycan to a mono-glycan with a reactive group. The reactive group on the sugar chain is then used to link to a "linker," thereby achieving site-specific coupling of the drug and antibody to obtain an antibody-drug conjugate. In a preferred embodiment, the N-glycosylation site of the antibody is in the antibody Fc domain, preferably a conserved N-glycosylation site in the CH2 domain, such as Asn297. Thus, in one embodiment, a "linker for linking to an antibody oligosaccharide" of the present invention is any linker that can achieve site-specific coupling with a reactive group of an N-glycan, particularly at a conserved N-glycosylation site (e.g., Asn297) in an antibody Fc domain, such as a linker described in PCT / NL2013 / 050744 or a linker described in PCT / EP2021 / 075401, which are incorporated herein in their entireties. In one embodiment, the reactive group of the present invention is an azide group, a ketone group, or an 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 azide group, a ketone group, or an alkynyl group, preferably an azide group, and the linker of the present invention is a linker comprising an alkynyl group. When such a linker is referred to in the present invention, the reactive group reacts with a group on the linker to form a new group, and therefore the group formed after reaction of the reactive group on the antibody-drug conjugate can also be defined as part of the "linker", for example, as shown in formula (II) of the present invention.

[0129] Examples of linkers applicable to the present invention include cathepsin-degradable linkers such as Val-Cit linkers (such as vc-PAB), cBu-Cit linkers, and CX linkers, non-cleavable linkers such as SMCC linkers or MD linkers, acid-sensitive linkers, silicone 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 applied to the present invention may be a combination of one or more linkers. For example, a cathepsin-degrading linker can be combined with other types of linkers to form a new linker. Therefore, the "linker" described in the present invention includes 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 a drug. Thus, in one embodiment, the linker applied to the present invention is MC-VC-PAB, vc-PAB, SMCC, or MC-GGFG.

[0130] D in formula (I) of the present invention may be any antitumor compound, and is not particularly limited, as long as it has an antitumor effect and a substituent or partial structure that can be linked to a linker structure. For example, the antitumor compound may be a pharmaceutically active compound that acts on tumors. In the case of an antitumor compound, a part or all of the linker is preferably cleaved in tumor cells, liberating the antitumor compound moiety and allowing it to exhibit antitumor effects. When the linker is cleaved at the linking moiety to the drug, the antitumor compound is released in an unmodified structure, allowing it to exert its inherent antitumor effect.

[0131] In some embodiments, the antitumor compound may be, for example, a cytotoxic or chemotherapeutic agent, such as a camptothecin-based compound such as ixitecan (the topoisomerase I inhibitor exatecan) or Dxd (a novel derivative of the topoisomerase I inhibitor exatecan), an auristatin-based compound such as monomethyl auristatin E (MMAE), or a maytansine-based compound such as the small molecule microtubule inhibitor DM1. The structures in the examples of the present invention show the structures of representative compounds of these antitumor compounds.

[0132] In one embodiment, the present invention provides an antibody-drug conjugate according to (II) or a pharmaceutically acceptable salt or solvate thereof, [ka] Ab represents an antibody as defined herein; L1 is a linker, E is a sugar or sugar derivative, for example a sugar or sugar derivative as defined above, GlcNAc is N-acetylglucosamine, Fuc is fucose, D and r are as defined in Formula I above; b is 0 or 1, for example 0; x is 1, 2, 3 or 4, preferably 1 or 2; The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided, wherein y is 1 to 20, for example, 1 to 10, more preferably 1, 2, 3, 4, 5, 6, 7, or 8, and even more preferably 1, 2, 3, or 4.

[0133] In one embodiment, Fuc is fucose, b is 0 or 1, for example 0, and / or x is 1 or 2, more preferably 2, and / or y is 1 or 2, more preferably 2.

[0134] In one embodiment, in formula (II), E is selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose and GalNAc, most preferably GalNAc, for example 6-deoxy-2-acetamidogalactose, and preferably linked to L1 via the C atom at position 6.

[0135] In one preferred embodiment, the GlcNAc linked to Ab is present at a native N-glycosylation site (e.g., a native conserved N-glycosylation site) of the antibody, e.g., a glycosylation site in the Fc region. In a further preferred embodiment, the antibody is an IgG, and the GlcNAc is present at a native N-glycosylation site (e.g., a native conserved N-glycosylation site) of IgG, e.g., a glycosylation site in the Fc region. In a further preferred embodiment, the native site is the Asn297-glycosylation site of IgG. The Asn297-glycosylation site is present in the Fc region of the heavy chain of an IgG antibody. In one preferred embodiment, the GlcNAc group is present at the Asn297-glycosylation site of the two heavy chains of the antibody.

[0136] In one embodiment, in Formula (II), L1 has the following structure: [ka] Q is —N(H)C(O)CH— or —CH—; R1 is independently hydrogen, halogen, -OR2, -NO2, -CN, -S(O)2R2, C1-C 12 Alkyl groups, aryl groups, heteroaryl groups, C1-C 12 Alkylaryl groups, C1-C 12 Alkylheteroaryl group, aryl C1-C 12 Alkyl groups and heteroaryls C1-C 12alkyl groups, and the alkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, arylalkyl and heteroarylalkyl groups are optionally substituted; two substituents R1 can be linked together to form a bridged or spiro-linked cycloalkyl group or a fused or spiro-linked aromatic or heteroaromatic hydrocarbon substituent; and R2 can independently be selected from hydrogen, halogen, C1-C 24 Alkyl groups, aryl groups, heteroaryl groups, C1-C 12 Alkylaryl groups, C1-C 12 Alkylheteroaryl group, aryl C1-C 12 Alkyl groups and heteroaryls C1-C 12 alkyl groups, R3 and R4 are each independently hydrogen, halogen, C1-C 24 Alkyl groups, aryl groups, heteroaryl groups, C1-C 12 Alkylaryl groups, C1-C 12 Alkylheteroaryl group, aryl C1-C 12 Alkyl groups and heteroaryls C1-C 12 alkyl groups, c is 0, 1, 2, 3 or 4; m is 0 or 1, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Y is O, S or NR1; L2 and L3 are each independently C1-C 12 an alkyl group, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms in the alkyl group are optionally replaced with heteroatoms selected from O, N, and S, provided that the O, N, and / or S are not directly linked to each other; Each L4 is an independently cleavable linker.

[0137] It should be understood that when m is 0, Q is absent, thereby meaning that the associated nitrogen atom of the triazole is directly linked to the E moiety by a single covalent bond.

[0138] In one embodiment, each L4 is independently [ka] and wherein R5 and R6 are each independently hydrogen and C1-C 12 alkyl groups, Ar is selected from aryl groups, preferably phenyl groups; In one embodiment, each L4 is independently [ka] is.

[0139] In one embodiment, -L1- has the following structure: [ka]

[0140] In one embodiment, the antibody-drug conjugate has an average DAR of 1-15, for example 1-10, 2-8, 2-6, or 3-5.

[0141] In one embodiment, the antibody-drug conjugate of the present invention comprises: [ka] [ka] is selected from In the formula, Ab is HB37A6, In the formula, q represents the average DAR value, In IEX019-02, IEX019-04, and IEX019-05, q is 2 to 5, for example, 3 to 5, 3 to 4, or 3.5 to 4.5;

[0142] In IEX019-03, q is 5 to 11, for example, 6 to 10, 7 to 9, or 7.5 to 8.5.

[0143] III. Production of the ADC Molecules of the Invention

[0144] One aspect of the invention is a method for preparing an antibody with altered glycosylation, comprising the steps of:

[0145] (1) Preparation of glycosylated antibodies: culturing a host cell containing a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid under conditions suitable for expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium), to obtain an antibody comprising a core N-acetylglucosamine substituent (core-GlcNAc substituent), wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated;

[0146] (2) Preparing a trimmed antibody: deglycosylating the antibody prepared in step (1) in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated;

[0147] (3) contacting the trimmed antibody obtained in (2) with a compound of formula E(A)xP in the presence of a suitable catalyst to obtain an antibody comprising a GlcNAc-E(A)x substituent, wherein the GlcNAc-E(A)x substituent is attached to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent, wherein the catalyst is a glycosyltransferase and wherein P is selected from uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP).

[0148] To carry out step (1), nucleic acid encoding an antibody (e.g., an antibody described above, e.g., any one polypeptide chain and / or multiple polypeptide chains) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced by conventional processes (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

[0149] The endoglycosidase in step (2) can be selected according to the properties of the glycosylated antibody, for example, selected from Endo S, Endo A, Endo F, Endo M, Endo D and Endo H enzymes and / or combinations thereof, and for example, Endo S, Endo S49, Endo F or combinations thereof. In a preferred embodiment, the endoglycosidase is an endoglycosidase described in PCT / EP2017 / 052792, most preferably Endo SH of PCT / EP2017 / 052792.

[0150] The glycosyltransferase in step (3) is preferably a β-(1,4)-N-acetylgalactosamine transferase glycosyltransferase or is derived therefrom, and more preferably is any of the β-(1,4)-GalNAcT enzymes described in PCT / EP2016 / 059194. In some embodiments, the β-(1,4)-GalNAcT enzyme is or is derived therefrom an invertebrate β-(1,4)-GalNAcT enzyme. The β-(1,4)-GalNAcT enzyme may be or be derived therefrom any invertebrate β-(1,4)-GalNAcT enzyme known to those of skill in the art. Preferably, the β-(1,4)-GalNAcT enzyme is or is derived from a β-(1,4)-GalNAcT enzyme from the phylum Nematoda, preferably from the class Chromadorea or Secernentea, or from a phylum Arthropoda, preferably from the class Insecta. More preferably, the β-(1,4)-GalNAcT enzyme is or is derived from a β-(1,4)-GalNAcT enzyme from Mylestine worm, Ascaris suum, C. nigricans, Drosophila melanogaster, saprophytic fruit nematode, Caenorhabditis briggsae, C. nigricans, Lobaria beetle, Piseor cricket, Wood termite, Hemitermes oryzae, B. japonica, and B. hamster. Preferably, a glycosyltransferase suitable for step (3) is the Stinging Oil Palm Oil β-(1,4)-GalNAcT enzyme (e.g., His-TnGalNAcT) disclosed in PCT / EP2016 / 059194 and designated as TnGalNAcT.

[0151] Another aspect of the present invention provides a method for preparing an ADC using an antibody of the present invention. An "ADC" in this invention is defined as an antibody coupled to an active agent (D) having biological and / or pharmaceutical activity via a linker (L). The method comprises coupling an antibody of the present invention to one or more active agents D via one or more linkers (L) defined in the present invention. Preferably, the linker-active agent is site-specifically coupled to the antibody.

[0152] In one embodiment, there is provided a method for preparing an ADC of the invention, comprising administering to a subject a glycosylated antibody of formula III: [ka] wherein the meaning of each variable or symbol is as defined above; reacting the compound with a linker-drug compound (linker-payload) comprising an alkynyl group and one or more (e.g., 1, 2, 3, or 4) drug molecules; forming an antibody-drug conjugate of formula II: [ka] wherein the meaning of each variable or symbol is as defined above.

[0153] In one embodiment, the linker-drug compound has the structure of the formula: [ka] wherein each variable is as defined above.

[0154] IV. Pharmaceutical Compositions

[0155] In some embodiments, the present invention provides a composition comprising any ADC molecule described herein or a pharmaceutically acceptable salt thereof, preferably a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical adjuvant. In one embodiment, the composition, e.g., pharmaceutical composition, comprises a combination of an ADC molecule of the present invention and one or more other therapeutic agents.

[0156] The present invention further includes compositions (including pharmaceutical compositions) comprising the ADC molecules of the present invention or pharmaceutically acceptable salts thereof. These compositions may also contain suitable pharmaceutical auxiliary materials, such as pharmaceutical vectors known in the art, pharmaceutical excipients including buffering agents.

[0157] As used herein, "medicinal vector" includes any or all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, and the like.

[0158] For a discussion of the use of pharmaceutical adjuvants and their applications, see also Handbook of Pharmaceutical Excipients, 8th Edition, RC Rowe, PJ Seskey and SCOwen, Pharmaceutical Press, London, Chicago.

[0159] The compositions of the present invention may be in a variety of forms, including, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form is determined by the desired mode of administration and therapeutic use.

[0160] Drugs of the ADC molecules described herein can be produced by mixing the ADC molecules of the invention having the desired purity with one or more optional pharmaceutical auxiliary materials, preferably in the form of a lyophilized formulation or an aqueous solution.

[0161] The pharmaceutical compositions or formulations of the present invention may further comprise one or more active ingredients, as needed for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. It may also be desirable to provide other therapeutic agents, including, for example, chemotherapeutic agents, antiangiogenic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). The active ingredients are present in an appropriate combination in amounts effective for the intended use.

[0162] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0163] VII. Pharmaceutical Combinations and Drug Kits

[0164] In some embodiments, the present invention further provides pharmaceutical combinations or pharmaceutical combination products comprising an ADC molecule of the invention and one or more other therapeutic agents (e.g., therapeutic agents including chemotherapeutic agents, antiangiogenic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immune checkpoint inhibitors or agonists), etc.).

[0165] Another object of the present invention is to provide a pharmaceutical kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, which can provide dosage units according to a dosing program or pharmaceutical administration interval.

[0166] In one embodiment, the pharmaceutical kit of the present invention comprises, in the same package: a first container containing a pharmaceutical composition comprising an ADC molecule of the invention; a second container containing a pharmaceutical composition comprising another therapeutic agent.

[0167] VIII. Uses and Methods

[0168] One aspect of the invention provides a method for preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of an ADC molecule, pharmaceutical composition, pharmaceutical combination, or drug kit of the invention.

[0169] In some embodiments, the tumor (e.g., cancer) patient has CLDN18.2 (e.g., elevated levels of nucleic acid, protein, etc.). In some embodiments, the tumor cells of the patient express CLDN18.2, e.g., moderately express CLDN18.2, and preferably highly express CLDN18.2.

[0170] In some embodiments, the tumor, e.g., cancer, includes solid tumors, hematological tumors, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer may be early-stage, intermediate-stage, or late-stage cancer, or metastatic cancer.

[0171] In one specific embodiment, the ADC molecules of the invention can kill tumor cells and / or inhibit the proliferation of tumor cells, e.g., tumor cells that express CLDN18.2, e.g., gastrointestinal tumor cells, e.g., gastric cancer cells, pancreatic cancer cells, colon cancer cells, or colorectal cancer cells.

[0172] In some embodiments, the tumor is tumor immune evasion.

[0173] In some embodiments, the tumor is a cancer, eg, an epithelial tumor, eg, a gastrointestinal tumor, eg, an epithelial cancer or a gastrointestinal cancer, eg, gastric cancer, gastroesophageal junction cancer, pancreatic cancer, colorectal cancer, or colon cancer.

[0174] The subject may be a mammal such as a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease described herein (e.g., cancer). In some embodiments, the subject will undergo or has undergone other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject will undergo or has undergone prior immunotherapy.

[0175] In another aspect, the invention provides the use of an ADC molecule, pharmaceutical composition, pharmaceutical combination, or drug kit in the production or manufacture of a medicament, wherein the medicament is for a use as described herein, e.g., for preventing or treating an associated disease or condition as described herein.

[0176] In some embodiments, the ADC molecules, pharmaceutical compositions, pharmaceutical combinations, or drug kits of the invention delay the onset of a condition and / or symptoms associated with the condition.

[0177] In some embodiments, the ADC molecules or pharmaceutical compositions of the invention can be further administered in combination with one or more other therapies, e.g., treatment regimens and / or other therapeutic agents, and used for the uses described herein, e.g., to prevent and / or treat the associated diseases or conditions described herein.

[0178] In some embodiments, the treatment modality comprises surgery, radiation therapy, localized or focused radiation, or the like.

[0179] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an anti-angiogenic agent, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immune checkpoint inhibitor or agonist).

[0180] Other exemplary antibodies include antibodies that specifically bind to immune checkpoints.

[0181] Combination therapies of the invention include co-administration (e.g., two or more therapeutic agents in the same formulation or in separate formulations), separate administration, in which case administration of an ADC molecule of the invention may occur prior to, simultaneously with, and / or after administration of another therapeutic agent and / or agent.

[0182] The pharmaceutical composition can be administered by known methods, for example, orally, intravenously, intraperitoneally, intracerebrally (intraparenchymal), intraventricularly, intramuscularly, intraocularly, intraarterially, intraportally, or intralesionally, by sustained release systems, or by implanted devices. In certain embodiments, the composition can be administered by bolus injection or continuous infusion or by implanted devices.

[0183] The compositions may be administered locally via an implant membrane, sponge, or another suitable material that absorbs or encapsulates the desired molecule. In certain embodiments, when an implant device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecule by diffusion, sustained release bolus, or continuous administration.

[0184] These and other aspects and embodiments of the present invention are described in the drawings (followed by a brief description of the drawings) and the following detailed description of the invention, and are illustrated in the following examples. Any or all of the features described above and throughout this application may be combined in various embodiments of the present invention. The present invention is further described using the following examples, which are given by way of illustration and not by way of limitation, and it should be understood that various modifications may be made by those skilled in the art. [Example]

[0185] Example 1.1. Construction of stable expression cell lines

[0186] Preparation of human CLDN18.2 overexpressing cell lines

[0187] A cell line stably expressing human Claudin18.2 (abbreviated as CLDN18.2, hereafter referred to as CLDN18.2) was constructed using the Freedom® CHO-S® Reagent Kit (Invitrogen, A1369601) according to the manufacturer's instructions. First, the full-length human CLDN18.2 gene (UniProt ID: P56856-2) was inserted into the vector pCHO1.0, and the resulting plasmid was then introduced into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) by chemical transfection and electrotransfection, respectively. The transfected cells underwent two rounds of press screening to obtain cell pools expressing CLDN18.2. Next, cells that highly expressed CLDN18.2 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter), and monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2 that stably expressed CLDN18.2 were obtained by the dilution method.

[0188] Preparation of human CLDN18.1 overexpressing cell line

[0189] A cell line stably expressing human Claudin18.1 (hereinafter referred to as CLDN18.1) was constructed using the Freedom® CHO-S® Reagent Kit (Invitrogen, A1369601) according to the manufacturer's instructions. First, the full-length human CLDN18.1 gene (UniProt ID: P56856-1) was inserted into the vector pCHO1.0 (Invitrogen, A1369601) to form a plasmid. The constructed plasmid was then introduced into CHO-S cells (Invitrogen, A1369601) by chemical transfection. The transfected cells were then subjected to two rounds of prescreening to obtain cell pools expressing CLDN18.1. Next, cells that highly expressed CLDN18.1 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter), and a monoclonal cell line, CHO-hCLDN18.1, that stably expressed CLDN18.1 was obtained by the dilution method.

[0190] Construction of tumor cell lines overexpressing CLDN18.2

[0191] The full-length human CLDN18.2 gene (UniProt ID: P56856-2) was inserted into the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence. This vector was then transfected into HEK293T (ATCC, CRL-3216) cells with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259). After 48 and 72 hours, the culture supernatants were collected and the lentivirus was concentrated using PEG8000. Pancreatic cancer DAN-G cells (CLS Cell Lines Service GmbH, 300162) and gastric cancer KATO III cells (ATCC, HTB-103) were transfected with the concentrated virus, and CLDN18.2-expressing cells were then sorted using a flow cytometer (MoFlo XDP, Beckman Coulter) to obtain the tumor cell lines DAN-G-hCLDN18.2 and KATO III-hCLDN18.2 stably transfected with CLDN18.2.

[0192] Example 1.2. Generation of CLDN18.2 Monoclonals

[0193] In the present invention, hybridoma technology was used to immunize mouse H2L2 fully human antibody recombinant mice (purchased from Harbour BioMed) with the cells (CHO-hCLDN18.2) obtained in Example 1. Then, mouse spleen cells were obtained and electrofused with myeloma cells. The supernatant was then collected and screened by flow cytometry (FACS) for hybridoma cells that specifically expressed anti-CLDN18.2 antibodies, and the secreted antibodies did not bind to CLDN18.1. The cells to be detected (HEK293-hCLDN18.2) obtained in Example 1 were counted, and 1 × 10 6The cells were diluted to 100 cells / mL and added to a 96-well plate with a U-bottom at 100 μL per well. The mixture was centrifuged at 500×g for 5 minutes, and the cell culture medium was removed. The supernatant from the 96-well hybridoma plate was added to the U-shaped plate, and the cells were resuspended. 100 μL was added per well and incubated on ice for 30 minutes. The mixture was incubated at 500×g for 5 minutes, and the supernatant was removed. The cells were washed once with PBS. 100 μL of anti-mouse Fab FITC-labeled secondary antibody (diluted 1:500 in PBS) was added per well. 100 μL of anti-human Fab FITC-labeled secondary antibody was added as a positive control antibody. The mixture was incubated on ice for 30 minutes, protected from light. The mixture was centrifuged at 500×g for 5 minutes, and the supernatant was removed. The cells were washed once with PBS. The cells were resuspended in 50 μL of 1x PBS and detected on-board by FACS. Positive clones were rescreened using CHO-hCLDN18.1 in the same manner as above, and after two rounds of screening, a fully human antibody clone, HB37A6, was obtained.

[0194] Example 1.3. Preparation of Recombinant CLDN18.2 Monoclonal Antibody

[0195] The anti-CLDN18.2 monoclonal antibody HB37A6 (see CN202010570517.X) and the control antibody zolbetuximab (abbreviated as Zmab, the sequence of which is derived from INN117) were expressed in HEK293 cells (Invitrogen, A14527) in the form of full-length monoclonal antibodies.

[0196] First, expression vectors were constructed, and the heavy and light chain variable regions of HB37A6 and a control antibody (see Sequence Listing information) were placed at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 5) and light chain kappa constant region (SEQ ID NO: 10), respectively. These were then constructed into a pcDNA3.1 expression vector with an N-terminal signal peptide to obtain the light and heavy chain expression vector. The resulting light and heavy chain expression vectors were co-transfected with PEI (Polysciences Inc., 23966) into HEK293 cells. After 7 days of culture, the culture supernatant was collected. The supernatant was purified using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), ultrafiltered, and then replaced with PBS (Gibco, 70011-044). The concentration was detected by the A280 method, and the purity was measured by SEC-HPLC. An antibody solution with a purity of over 95% was obtained, yielding the recombinant CLDN18.2 monoclonal antibody HB37A6.

[0197] The specific transfection and purification procedures are as follows:

[0198] Depending on the required transfection volume, passage Expi293 cells (Invitrogen, A14527) to a cell density of 1.5 × 10 the day before transfection. 6 The cell density was adjusted to approximately 3 × 10 cells / mL on the day of transfection. 6 The transfection buffer was 1 / 10 the final volume of Opti-MEM medium (Gibco, 31985-070), and the appropriate plasmid was added to the transfection buffer at a concentration of 1.0 μg / mL. The appropriate polyethylenimine (PEI) (Polysciences, 23966) was added to the plasmid (the ratio of plasmid to PEI was 1:3 for 293F cells). After mixing, the mixture was incubated at room temperature for 20 minutes to obtain the DNA / PEI mixture. The flask was gently shaken while the DNA / PEI mixture was gradually added to the cells, and the cells were cultured at 36.5°C in an 8% CO2 incubator. After 7 days, the cell suspension was obtained, and the cell supernatant was collected and purified.

[0199] The Protein A column (Hitrap Mabselect Sure, GE, 11-0034-95) used for purification was treated with 0.1 M NaOH for 2 h, washed with distilled water, and then dried at 180 °C for 4 h. Prior to purification, the collected cell solution was centrifuged at 4500 rpm for 30 min, and the supernatant was filtered through a 0.22 μm filter. The Protein A column was equilibrated with 10 column volumes of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). The filtered supernatant was applied to the purification column, which was then equilibrated with 10 column volumes of binding buffer. 5 mL of elution buffer (citric acid + 0.1 M sodium citrate, pH 3.5) was added, and the eluate was collected. 80 μL of 2 M Tris-HCl was added per mL of eluate. The collected antibody was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the concentration was detected.

[0200] Example 1.4: Measurement of affinity of CLDN18.2 antibody by SPR method

[0201] Surface plasmon resonance (SPR) was used to measure the equilibrium dissociation constant (K ) of HB37A6 binding to human CLDN18.2. D) was measured. Human Claudin18.2 (GenScrip, P50251802) was coupled to the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino coupling reagent kit (GE Healthcare, BR-1006-33) according to the manufacturer's instructions. After coupling, 1 M ethanolamine was injected to block any remaining activated sites. Affinity and kinetic constants were obtained by detecting binding and dissociation between the chip surface antigen and the antibody in the mobile phase using a Biacore (GE Healthcare, T200) according to the manufacturer's instructions. Gradient dilutions of antibody (0 nM to 100 nM) were flowed over the chip surface, from lowest to highest concentration, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM glycine pH 1.5 (GE Healthcare, BR-1003-54). The data results were analyzed using Biacore T200 analysis software for kinetic analysis under a 1:1 binding model. As shown in Table 1, the affinity of HB37A6 was superior to that of the control antibody Zmab.

[0202] [Table 2]

[0203] Example 1.5: Binding specificity of CLDN18.2 antibody to CLDN18 cells

[0204] The binding of the anti-CLDN18.2 monoclonal antibody HB37A6 and the control antibody Zmab to CHO-S cell lines stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1, respectively (i.e., CHO-hCLDN18.2 and CHO-hCLDN18.1 prepared as described in Example 1) was measured by flow cytometry (FACS).

[0205] Specifically, the cells to be detected (CHO-hCLDN18.2 and CHO-hCLDN18.1) obtained in Example 1 were counted, and 1 × 10 6The cells were diluted to 100 cells / mL and added to a 96-well U-bottom plate at 100 μL per well. The plate was centrifuged at 500×g for 5 min, and the cell culture medium was removed. The anti-CLDN18.2 monoclonal antibody HB37A6 and control antibody Zmab were added to the U-bottom plate at 100 μL per well, and the cells were resuspended. The antibody was initially concentrated at 900 nM, and then serially diluted 3-fold for a total of 10 concentration points. The plate was then incubated on ice for 30 min. The supernatant was removed, and the cells were washed once with PBS. 100 μL of goat anti-human Fc PE-labeled secondary antibody (SouthernBiotech, J2815-5H87B) was added per well. The plate was then incubated on ice for 30 min, protected from light. The supernatant was removed, and the cells were washed once with PBS. The cells were resuspended in 50 μL of 1x PBS, and on-board detection was performed using a FACS machine. The experimental data were analyzed using GraphPad Prism software, and the results are shown in Figures 1 and 2. As shown in Figures 1 and 2, all of the above antibodies specifically bound to human CLDN18.2 (Figure 1), but did not bind to human CLDN18.1 (Figure 2).

[0206] Example 1.6. Binding of CLDN18.2 antibody to tumor cell lines

[0207] As shown in Example 1.5, the binding of HB37A6 to the gastric cancer cell line NUGC-4 (JCRB, JCRB0834), the gastric cancer cell line KATO III-hCLDN18.2, and the pancreatic cancer cell line DAN-G-hCLDN18.2 was measured by FACS. Figure 3 shows that the fully human antibody HB37A6 has relatively good specific binding to tumor cells, and is superior to the control antibody Zmab.

[0208] Example 1.7: In vivo antitumor effect of CLDN18.2 antibody

[0209] 1. Antibody activity against DAN-G-CLDN18.2 tumor-bearing mouse model

[0210] The antitumor effect of the HB37A6 antibody was tested in NOD-SCID mice bearing human pancreatic cancer (female NOD-SCID mice (15-18g), purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). The human pancreatic cancer cells DAN-G-hCLDN18.2 constructed in Example 1 were subjected to regular subculture and used for subsequent in vivo experiments. The cells were collected by centrifugation and dispersed in PBS (1x) at a concentration of 12 x 10 5 The cell suspension and Matrigel gel were mixed at a 1:1 ratio to obtain a suspension with a cell density of 6 × 10 cells / mL. 5 A cell suspension with a cell concentration of 100 / mL was prepared. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of NOD-SCID mice to establish a DAN-G-CLDN18.2 tumor-bearing mouse model.

[0211] After 5 days of tumor cell inoculation, the tumor volume of each mouse was detected, and the tumor volume was 43.36 mm 3 ~89.47mm 3 The mice were divided into S-shaped groups (8 mice per group) according to the size of the tumor volume.

[0212] Mice were administered hIgG (Equitech-Bio, Lot No. 160308-02), HB37A6, and the control antibody Zmab at a dose of 10 mg / kg each on days 5, 9, 12, and 16 after inoculation, respectively. Tumor volume was monitored two to three times a week. Tumor volume measurement: The longest axis (L) and widest axis (W) of the tumor were measured with a vernier caliper, and tumor volume was calculated as V = L × W. 2 The weight was measured using an electronic balance.

[0213] 2. Antibody activity against NUCG-4 tumor-bearing mouse model

[0214] The HB37A6 antibody was selected to test its antitumor effect in NOG mice (female NOG mice, 15-18 g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) bearing human gastric cancer. PBMC cells (Allcells) were resuscitated and collected by centrifugation. 2.5 × 10 6 PBMC cells were dispersed in PBS (1x) to obtain a cell suspension with a cell density of 100 / mL. On day 0, 0.2 mL of the cell suspension was injected into the ocular vein of NOG mice to establish the NOG humanized mouse model.

[0215] NUGC-4 cells were subjected to the usual resuscitation subculture and used for subsequent in vivo experiments. Cells were collected by centrifugation and collected at 12 × 10 6 Disperse NUGC-4 cells in PBS (1x) to a cell density of 6x10 cells / mL and mix with Matrigel gel at a 1:1 ratio. 6 A cell suspension with a cell concentration of 100 / mL was prepared. On day 5, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of NOG humanized mice to establish a NUCG-4 tumor-bearing mouse model.

[0216] On the first day after tumor cell inoculation, mice were randomly divided into groups (7 mice per group). Each group received hIgG (Equitech-Bio, Lot No. 160308-02, control group), HB37A6, and the positive control antibody Zmab (treatment group) at a dose of 10 mg / kg per administration on days 1, 5, 8, and 12 after inoculation, respectively. Mouse tumor volume and body weight were monitored two to three times a week. Tumor volume measurement: The longest axis (L) and widest axis (W) of the tumor were measured with a vernier caliper, and tumor volume was calculated as V = L × W. 2 The relative tumor inhibition rate (TGI%) was calculated on the 26th day after inoculation using the formula:

[0217] TGI% = 100% × (tumor volume in the control group − tumor volume in the treatment group) / (tumor volume in the control group − tumor volume before administration in the control group).

[0218] 3, results

[0219] As shown in Figure 4, both HB37A6 and the control antibody Zmab were able to inhibit tumor growth in the human pancreatic cancer DAN-G-CLDN18.2 mouse model, with a TGI of 28% for HB37A6 and 24% for Zmab. As shown in Figure 5, HB37A6 exhibited superior antitumor activity to the control antibody Zmab in the human gastric cancer NUGC-4 mouse model, with a TGI of 31% for HB37A6 and 0% for Zmab.

[0220] Example 2.1 Synthesis of IEX019 ADC Molecule

[0221] IEX019

[0222] Based on HB37A6, we further designed the synthesis of ADCs coupled to different small molecule compounds. The specific process is as follows:

[0223] Example 2.1.1 Preparation of IEX019-02

[0224] 1). Preparation of Compound 6 [ka]

[0225] Under N2 atmosphere, a solution of BCN-OH (5, 1.5 g, 10 mmol) in DCM (150 mL) was added with chlorosulfonyl isocyanate (CSI) (0.87 mL, 1.4 g, 10 mmol), Et3N (2.8 mL, 2.0 g, 20 mmol), and 2-(2-aminoethoxy)ethanol (1.2 mL, 1.26 g, 12 mmol). The mixture was stirred for 10 min and quenched by adding aqueous NH4Cl (saturated, 150 mL). After separation, the aqueous layer was extracted with DCM (150 mL). The combined organic layers were dried (Na2S O4 ) and concentrated. The residue was purified by column chromatography to give pale yellow thick oily product 6 (2.06 g, 5.72 mmol, 57%).

[0226] 1 H NMR (400MHz, CDCl3)δ(ppm)6.0(bs, 1H), 4.28(d, J=8.2Hz, 2H), 3.78-3.73(m, 2H), 3.66-3.61(m, 2H), 3.61-3.55(m , 2H), 3.34(t, J=4.9Hz, 2H), 2.37-2.15(m, 6H), 1.64-1.48(m, 2H), 1.40(quintet, J=8.7Hz, 1H), 1.05-0.92(m, 2H).

[0227] 2) Preparation of Compound 7 [ka]

[0228] To a stirred solution of 6 (47 mg, 0.13 mmol) in DCM (10 mL) was added CSI (11 μL, 18 mg, 0.13 mmol). After 30 min, a solution of EtN (91 μL, 66 mg, 0.65 mmol) and diethanolamine (16 mg, 0.16 mmol) in DMF (0.5 mL) was added. After 30 min, p-nitrophenyl chloroformate (52 mg, 0.26 mmol) and EtN (54 μL, 39 mg, 0.39 mmol) were added. After an additional 4.5 h, the reaction mixture was concentrated, and the residue was purified by gradient column chromatography (33→66% EtOAc / heptane (1% AcOH)) to give 7 (88 mg, 0.098 mmol, 75%) as a colorless oil.

[0229] 1 H NMR(400MHz, CDCl3)δ(ppm)8.28-8.23(m, 4H), 7.42-7.35(m, 4H), 4.52(t, J=5.4Hz, 4H), 4.30(d, J=8.3Hz, 2H), 4.27-4.22(m, 2H), 3.86(t, J=5.3Hz, 4H), 3.69-3.65(m, 2H), 3.64-3.59(m, 2H), 3.30-3.22(m, 2H), 2.34-2.14( m, 6H), 1.62-1.46(m, 2H), 1.38(quintet, J=8.7Hz, 1H), 1.04-0.92(m, 2H).

[0230] 3) Preparation of Compound 9 [ka]

[0231] Compound 8 (163 mg, 240 μmol) was added to a mixture of ixanotecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in dry DMF (0.9 mL). After 20 h, the reaction mixture was diluted with 9 mL of DCM and purified by gradient column chromatography (0 to 40% MeOH / DCM) to give 9 (155 mg, 159 μmol, 68%). LCMS (ESI+) C 55 H 54 FN6O 10 + (M+H) + Calculated value: 977.39, measured value: 977.80.

[0232] 4) Preparation of Compound 1

[0233] To a solution of compound 9 (155 mg, 159 μmol) in DMF (1.6 mL) was added EtN (73 mg, 101 μL, 0.72 mmol) and a solution of compound 7 (65 mg, 72 μmol) in DMF (1.4 mL). The reaction mixture was stirred for 24 h, diluted with DCM (20 mL), and purified by gradient column chromatography (0 to 40% MeOH / DCM) to give compound 1 (94 mg, 44 μmol, 28%) as a pale yellow solid. LCMS (ESI+) C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + Calculated value: 1066.88, measured value: 1067.12.

[0234] 5) Enzymatic reconstitution of HB37A6 to HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2

[0235] HB37A6 was expressed in HEK293 cells and purified according to Example 1.3. The resulting HB37A6 (16.4 mg / mL) was incubated with EndoSH (1% w / w) as described in PCT / EP2017 / 052792 to obtain trimmed HB37A6 bearing -GlcNAc or GlcNAc(Fuc) at Asn297. The trimmed HB37A6 was then incubated with the enzyme His-TnGalNAcT (4.5% w / w) disclosed in PCT / EP2016 / 059194 and 6-azido-GalNAc-UDP (25 eq (equivalents) relative to antibody) disclosed in PCT / EP2016 / 059194 in a 6 mM solution containing MnCl, histidine (20 mM), and NaCl (150 mM) at 30°C for 16 hours.

[0236] The incubation mixture was then purified using a 50 mL protA column (Hitrap Mabselect Sure, GE, 11-0034-95). The incubation mixture was applied to the column and washed with TBS + 0.2% Triton and TBS. The column was then eluted with 0.1 M acetate buffer, pH 2.9, and neutralized with 2.5 M Tris-HCl, pH 7.2. After dialysis three times against PBS, the resulting (modified) glycosylated antibody was concentrated to 32.6 mg / mL using a Vivaspin Turbo 15 ultrafiltration device (Sartorius).

[0237] The resulting glycosylated antibody was analyzed by mass spectrometry. The main steps are as follows: Prior to mass spectrometry, the glycosylated antibody was treated with IdeS to enable analysis of the Fc / 2 fragment. 20 μg of the (modified) glycosylated antibody solution and IdeS (Fabricator TM) (1.25 U / μL) was incubated in a total volume of 10 μL in PBS, pH 6.6, for 1 h at 37 °C. Samples were diluted to 80 μL and then analyzed on a JEOL AccuTOF (ESI-TOF) instrument, and deconvoluted spectra were acquired using Magtran software. Mass spectrometry analysis of the IdeS-digested sample showed a major product corresponding to the resulting HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 with an observed mass of 24330.4.

[0238] Therefore, the above results demonstrated that HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 was obtained, in which the GlcNAc at Asn297 in both heavy chains was substituted with 6-azido-GalNAc (4-position substitution).

[0239] 6) Preparation of HB37A6-SYNtecan E conjugate (IEX019-02)

[0240] The bioconjugate IEX019-02 of the present invention was prepared by coupling the linker-conjugate compound 1 (linker-payload) to the azide-modified biomolecule HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2. Thus, to a solution of HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 (10.8 mL, 350 mg, 32.6 mg / mL, PBS pH 7.4) was added PBS pH 7.4 (808 μL), 1,2-propylene glycol (11.3 mL), and compound 1 (350 μL, 40 mM DMF solution). The reaction was incubated overnight at room temperature, then filtered and dialyzed against PBS pH 7.4. After adding charcoal (350 mg), the mixture was rotated overnight at room temperature to remove any remaining free payload. The charcoal was removed by centrifugation and filtration, and the ADC was purified with an AKTA Purifier-10 (GE Healthcare) equipped with a Superdex200 26 / 600SEC (GE Healthcare) column.

[0241] Mass spectrometry of the IdeS-digested sample showed two major products, each corresponding to the resulting ADC, i.e., the HB37A6-SYNtecan E conjugate. The first peak had an observed mass of 26,469 Da (calculated mass: 26,465 Da), corresponding to the coupled Fc / 2 fragment (2x closed lactone form of the payload). The second peak had an observed mass of 26,499 Da (calculated mass: 26,501 Da), corresponding to the coupled Fc / 2 fragment (2x open carboxylate form of the payload).

[0242] The structure of the prepared product was as follows: The concentration, DAR, and SEC purity of the ADC product were determined using UV, SEC, RP-HPLC, and LC-MS. The monomer purity detected by SE-HPLC was over 99% and the concentration was 6.12 mg / mL.

[0243] [ka] In the formula, q represents the average DAR value, for example, 3 to 5, 3.2 to 4.8, or 3.5 to 4.5, for example, 3.52 measured in Table 2.

[0244] In the formula, Ab is It was HB37A6.

[0245] Example 2.1.2 Preparation of IEX019-03 [ka]

[0246] In the formula, q represents the average DAR value, for example, 5 to 11, 6 to 10, 7 to 9, or 7.5 to 8.5, for example, 7.9 measured in Table 2.

[0247] In the formula, Ab is It was HB37A6.

[0248] (a) A reducing agent solution (TCEP (Sigma, C4706) dissolved in water) was added to an HB37A6 solution (antibody HB37A6 dissolved in PBS buffer (Thermo, 10010023)), and the reaction mixture was placed on a shaker for 2 to 4 hours. (i) The optimal concentration of HB37A6 was 5 mg / mL to 10 mg / mL. (ii) The optimal molar ratio of TCEP / mAb was 4.5-6.5. (iii) The optimum temperature for the reaction was 20℃-37℃. (iv) The optimum pH value for the reaction was generally between 6.5 and 8.0.

[0249] (b) Excess linker-toxin MC-GGFG-DXD (purchased from Levena biopharma, SET0218, structure shown below) was dissolved in DMSO and reacted with the thiol groups of the antibody reduced in step (a). The reaction mixture was placed on a shaker for 1 to 2 hours. (i) The optimal molar ratio of DXD / mAb was 10.0-12.0. (ii) The optimum temperature for the reaction is 20°C to 37°C; A crude ADC product was obtained.

[0250] (c) The resulting crude ADC product was purified by spin desalting, ultrafiltration or dialysis to give the final ADC product IEX019-03.

[0251] (d) HIC, LC-MS, and SEC HPLC were used to detect the ADC products and determine the mean DAR and SEC purity.

[0252] [ka]

[0253] Example 2.1.3 Preparation of IEX019-04 [ka] In the formula, q represents the average DAR value, for example, 3 to 5, 3.2 to 4.8, or 3.0 to 4.0, for example, 3.5 as measured in Table 2.

[0254] In the formula, Ab is It was HB37A6.

[0255] This molecule was prepared according to the following method.

[0256] (a) A reducing agent solution (TCEP (Sigma, C4706) dissolved in water) was added to an antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer), and after the addition was complete, the reaction mixture was placed on a shaker for 2 to 4 hours. (i) The optimal concentration of antibody HB37A6 was 5 mg / mL to 10 mg / mL. (ii) The optimal molar ratio of TCEP / mAb was 1.9-2.7. (iii) The optimum temperature for the reduction reaction was 20℃-37℃. (iv) The optimum pH value for the reaction was generally between 6.5 and 8.0.

[0257] (b) Excess linker-payload MC-VC-PAB-MMAE (purchased from Levena Biopharma, SET0201) containing a reactive group (maleimide-conjugated drug) was dissolved in the organic solvent DMSO and reacted with the reduced thiol group generated in step (a). The reaction mixture was placed on a shaker for 1–2 h. (i) The optimal molar ratio of MC-VC-PAB-MMAE / mAb was 8.0-10.0. (ii) The optimum temperature for the binding reaction was 20°C to 37°C.

[0258] (c) After the binding reaction was completed, the reaction in step (b) was stopped by adding acetylcysteine ​​solution. After mixing, the mixture was incubated at 20°C to 25°C for 5 to 15 minutes.

[0259] (d) The resulting crude ADC product was purified by spin desalting, ultrafiltration or dialysis to give the final ADC product IEX019-04.

[0260] (e) The ADC product was detected using HIC and SEC-HPLC, and the average DAR and SEC purity were determined.

[0261] [ka]

[0262] Example 2.1.4 Preparation of IEX019-05 [ka]

[0263] In the formula, q represents the average DAR value, for example, 3 to 5, 3.2 to 4.8, or 3.0 to 4.5, for example, 3.3 measured in Table 2.

[0264] where Ab was HB37A6.

[0265] This molecule was prepared according to the following method.

[0266] (a) Add the linker-payload SMCC-DM1 solution (purchased from Levena Biopharma and dissolved in an organic solvent such as SET0101 or DMSO) to the antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer). After addition, place the reaction mixture on a shaker for 2 to 5 hours. (i) the optimal concentration of antibody HB37A6 is 5 mg / mL to 10 mg / mL; (ii) the optimal molar ratio of linker-payload / mAb is 5.5 to 6.5; (iii) The optimum pH value for the reaction is generally between 6.5 and 8.0; (iv) The optimum temperature for the reaction is 20°C to 37°C; A crude ADC product was obtained.

[0267] (b) The resulting crude ADC product was purified by spin desalting, ultrafiltration or dialysis to give the final ADC product IEX019-05.

[0268] (c) The average DAR and SEC purity of the ADC product were determined using ultraviolet spectrophotometry and SEC high-performance liquid chromatography.

[0269] [ka]

[0270] Example 2.1.5 Preparation of IEX019-06

[0271] The preparation process was similar to IEX019-02, except that the control antibody IgG was substituted for the HB37A6 monoclonal antibody.

[0272] Example 2.1.6 Preparation of IEX019-07

[0273] The preparation process was similar to IEX019-04, except that the control antibody IgG was substituted for the HB37A6 monoclonal antibody.

[0274] Information on all monoclonal antibodies and ADCs is summarized in Table 2.

[0275] [Table 3]

[0276] Example 2.2 Cell binding experiments of IEX019 molecules

[0277] To detect whether small molecule coupling alters the binding properties of IEX019-01 monoclonal antibody to target cells, the inventors used the DAN-G cell line (hCLDN18.2 negative) and the DAN-G-hCLDN18.2 cell line (hCLDN18.2 overexpression) prepared in Example 1, and detected the affinity of IEX019-01 and IEX019-02 to the target by flow cytometry technology. The experimental method was the same as in Example 1.5.

[0278] Neither IEX019-01 nor IEX019-02 bound to non-target cells, DAN-G, but showed very high affinity for DANG-hCLDN18.2, indicating that antibody binding depends on target expression specificity and that coupling with Exatecan does not affect antibody binding. At the same time, the control molecule IEX019-06 (a monoclonal antibody, a negative control of IgG coupled to Exatecan toxin using the same technology) did not bind to target cells (Figure 6).

[0279] Example 2.3 Endocytosis experiment of IEX019 molecule

[0280] Strong endocytosis is one of the important properties of ADC drugs. After ADC binds to antigens on the cell membrane surface, the ADC-antigen complex enters the cell through endocytosis and kills the target cell. Therefore, the endocytosis efficiency of ADC is one of the important indicators for determining the tumor suppression effect.

[0281] To detect the endocytosis efficiency of antibodies coupled to small molecule compounds, the inventors used flow cytometry to detect the endocytosis of different IEX019 molecules in DANG-hCLDNA18.2 cells. After digesting DANG-hCLDNA18.2 cells, the cell density was adjusted to 1 × 10 5Cells were seeded at 100 μL per well into a 96-well plate. The cells were centrifuged at 500 g for 3 min and the supernatant was discarded. 100 μL of the target molecule was resuspended (molecule concentration: 50 nM). Five replicates were set for each sample (i.e., endocytosis times: 0 h, 1 h, 2 h, 3 h, and 4 h). The cells were placed on ice and incubated for 1 h. After 1 h, the cells were centrifuged at 500 g for 3 min and the supernatant was discarded. 200 μL of FACS buffer (1% FBS, 1x PBS) was added to each well and washed twice. One set of samples was transferred to a new 96-well plate and incubated at 37°C for 4 h. The remaining samples continued to be incubated on ice. The above procedure was repeated, with the samples incubated at 37°C for 3 h, 2 h, 1 h, and 0 h, respectively. After the specified incubation times were completed, the cells were centrifuged at 500 g for 3 min and the supernatant was discarded. 100 μL of anti-hFC-PE antibody (SouthernBiotech) diluted 1:400 was added to each well and incubated on ice for 30 minutes, protected from light. After the secondary antibody incubation was complete, the cells were centrifuged at 500 g for 3 minutes and the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. Finally, the cells were resuspended in 100 μL of PBS and subjected to on-chip detection.

[0282] As shown in Figure 7, the experimental results showed that, with 0 hours of incubation at 37°C as the zero point of endocytosis, after 2 hours of incubation, all molecules reached the maximum endocytosis level of approximately 60%, indicating that the ADC molecules designed and synthesized based on IEX019-01 maintained a strong endocytosis ability consistent with that of a monoclonal antibody after binding to tumor cells.

[0283] Example 2.4: In vitro cell killing effect of IEX019 molecules

[0284] The CellTiter-Glo (Promega, G9242) detection reagent kit was used to detect the effect of ADCs on cell viability in various cell lines expressing different levels of hCLDNA8.2 (Table 3).

[0285] [Table 4]

[0286] The cells used were digested with EDTA / Trypsin, adjusted to a uniform density, and uniformly seeded onto a 96-well plate (Table 4). A specific concentration of diluted IEX019 molecules (IEX019-02, IEX019-03, IEX019-04, starting at 100 nM, diluted 3 times) was added. Control wells were used without IEX019 molecules. The wells were incubated at 37°C for 5 days. After 5 days, 100 μL of CellTiter-Glo detection reagent was added to each well, incubated at room temperature for 30 min, and detected using a microplate reader. Relative cell viability (relative cell viability = sample / control × 100%) was calculated, and curve fitting was performed using GraphPad Prism 8.0.

[0287] As shown in Figure 8, the killing of the ADC molecules against the cell lines depended on the expression level of surface hCLDN18.2. In hCLDN18.2-negative DANG, the IEX019 molecule had no significant effect on cell viability (Figure 8A). In cell lines with moderate hCLDN18.2 expression levels (NUGC-4, SNU620), the IEX019 molecule exhibited a certain degree of cell killing (Figure 8B). In the highly expressing cell line DAN-G-hCLDN18.2, both IEX019 molecules exhibited significant killing effects (Figure 8C). This indicates that the IEX019 molecule has good selectivity and efficacy.

[0288] [Table 5]

[0289] Example 2.5: Bystander killing effect

[0290] In the synthesis of ADC drugs, small molecule compounds are linked to antibodies via cleavable linkers. After endocytosis into the cell membrane, the linker is cleaved, releasing the small molecule to kill the target cell. After the target cell is killed, the small molecule compound is released from the target cell into the intercellular space and further kills non-target cells within a certain range, an effect known as bystander killing. Because cells within a tumor vary greatly in target expression levels (tumor heterogeneity), bystander killing is crucial for effective tumor cell killing and tumor growth inhibition.

[0291] The present invention detected the bystander killing effect of the IEX019 molecule using non-target cells DAN-G and target cells DANG-hCLDN18.2.

[0292] After digesting the cells with EDTA / Trypsin, the density was adjusted to prepare a 6-well cell culture plate. DANG cells and DANG-hCLDN18.2 were cultured at 7.5 × 10 4The two types of cells were co-cultured. 200 μL of the sample to be measured (IgG (SEQ ID NO: 21, SEQ ID NO: 22), IEX019-02, IEX019-05, IEX019-06, Exatecan (Macklin, E881532)) was added to each well to a final concentration of 50 nM. Three replicates were used for each sample. The cell culture plate was placed in a 37°C incubator and cultured for 5 days. After 5 days, the culture supernatant was discarded and washed with PBS. Trypsin-EDTA was added to digest the cells, and all digested cells were collected and transferred to a 96-well plate. Following the antibody incubation process for flow cytometry, the cells were incubated with a primary antibody (IEX019-01, 100 nM) and a secondary antibody (anti-hFc-PE, SouthernBiotech) at 4°C for 1 h and 0.5 h, respectively. After washing with PBS, live / dead cells were cultured. Violet dye (Thermo, L34964) was diluted 1:1000, and 100 μL was added to each well and incubated at 4°C for 20 min. After washing with PBS, cells were resuspended in 100 μL of PBS and subjected to on-board detection. Each sample was divided into populations using live / dead dye, where the IEX019-01-negative (i.e., hCLDN18.2-negative) population was designated DAN-G cells, and the IEX019-01-positive (i.e., hCLDN18.2-positive) population was designated DAN-G-hCLDN18.2 cells. The numbers of the two types of cells in each sample were separately calculated, and the relative cell viability of each type of cell was calculated according to the following equation. Curves were fitted using GraphPad Prism 8.0.

[0293] Relative viability of DNAG cells = Number of DNAG cells in the sample group / Number of DNAG cells in the IgG group × 100%

[0294] Relative viability of DANG-hCLDN18.2 cells = number of DNAG-18.2 cells in the sample group / number of DNAG-18.2 cells in the IgG group × 100%

[0295] The cell viability of the IgG group was set as 100%.

[0296] As shown in Figure 9, the negative control sample IEX019-06 had no killing effect on either of the two cell types. The IEX019-05 molecule, which links the DM1 toxin to the IEX019-01 monoclonal antibody via a non-cleavable linker and has no ability to kill surrounding non-target cells, was able to kill only DANG-hCLDNA8.2 cells without affecting DANG cells. Only IEX019-02 had a significant bystander killing effect, capable of simultaneously killing target and non-target cells.

[0297] Example 2.6: Antitumor efficacy of IEX019 molecule in DAN-G-hCLDN18.2 mouse xenograft tumor model

[0298] To demonstrate the in vivo efficacy of the IEX019 molecule, the inventors inoculated DANG-hCLDN18.2 cells into CB-17-SCID mice to measure the antitumor efficacy of the antibody molecule of the present invention. SPF female CB-17-SCID mice (14-17g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) with a qualification certificate number of 110011201108225246 were used in the experiment.

[0299] DANG-hCLDN18.2 cells were subcultured periodically and used for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1x). 3x10 6 A cell suspension with a cell concentration of 100 / mL was prepared. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of CB-17 SCID mice to establish a DANG-hCLDN18.2 tumor-bearing mouse model.

[0300] On the fifth day after tumor cell inoculation, the tumor volume was 50.16 mm 3 ~136.68mm 3 All mice were divided into snake-shaped groups (6 mice per group) and administered the doses and methods shown in Table 5 on the 5th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 10a and 10b, and monitoring ended after 92 days.

[0301] On the 50th day after inoculation, the relative tumor inhibition rate (TGI%) was calculated using the following formula: TGI% = 100% × (tumor volume in the control group − tumor volume in the treatment group) / (tumor volume in the control group − tumor volume before administration in the control group).

[0302] Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a caliper, and the tumor volume was calculated as V = L × W. 2 The weight was measured using an electronic balance.

[0303] [Table 6]

[0304] The tumor inhibition rate results are shown in Table 6 and Figure 10A. 50 days after inoculation, the tumor inhibition rate after a single administration of IEX019-02 reached 103.60% compared to the IEX019-01 monoclonal antibody, significantly superior to IEX019-03 and IEX019-04, with tumor inhibition rates of 93.70% and 35.20%, respectively. 82 days after inoculation, 100% of the mouse tumors in the IEX019-02 group had completely regressed. At the same time, the inventors monitored the mouse weights. The results are shown in Figure 10B. There was no significant difference in mouse weights.

[0305] [Table 7]

[0306] Example 2.7: Antitumor efficacy of IEX019 molecule in NUGC-4 mouse xenograft tumor model

[0307] To demonstrate the in vivo efficacy of the IEX019 molecule, the inventors inoculated NUGC-4 cells into CB-17-SCID mice and measured the antitumor efficacy of the antibody molecule of the present invention.

[0308] In the experiment, SPF-grade female CB-17-SCID mice (14 g–17 g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, and the qualification certificate number was NO. 110011201109348141.

[0309] NUGC-4 cells were subcultured periodically and used for subsequent in vivo experiments. Cells were collected by centrifugation, dispersed in PBS (1x), and diluted to 3x10 7 A cell suspension with a cell concentration of 100 / mL was prepared. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of CB-17 SCID mice to establish a NUGC-4 tumor-bearing mouse model.

[0310] On the fifth day after tumor cell inoculation, the tumor volume was 72.25 mm 3 ~140.50mm 3 All mice were divided into snake-shaped groups (6 mice per group) and administered the doses and methods shown in Table 7 on the 5th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 11A and 11B, and monitoring ended after 40 days.

[0311] On the 33rd day after inoculation, the relative tumor inhibition rate (TGI%) was calculated using the following formula: TGI% = 100% × (tumor volume in the control group − tumor volume in the treatment group) / (tumor volume in the control group − tumor volume before administration in the control group).

[0312] Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a caliper, and the tumor volume was calculated as V = L × W. 2 The weight was measured using an electronic balance.

[0313] [Table 8]

[0314] The tumor inhibition rates were as shown in Tables 8 and 11A. On day 33 after inoculation, the tumor inhibition rates for the negative controls IEX019-06, IEX019-02, and IEX019-03 were 80.04% and 54.31%, respectively. Mouse weights were also measured, and the results, shown in Figure 11B, showed no significant differences in mouse weight.

[0315] [Table 9]

[0316] Example 2.8: Antitumor efficacy of IEX019 molecule in SNU620 mouse xenograft tumor model

[0317] To demonstrate the in vivo efficacy of the IEX019 molecule, we inoculated SNU620 cells into CB-17-SCID mice and measured the antitumor efficacy of the molecule of the present invention (IEX019-02). SPF female CB-17-SCID mice (18-20g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used in the experiment, with the qualification number of No. 110011211102179364.

[0318] SNU620 cells were subcultured periodically and used for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1x). 3x10 7 A cell suspension with a cell concentration of 100 / mL was prepared. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of CB-17 SCID mice to establish an SNU620 tumor-bearing mouse model.

[0319] On the 7th day after tumor cell inoculation, the tumor volume was 58.1 mm 3 ~117.3mm 3 All mice were divided into snake-shaped groups (6 mice per group) and administered the doses and methods shown in Table 9 on day 7 after inoculation. The tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 12a and 12b, and monitoring ended after 39 days.

[0320] On the 39th day after inoculation, the relative tumor inhibition rate (TGI%) was calculated using the following formula: TGI% = 100% × (tumor volume in the control group − tumor volume in the treatment group) / (tumor volume in the control group − tumor volume before administration in the control group).

[0321] Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a caliper, and the tumor volume was calculated as V = L × W. 2 The weight was measured using an electronic balance.

[0322] [Table 10]

[0323] As shown in Table 10 and Figure 12A, on day 39 after inoculation, the tumor inhibition rate after a single administration of 10 mg / kg of IEX019-02 was 143.77% compared to hIgG, with 100% of mouse tumors completely regressing. There was no significant difference in mouse weight between each administration group and the hIgG control group (Figure 12B).

[0324] [Table 11]

[0325] Sequence information: [Table 12-1] [Table 12-2] [Table 12-3]

Claims

1. The following formula: 【Chemistry 1】 an antibody-drug conjugate comprising During the ceremony, The Ab is an anti-Claudin18.2 antibody comprising HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively; and where q is a drug-antibody ratio (DAR) of 3 to 5; The antibody-drug conjugate.

2. The antibody-drug conjugate of claim 1, wherein the Ab is an anti-Claudin18 antibody comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 4; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

9.

3. the Ab comprises a heavy chain constant region from IgG1; The antibody-drug conjugate of claim 2.

4. The antibody-drug conjugate of claim 3 , wherein the Ab comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 11 and a light chain having the amino acid sequence shown in SEQ ID NO:

12.

5. The antibody-drug conjugate described in claim 1, wherein q is a DAR in the range of 3 to 4.

6. The antibody-drug conjugate described in claim 1, wherein q is a DAR in the range of 3.2 to 4.

8.

7. The antibody-drug conjugate described in claim 4, wherein coupling to the Ab occurs at position Asn297.

8. The following formula: 【Chemistry 2】 an antibody-drug conjugate of the formula: wherein Ab is an anti-Claudin18.2 antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain having the amino acid sequence set forth in SEQ ID NO: 12; coupling to the Ab occurs at position Asn297; and wherein q is a DAR of 3 to 4. Antibody-drug conjugates.

9. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 1.

10. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 2.

11. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 3.

12. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 4.

13. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 5.

14. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 6.

15. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 7.

16. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 8.

17. The antibody-drug conjugate described in claim 8 for treating Claudin18-expressing cancer in a subject.

18. The pharmaceutical composition of claim 16 for treating Claudin18-expressing cancer in a subject.

19. An antibody-drug conjugate described in claim 8 for treating gastric cancer in a subject.

20. An antibody-drug conjugate described in claim 8 for treating pancreatic cancer in a subject.

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