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) targeting 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 cellular tight junctions. They can establish an intercellular barrier that controls the movement of molecules between cells. Claudins family proteins have four transmembrane domains, and both their N- and C-termini are contained in the cytoplasm. Different Claudins proteins are expressed in different tissues, and their functional changes are related to the carcinogenesis of 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 cancer, 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 not expressed 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. In addition to gastric cancer, CLDN18.2 is also expressed in pancreatic cancer, making it an ideal target molecule for treating these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as 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 of gastric cancer and approximately 290,000 deaths nationwide, accounting for nearly half of the total number of cases and deaths in the world, and the number is still increasing. However, there is a large unmet need for clinical tumor treatment, so there is a great need to develop drugs that target Claudin18.2.

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

[0006] Owing to technological advances made 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 an antibody-drug conjugate (ADC) targeting Claudin18.2, which has 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 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) It has good stability. (7) It has good drug-developability.

[0010] In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface, in some embodiments, the target cell is a CHO cell or 293 cell expressing CLDN18.2, such as CHO-S cell or HEK293 cell, in some embodiments, the target cell is a cancer cell expressing CLDN18.2, such as a cell that naturally expresses CLDN18.2, a cell that expresses CLDN18.2 by artificial transfection, or a cell that has an increased expression level of CLDN18.2 by artificial transfection, such as a gastric cancer cell, a pancreatic cancer cell line, or a colon cancer or colorectal cancer cell line expressing CLDN18.2. In some embodiments, the target cell is a cell line with a moderate expression level of hCLDN18.2, such as NUGC-4, SNU620. In some embodiments, the target cell is a cell line with a high expression level, such as DAN-G cell that overexpresses hCLDN18.2. [Brief description of the drawings]

[0011] [Figure 1] This shows that the HB37A6 antibody specifically binds to CLDN18.2 on the cell surface. [Diagram 2] This shows that the HB37A6 antibody does not bind to CLDN18.1 on the cell surface. [Diagram 3] 1 shows the binding of HB37A6 antibody to gastric cancer cell line NUGC-4, gastric cancer cell line KATO III-hCLDN18.2, and 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. [Diagram 5] 1 shows the antitumor effect of HB37A6 antibody in a mouse model of gastric cancer. [Figure 6] 4 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 levels (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 levels (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 levels (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 PREFERRED EMBODIMENTS

[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, forms, or reagents described herein, as they may be modified. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments, 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 of ordinary skill in the art.

[0014] The following definitions will be used to interpret the 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% more than the number or numerical value specified as the upper limit.

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

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

[0018] The term "CLAUDIN" or "CLDN" as used herein is the most important scaffold protein that determines the structure of tight junctions between cells, and is involved in adherens junctions and plays an important role in the metastasis and invasion of tumor cells. Claudin proteins are widely present in mammalian epithelial and endothelial cells, and are mainly distributed on the epithelial cell lateral and basal plasma membranes. Different Claudin proteins have specific expression in different tissues, among which the Claudin18 (CLDN18) gene is located at 3q22.3, has a molecular weight of 24 kDa, has 261 amino acid residues, belongs to the Claudins superfamily, and its protein composition has 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, and in the primary structure sequences of both proteins, only amino acid residues at certain positions from the N-terminal signal peptide to the extracellular loop 1 (Loop1) structure are different, especially on the extracellular loop 1, CLDN18.1 and CLDN18.2 differ by only eight amino acids. The interspecies sequence homology of the two subtype proteins of CLDN18 is also very high. Among them, the extracellular loop 1 of CLDN18.2 has a perfect sequence match in different organisms such as human, mouse, and rhesus monkey, 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 variants and isotypes thereof can be isolated from cells or tissues that naturally express them, or recombinantly produced by techniques well known in the art and / or described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0019] The term "anti-CLDN18.2 antibody", "anti-CLDN18.2", "CLDN18.2 antibody", "antibody that binds to CLDN18.2" or "antibody that specifically binds to CLDN18.2" as used herein refers 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, for example, by radioimmunoassay (RIA), biolayer interferometry (BLI), MSD assay or surface plasmon resonance (SPR) or flow cytometry.

[0020] Various means, such as anti-CLDN18.2 antibody, can determine the expression of CLDN18.2 in cells.For example, the binding strength (e.g., measured by FACS) between "highly expressing CLDN18.2" cells and anti-CLDN18.2 antibody can be 500 times, 600 times, 700 times, 800 times, 900 times, or preferably 1000 times or more than the binding strength between anti-CLDN18.2 antibody and cells that do not express CLDN18.2, for example 1100 times, 1200 times, 1300 times, 1400 times or more or more. 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, for example, 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 that has only heavy chains and lacks 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 includes a portion of an intact antibody and binds to an antigen bound by the intact antibody, but is distinct 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 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, may serve as an antigen. Additionally, antigens may be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0026] "Complementarity determining regions", "CDR regions" or "CDRs" are the regions in an antibody variable domain 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 an antigen epitope. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2 and CDR3 and are numbered sequentially from the N-terminus. The CDRs in the heavy chain variable domain of an antibody are referred to as HCDR1, HCDR2 and HCDR3, and the CDRs in the light chain variable domain of an antibody are referred to as LCDR1, LCDR2 and LCDR3.For a given light 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 a number of known antibody CDR assignment systems, including, for example, 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)), which is based on the variability of antibody sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics (International database (IMGT) (available on the World Wide Web at imgt.cines.fr / ), and the North CDR definition, which is based on affinity propagation clustering utilizing 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, references to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues) refer to numbered positions 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 according to 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 boundaries of the CDRs of the variable regions of the same antibody obtained by different allocation systems may differ. That is, there are differences in the CDR sequences of the variable regions of the same antibody defined by different allocation systems. Thus, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of said antibody also includes antibodies whose variable region sequences include said specific CDR sequences, but whose CDR boundaries differ from the specific CDR boundaries defined in the present invention because a different approach (e.g., the rules or combinations of different allocation systems) is used.

[0037] Antibodies with different specificities (i.e., directed to different binding sites of different antigens) have different CDRs (in the same allocation system). However, even though CDRs differ between antibodies, there are a limited number of amino acid positions in the CDRs that are directly involved in binding to the antigen. The minimum overlapping region can be determined to provide a "minimal binding unit" for antigen binding by at least two of the Kabat, Chothia, AbM, Contact and North methods. The minimal binding unit may be a subpart of one of the CDRs. As known to those skilled in the art, the structure of the antibody and protein folding can determine the residues of the remaining part of the CDR sequence. Thus, 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 minimal binding unit are kept unchanged, but the remaining CDR residues defined according to Kabat or Chothia may be replaced with conservative amino acid residues.

[0038] The term "Fc region" is used herein to define a 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, namely, a CH2 domain, a CH3 domain and an optional CH4 domain. For example, in a native antibody, 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 called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 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, and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, 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 heavy chain constant region of the antibody belongs. All antibodies of the same type have the same heavy chain constant region, and antibodies of different types have different heavy chain constant regions. For example, an antibody of IgG4 type refers to an antibody whose heavy chain constant region is derived from IgG4, or an antibody of IgG1 type refers to an antibody whose heavy chain constant region is 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 non-specific 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 with a drug.

[0042] The general term "sugar" as used herein refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" as used herein refers to a derivative of a monosaccharide, i.e., a monosaccharide that includes 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 includes x functional groups A.

[0043] A core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as a GlcNAc that is attached to the antibody via C1, preferably via an N-glycosidic bond of the amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. The core-GlcNAc substituent may be present at a native glycosylation site of the antibody, but may also be introduced into a different site of the antibody. As used herein, a core-N-acetylglucosamine substituent is a monosaccharide 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 modifying the carbohydrate moiety 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 remove FcγR affinity and complement binding / effector function, 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 of glycosylation modification known in the art include, for example, increasing or decreasing carbohydrate moieties on the antibody surface by altering the glycosylation site of the antibody, or chemically or enzymatically modifying carbohydrate moieties in vitro, or modifying the glycosylation pathway of the expression system (e.g., composed of enzymes such as glycosidases and glycosyltransferases) to catalyze the glycosylation of the antibody, as well as affecting cell culture conditions to modify the glycosylation of the antibody. In some embodiments, the "glycosylation modification" of the present invention is performed by enzymatic modification of carbohydrate moieties in vitro. Preferably, the glycosylation alteration of the present invention is carried out by modification of the sugar chain with a glycosidase (such as an endoglycosidase or a glycosyltransferase).

[0045] The antibody with modified glycosylation of the present invention refers to an antibody with a modified glycosylation mode compared to an antibody with a native glycosylation mode. Preferably, the antibody with modified glycosylation as described above refers to an antibody obtained after expression in an expression system (e.g., mammalian cells) and enzymatic modification of the glycan in vitro (e.g., modification of the glycan by glycosidases (such as endoglycosidases or glycosyltransferases)). More preferably, the antibody with modified glycosylation of the present invention refers to an antibody comprising a core-GlcNAc and a sugar derivative E(A)x linked thereto, where GlcNAc is linked to the antibody via C1, preferably via an N-glycosidic bond of 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, generally the fucose is 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 that specifically links a drug / active agent 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, for example 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, sulfonamide, 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, for example, 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-imidazolyl, 4- or 5-imidazolyl, 3- or 4-pyrazolyl, 2- or 4-thiazolyl, 3- or 4-isothiazolyl, 2- or 4-oxazolyl, 4- or 5-oxazolyl. , 3-, 4- or 5-isoxazolyl, 3- or 5-triazolyl, 4- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-pyridyl, 3- or 4-pyridyl, 3- or 4-pyridazinyl, 3-pyrazinyl, 4- or 5-pyrazinyl, 2-pyrazinyl, 2-pyrimidinyl, 4- or 5-pyrimidinyl.

[0051] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and performance of the ADC conjugate of the present invention, and which is not biologically or otherwise undesirable. The ADC conjugate 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 conjugate 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 pharma- ceutically acceptable non-toxic base addition salt refers to a salt formed between the ADC complex 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 complex of the invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethylsulfoxide, and the like.

[0053] Unless contradictory 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, for example, the DAR can be 1-20, for example, 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example, 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 measured by the 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 complex according to the 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] The term "therapeutic agent" as used herein includes any substance effective in the treatment or prevention of a tumor, such as a 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, 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] The term "immunomodulatory agent" as used herein refers to a natural or synthetic active agent or agent 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 present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0060] The term "effective amount" refers to an amount or dosage that provides 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 a required dosage for a required 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 are not offset by 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 more preferably 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 to effectively 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 a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and progeny derived therefrom regardless of the number of passages. The progeny may not be identical to the parent cell in nucleic acid content and may contain mutations. As used herein, includes mutant progeny having the same function or biological activity as screened or selected from the primary transformed cell.

[0064] The term "label" as used herein refers to a compound or composition that is directly or indirectly attached or fused to a reagent (e.g., a polynucleotide probe or an antibody) and facilitates detection by the reagent to which it is attached or fused. The label may be detectable itself (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, and 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 reduction in tumor volume, a reduction in tumor cell number, a decrease in tumor cell proliferation, or a reduction 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 mammalian physiological disorder generally 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 "auxiliary pharmaceutical material" 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, drug kits and pharmaceutical compositions. The term "non-fixed combination" refers to active ingredients (e.g., (i) the ADC molecule of the present invention, and (ii) other therapeutic agents) administered to a patient simultaneously in 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 other therapeutic agents used in the pharmaceutical combination are administered at levels that do not exceed the levels at which they are used 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 single formulation, which may be the same or different.

[0074] The term "combination therapy" refers to the administration of two or more therapeutic agents or forms of treatment (e.g., radiation therapy and surgery) to treat a disease as described herein. 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. It should be noted that such administration includes the use of each type of therapeutic agent at substantially the same time or at different times in a sequential manner. In either case, the treatment program provides the beneficial effect of the pharmaceutical combination in treating the disease or condition as described herein.

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

[0076] As used herein, "prevention" includes the inhibition of 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. In the context of cancer generally, the term "prevention" refers to the administration of a drug before symptoms or symptoms of cancer arise, particularly before cancer occurs in a subject at risk of suffering from 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] "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 or aspirate samples, 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 mixed 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 pharma- ceutically 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 prodrug, preferably a drug, including 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; The present invention provides an antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein 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, F(ab')2, a domain antibody (dAb), 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, 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) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 from the light chain variable region.

[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, 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, said amino acid changes do not occur in the CDR regions.

[0088] In some embodiments, the V 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, 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, said 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 the VH shown in SEQ ID NO: 4; or (ii) A sequence that contains 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 that contains 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, or 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 having 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, or alternatively, the HCDR3 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: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 invention is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, such as a wild-type IgG1 heavy chain constant region. In some embodiments, the antibody light chain constant region LC of the 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, a heavy chain of the invention comprises or consists of a heavy chain variable region and a heavy chain constant region. In some specific embodiments of the invention, a light chain of the invention comprises or consists 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 The VL comprises or consists of the amino acid sequence shown 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., one that contains 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 a conservative substitution. A conservative substitution refers to the replacement of one amino acid with another amino acid of the same kind, for example, an acidic amino acid is replaced with another acidic amino acid, a basic amino acid is replaced with another basic amino acid, or a neutral amino acid is replaced 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 has substantially retained certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody.

[0113] The antibody Ab in formula (I) of the present invention may be an antibody having modified glycosylation. In some embodiments, the antibody is an antibody obtained after in vitro enzymatic modification of the sugar chain (e.g., modification of the sugar chain by glycosidase (endoglycosidase or glycosyltransferase, etc.)). In some embodiments, the antibody having modified glycosylation refers to an antibody in which the sugar chain at the glycosylation site of the antibody has been modified from a heterogeneous N-sugar chain to a single structure N-sugar chain having a reactive group (e.g., any reactive group that can react with a linker moiety, such as an azide group, a ketone group, and 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, such as 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, the above 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 and wherein E(A)x is a sugar derivative comprising x functional groups A, where A is independently selected from an azide group, a ketone group, and an alkynyl group, 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, 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 invention is an antibody of formula (III), where Ab represents an antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, b is 0 or 1, y is 1-20, and E(A)x is a sugar derivative containing x functional groups A, where A is an azide group, a ketone group and an alkynyl group, and x is 1, 2, 3 or 4. In one preferred embodiment, y is 1-10, more preferably y is 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably y is 1, 2, 3 or 4, and most preferably y is 1 or 2. [ka]

[0117] The sugar derivative E(A)x in the GlcNAc-E(A)x substituent of the antibody with altered glycosylation can be, for example, linked to C4 of the GlcNAc via a β(1,4)-glycosidic bond or linked 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)x substituent is linked to the antibody via C1, preferably linked 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)x substituent is optionally fucosylated. Correspondingly, the GlcNAc-E in the antibody-drug conjugate, if present, 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)x 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 where 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, most preferably GalNAc.

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

[0121] Unless inconsistent, the above descriptions and explanations regarding saccharides (including but not limited to the glycosidic linkage of GlcNAc-E(A)x) should be understood to apply equally to the corresponding saccharides, e.g., saccharide linkages, of the conjugates of formula (II) below. In some embodiments, a reactive group (e.g., functional group A) is linked to the glycan of an antibody in the glycosylation modification of the antibody, so that one of skill in the art will understand that, when the antibody-drug conjugate is formed, the reactive group reacts with the linker moiety to form a new group with the linker moiety, such that in the antibody-drug conjugate, the new group is considered to be part of the linker.

[0122] Preferably, the antibody of the 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 complete 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 a Fab or Fc fragment, the antibody may only have 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 the substituent does not interfere with the binding of an antigen to the antigen-binding site of the antibody. 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 a preferred embodiment, the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (e.g., a native conservative N-glycosylation site) of the antibody, such as a glycosylation site in the Fc region (more preferably in 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 conservative 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 a preferred embodiment, the GlcNAc-E(A)x substituent is present at the Asn297-glycosylation site of the two 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 capable of coupling an antibody to a drug. In some embodiments, the linker may be a linker used in a technology capable of achieving site-specific coupling.

[0128] In a preferred embodiment, the linker of the present invention is a linker that links to the oligosaccharide of an antibody. As defined herein, a "linker that links to the oligosaccharide of an antibody" refers to any linker that links to a reactive group of 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 so as to change an N-glycan of a heterogeneous structure to an N-glycan of a single structure having a reactive group, and is further linked to a "linker" using the reactive group of the sugar chain to realize site-specific coupling of the drug and the antibody to obtain an antibody-drug conjugate. In a preferred embodiment, the N-glycosylation site of the antibody is the antibody Fc domain, preferably a conserved N-glycosylation site of the CH2 domain, such as Asn297. Thus, in one embodiment, the "linker for linking to an oligosaccharide of an antibody" of the present invention is any linker capable of site-specific coupling with a reactive group of an N-glycan, particularly at a conserved N-glycosylation site (e.g., Asn297) of an antibody Fc domain, such as a linker described in PCT / NL2013 / 050744 or a linker of PCT / EP2021 / 075401, which are incorporated herein in their entirety. In one embodiment, the reactive group of the present invention is an 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 group formed after reaction of the reactive group in the antibody-drug conjugate can also be defined as part of the "linker", since the reactive group reacts with the group of the linker to form a new group, e.g., as shown in formula (II) of the present invention.

[0129] Linkers applicable to the present invention further include, for example, 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-structured linkers, disulfide-carbamate linkers, MC-GGGFG 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 in the present invention may be a combination of one or more linkers, for example, a cathepsin decomposition 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 in 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 has a substituent or partial structure that can be linked to a linker structure. For example, the antitumor compound may be a pharmacologic 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, and the antitumor compound portion is released to exhibit an antitumor effect. When the linker is cleaved at the linking portion with the drug, the antitumor compound is released in an unmodified structure, and the original antitumor effect can be exhibited.

[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), Dxd (a novel derivative of the topoisomerase I inhibitor Exatecan), an auristatin-based compound, such as monomethylauristatin 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 relates to an antibody-drug conjugate according to (II) or a pharma- ceutically acceptable salt or solvate thereof, [ka] Ab stands for antibody as defined herein; L1 is a linker, E is a sugar or a sugar derivative, for example a sugar or a 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 present invention provides an antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof, wherein y is 1 to 20, for example, y is 1 to 10, more preferably y is 1, 2, 3, 4, 5, 6, 7, or 8, and even more preferably y is 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, 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, such as 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, such as 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)CH2- or -CH2-; 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 heteroaryl groups C1-C 12alkyl groups, and the alkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, arylalkyl and heteroarylalkyl groups are optionally substituted; two substituents R1 may 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 is independently 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 heteroaryl groups 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 heteroaryl groups 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 are optionally replaced with a heteroatom 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 selected from hydrogen and C1-C 12 alkyl groups, Ar is selected from aryl groups, preferably phenyl groups; In one embodiment, each L4 is independently [ka] It is.

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

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

[0141] In one embodiment, the antibody-drug conjugate of the 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 comprising a nucleic acid encoding the antibody (e.g., any one and / or more than one polypeptide chain) or an expression vector comprising said 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 medium), to obtain an antibody comprising a core N-acetylglucosamine substituent (core-GlcNAc substituent), wherein said core N-acetylglucosamine and said 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 said core N-acetylglucosamine and said 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, said GlcNAc-E(A)x substituent being attached to said antibody via C1 of the N-acetylglucosamine of said GlcNAc-E(A)x substituent, wherein said 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 as 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 routine 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 nature of the glycosylated antibody, e.g. selected from Endo S, Endo A, Endo F, Endo M, Endo D and Endo H enzymes and / or combinations thereof, e.g. Endo S, Endo S49, Endo F or combinations thereof. In a preferred embodiment, said 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, more preferably 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 from an invertebrate β-(1,4)-GalNAcT enzyme. The β-(1,4)-GalNAcT enzyme may be or be derived from 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 the 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 Mylestinea nematode, Ascaris suum, C. nigricans, Drosophila melanogaster, Saprophytic fruit nematode, Caeno rhabditis briggsae, C. nigricans, Lobaria mites, Piseor crickets, Wood termites, Hemiptera pedunculida, Biki and Hamster butterfly. Preferably, a glycosyltransferase suitable for step (3) is the urticaria vine 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 the present 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) as 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 patient a glycosylated antibody of formula III: [ka] In the formula, the meaning of each variable or symbol is as defined above. reacting the linker-drug compound with a linker-payload that includes 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 invention provides a composition comprising any of the ADC molecules described herein or a pharma- ceutically acceptable salt thereof, preferably a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical auxiliary material. In one embodiment, the composition, e.g., the pharmaceutical composition, comprises a combination of an ADC molecule of the invention and one or more other therapeutic agents.

[0156] The present invention further includes compositions (including pharmaceutical compositions) comprising the ADC molecules of the invention or pharma- ceutically 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, a "medicinal vector" includes any or all of physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, and the like.

[0158] For the use of pharmaceutical adjuvants and their applications, reference is also made to Handbook of Pharmaceutical Excipients, Eighth Edition, R. C. Rowe, P. J. Eskey and S. C. Wen, Pharmaceutical Press, London, Chicago.

[0159] The compositions of the present invention may be in a variety of forms. These forms include, 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 composition or formulation of the present invention may further comprise one or more active ingredients, which are necessary for the particular indication being treated, and preferably have complementary activities that do not adversely affect each other.It may also be desirable to further provide other therapeutic agents, including, for example, chemotherapeutic agents, angiogenesis inhibitors, 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 an amount 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 in microcapsule form.

[0163] VII. Pharmaceutical Combinations and Drug Kits

[0164] In some embodiments, the 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, angiogenesis inhibitors, 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 said pharmaceutical kit being in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing program or pharmaceutical dosing 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 of 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, preferably highly express CLDN18.2.

[0170] In some embodiments, the tumors, e.g., cancers, include solid tumors, hematological tumors, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. The cancer may be early, intermediate, 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, such as an epithelial tumor, such as a gastrointestinal tumor, such as an epithelial cancer or a gastrointestinal cancer, such as gastric cancer, gastroesophageal junction cancer, pancreatic cancer, colorectal cancer or colon cancer.

[0174] The subject may be a mammal, such as a primate, and is preferably a higher primate, such as 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 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 immunotherapy before.

[0175] In other aspects, 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 said 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 disease state and / or symptoms associated with the disease state.

[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 includes surgery, radiation therapy, localized or focused radiation, and the like.

[0179] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, angiogenesis inhibitor, 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 route of administration of the pharmaceutical composition is by known methods, for example, oral, intravenous injection, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes, by sustained release systems, or by implanted devices. In certain embodiments, the compositions 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 can 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. EXAMPLES

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

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

[0187] A cell line stably expressing human Claudin18.2 (abbreviated as CLDN18.2, hereinafter the same) was constructed using the Freedom® CHO-S® Reagent Kit (Invitrogen, A1369601) according to the manufacturer's instructions. First, the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed in the vector pCHO1.0, a plasmid was constructed, and the constructed plasmid was introduced into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) by chemical transfection and electrical transfection, respectively, and the transfected cells underwent two rounds of press screening to obtain cell pools that expressed CLDN18.2, respectively. 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 cell lines overexpressing human CLDN18.1

[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 gene of human CLDN18.1 (UniProt ID: P56856-1) was constructed in the vector pCHO1.0 (Invitrogen, A1369601) to form a plasmid, and the constructed plasmid was introduced into CHO-S cells (Invitrogen, A1369601) by chemical transfection. The transfected cells were subjected to two rounds of press screening 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 gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed in the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence therein, and transfected into HEK293T (ATCC, CRL-3216) cells together with the lentivirus packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), and virus packaging was performed. Culture supernatants after 48 and 72 hours were collected, respectively, and lentivirus was concentrated using PEG8000. The concentrated virus was transfected into pancreatic cancer DAN-G cells (CLS Cell Lines Service GmbH, 300162) and gastric cancer KATO III cells (ATCC, HTB-103), and then CLDN18.2-expressing cells were 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, the hybridoma technology was used to immunize mouse H2L2 fully human antibody gene recombinant mice (purchased from Harbour BioMed) with the cells (CHO-hCLDN18.2) obtained in Example 1. Then, the spleen cells of the mice were obtained and electrofused with myeloma cells. The supernatant was then collected and screened for hybridoma cells that specifically expressed anti-CLDN18.2 antibodies by flow cytometry (FACS), 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-shaped bottom at 100 μL / well. Centrifuged at 500 g for 5 min to remove the cell medium. The supernatant of the hybridoma cultured in the 96-well plate was added to the U-shaped plate, and the cells were resuspended and added at 100 μL per well and left on ice for 30 min. Centrifuged at 500 g for 5 min, the supernatant was removed, and the cells were washed once with PBS. 100 μL of anti-mouse Fab FITC-labeled secondary antibody (diluted in PBS at 1:500) was added per well, and 100 μL of anti-human Fab FITC-labeled secondary antibody was added as a positive control antibody. Incubated on ice for 30 min in the dark. Centrifuged at 500 g for 5 min to remove the supernatant, and the cells were washed once with PBS. The cells were resuspended in 50 μL of 1×PBS and detected on-board by FACS. Positive clones were rescreened using CHO-hCLDN18.1 in the same manner as above. 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 is derived from INN117) were expressed in HEK293 cells (Invitrogen, A14527) in the form of full-length monoclonal antibodies.

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

[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 final volume of Opti-MEM medium (Gibco, 31985-070) was taken as transfection buffer, and the appropriate plasmid was added to the transfection cells at 1.0 μg / mL, and mixed uniformly. The appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the ratio of plasmid to PEI is 1:3 for 293F cells), mixed uniformly, and incubated at room temperature for 20 min to obtain a DNA / PEI mixture. The DNA / PEI mixture was gradually added to the cells, while the flask was gently shaken, and then cultured at 36.5°C in an 8% CO2 incubator. After 7 days, the cell solution 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, and glass bottles, etc. were washed with distilled water and then dried at 180 ° C for 4 h. Before purification, the collected cell fluid 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 (sodium phosphate 20 mM, NaCl 150 mM, PH 7.0). The filtered supernatant was added to the purification column, and then equilibrated with 10 column volumes of binding buffer. 5 mL of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5) was added to collect the eluate, and 80 μL of Tris-HCl with a concentration of 2 M was added per 1 mL of eluate. The collected antibodies were concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044) to detect the concentration.

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

[0201] Using surface plasmon resonance (SPR), the equilibrium dissociation constant (K 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, and after coupling, 1 M ethanolamine was injected to seal the remaining activation sites. The affinity and kinetic constants were obtained by detecting the 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 diluted antibodies (0 nM to 100 nM) were flowed over the chip surface in order from low to high 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 subjected to kinetic analysis using Biacore T200 analysis software with 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 above-mentioned 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 plate with a U-shaped bottom at 100 μL / well. Centrifuged at 500g for 5 min and the cell medium was removed. Anti-CLDN18.2 monoclonal antibody HB37A6 and control antibody Zmab were added to the U-shaped plate at 100 μL per well and the cells were resuspended, with the starting concentration of the antibodies being 900 nM, and then serially diluted 3-fold for a total of 10 concentration points. Incubated on ice for 30 min. Centrifuged at 500g for 5 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. Incubated on ice for 30 min, protected from light. Centrifuged at 500g for 5 min, the supernatant was removed, and the cells were washed once with PBS. The cells were resuspended in 50 μL of 1×PBS and detected on-board by FACS. The experimental data was analyzed using GraphPad Prism software, and Figures 1 and 2 were obtained. 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] Referring to Example 1.5, the binding of HB37A6 to gastric cancer cell line NUGC-4 (JCRB, JCRB0834), gastric cancer cell line KATO III-hCLDN18.2 and 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: Antitumor effect of CLDN18.2 antibody in vivo

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

[0210] The HB37A6 antibody was used to test the antitumor effect in NOD-SCID mice bearing human pancreatic cancer (female NOD-SCID mice (15g-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 normal subculture and used for subsequent in vivo experiments. The cells were collected by centrifugation, and the DAN-G-hCLDN18.2 was dispersed in PBS (1x) and diluted to 12x10 5 The cell suspension and Matrigel gel were mixed in a 1:1 ratio to obtain a suspension with a cell density of 6 × 10 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 inoculation of tumor cells for 5 days, the tumor volume of each mouse was detected, and the tumor volume was 43.36 mm 3 ~89.47mm 3 Mice ranging from 0.1 to 1.0 mm in diameter were divided into S-shaped groups (8 mice per group) according to the size of the tumor volume.

[0212] Each mouse was administered hIgG (Equitech-Bio, Lot No. 160308-02), HB37A6, and the control antibody Zmab at a dose of 10 mg / kg each on the 5th, 9th, 12th, and 16th days after inoculation, respectively, and the tumor volumes of the mice were monitored two to three times a week. 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.

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

[0215] NUGC-4 cells were subjected to normal resuscitation subculture and used for subsequent in vivo experiments. Cells were collected by centrifugation and diluted to 12 × 10 6 Disperse NUGC-4 cells in PBS (1x) to a cell density of 6x10 cells / mL, mix with Matrigel gel at a 1:1 ratio, and add 6x10 cells. 6 A cell suspension with a cell concentration of 100 / mL was prepared. On the fifth day, 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, the mice were randomly divided into groups, with seven mice per group. Each mouse was administered with hIgG (Equitech-Bio, Lot No. 160308-02, control group), HB37A6, and positive control antibody Zmab (treatment group) at a dose of 10 mg / kg per administration on the first, fifth, eighth, and twelfth days after inoculation, respectively. Mouse tumor volume and body weight were monitored two to three times a week. 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 relative tumor inhibition rate (TGI%) was calculated on the 26th day after inoculation using the formula:

[0217] TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group).

[0218] 3, results

[0219] The results are shown in Figure 4, where HB37A6 and the control antibody Zmab were both 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 showed superior antitumor effects 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 molecules

[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] To a solution of BCN-OH (5, 1.5 g, 10 mmol) in DCM (150 mL) under N2 atmosphere, 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) were added. 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. A pale yellow thick oily product 6 (2.06 g, 5.72 mmol, 57%) was obtained.

[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 Et3N (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 Et3N (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→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, actual 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→40% MeOH / DCM) to give a pale yellow solid compound 1 (94 mg, 44 μmol, 28%). LCMS (ESI+)C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + Calculated value: 1066.88, actual 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) to obtain trimmed HB37A6 with -GlcNAc or GlcNAc(Fuc) at Asn297 position as described in PCT / EP2017 / 052792. The trimmed HB37A6 was incubated with the enzyme His-TnGalNAcT (4.5% w / w) disclosed in PCT / EP2016 / 059194 and 6-azido-GalNAc-UDP (25 eq (equivalent) relative to antibody) disclosed in PCT / EP2016 / 059194 in a solution containing 6 mM of MnCl2 histidine (20 mM) + NaCl (150 mM) at 30°C for 16 hours.

[0236] The incubation mixture obtained above was then purified using a 50 mL protA column (Hitrap Mabselect Sure, GE, 11-0034-95). The incubation mixture obtained above was applied to the column, and the column was washed with TBS + 0.2% Triton and TBS. The column was then eluted with 0.1 M acetate buffer at pH 2.9 and neutralized with 2.5 M Tris-HCl at 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 obtained 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 allow the 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) and deconvoluted spectra were acquired using Magtran software. Mass spectrometry of the IdeS digested sample showed the major product obtained corresponding to 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 according to the 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), PBS pH 7.4 (808 μL), 1,2-propylene glycol (11.3 mL) and compound 1 (350 μL, 40 mM in DMF) were added. The reaction was incubated overnight at room temperature, then filtered and dialyzed against PBS pH 7.4. Residual free payload was removed by adding charcoal (350 mg) and rotating overnight at room temperature. 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, both of which corresponded to the resulting ADC, i.e., HB37A6-SYNtecan E conjugate. First peak: observed mass was 26469 Da (calculated mass 26465 Da), corresponding to the coupled Fc / 2 fragment (2x closed lactone form of the payload). Second peak: observed mass was 26499 Da (calculated mass 26501 Da), corresponding to the coupled Fc / 2 fragment (2x open carboxylate form of the payload).

[0242] The prepared structure was as follows: The concentration, DAR value and SEC purity of the ADC product were measured using UV, SEC, RP-HPLC and LC-MS. The monomer purity detected by SE-HPLC was over 99% with a concentration of 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 as measured in Table 2.

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

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

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

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

[0248] (a) A reducing agent solution (TCEP (Sigma, C4706) dissolved in water) was added to the HB37A6 solution (antibody HB37A6 dissolved in PBS buffer (Thermo, 10010023)) and the reaction mixture was placed on a shaker for 2 h to 4 h, where (i) The optimal concentration of HB37A6 was 5 mg / mL-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) An excess of 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 1-2 h, where (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; The 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) The ADC products were detected using HIC, LC-MS and SEC HPLC to 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 The bill was HB37A6.

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

[0256] (a) A reducing agent solution (TCEP (Sigma, C4706) dissolved in water) is added to an antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer), and the reaction mixture is placed on a shaker for 2-4 h after the addition is complete. (i) The optimal concentration of antibody HB37A6 was 5 mg / mL-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 1h-2h. (i) The optimal molar ratio of MC-VC-PAB-MMAE / mAb was 8.0-10.0. (ii) The optimal temperature for the binding reaction was 20℃~37℃.

[0258] (c) After the binding reaction was completed, acetylcysteine ​​solution was added to stop the reaction in step (b). 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 products were 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 as measured in Table 2.

[0264] where Ab was HB37A6.

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

[0266] (a) Linker-payload SMCC-DM1 solution (purchased from Levena Biopharma, dissolved in an organic solvent such as SET0101, DMSO) is added to antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer), and the reaction mixture is placed 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 5.5-6.5; (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; The 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 products 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] All monoclonal antibody and ADC information 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 changes the binding properties of IEX019-01 monoclonal antibody to target cells, the inventors used DAN-G cell line (hCLDN18.2 negative) and DAN-G-hCLDN18.2 cell line (overexpressing hCLDN18.2) 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] Both IEX019-01 and IEX019-02 did not bind to non-target cells DAN-G, but had very high affinity for DANG-hCLDN18.2, indicating that the binding of the antibodies depends on the expression specificity of the target and that the coupling of Exatecan does not affect the binding of the antibodies. At the same time, the control molecule IEX019-06 (a monoclonal antibody is a negative control of IgG coupled to Exatecan toxin by the same technology) did not bind to the 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 that determine the tumor suppression effect.

[0281] To detect the endocytosis efficiency of the antibody coupled to the small molecule compound, the inventors detected the endocytosis of different IEX019 molecules in DANG-hCLDNA18.2 cells by flow cytometry technology. After digesting the DANG-hCLDNA18.2 cells, the cell density was adjusted to 1 × 10 5100μL of the molecules to be detected were taken and the cells were resuspended (molecule concentration of 50nM), and 5 replicates were set for each sample (i.e., endocytosis time: 0h, 1h, 2h, 3h, 4h). The wells were placed on ice and incubated for 1h. After 1h, the wells were centrifuged at 500g for 3min and the supernatant was discarded. Each well was added with 200μL of FACS buffer (1% FBS, 1xPBS) and washed twice. One set of samples was taken and transferred to a new 96-well plate and incubated at 37℃ for 4h. The remaining samples were continued to be incubated on ice. The above procedure was repeated, and the samples were incubated at 37℃ for 3h, 2h, 1h, and 0h, respectively. After the completion of the specific incubation time, the wells were centrifuged at 500g for 3min 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 in the dark. After the secondary antibody incubation was completed, the wells 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 detected on the instrument.

[0282] The experimental results are shown in Figure 7. 0 h incubation at 37°C was taken as the zero point of endocytosis. After 2 h incubation, all molecules reached the maximum endocytosis level, about 60%, indicating that the ADC molecules designed and synthesized based on IEX019-01 maintained strong endocytosis ability consistent with monoclonal antibodies 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, and then the density was adjusted and uniformly seeded on a 96-well plate (Table 4). Sample IEX019 molecules (IEX019-02, IEX019-03, IEX019-04, starting dilution concentration was 100 nM, dilution factor was 3) were added at specific concentrations after dilution, and wells to which IEX019 molecules were not added were used as controls. The wells were left in a 37°C incubator and incubated 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 the curve was fitted using Graph Pad Prism 8.0.

[0287] As shown in Figure 8, the killing of ADC molecules against cell lines was dependent on the expression level of surface hCLDN18.2. In hCLDNA8.2 negative DANG, IEX019 molecules had no significant effect on cell viability (Figure 8A), in cell lines with moderate expression levels (NUGC-4, SNU620), IEX019 molecules showed a certain degree of cell killing (Figure 8B), and in the high expression cell line DAN-G-hCLDN18.2, both IEX019 molecules showed a significant killing effect (Figure 8C). This indicates that IEX019 molecules have good selectivity and efficacy.

[0288] [Table 5]

[0289] Example 2.5: Bystander killing effect

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

[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 and 6-well cell culture plates were prepared. DANG cells and DANG-hCLDN18.2 were cultured at 7.5 × 10 4100 nM) and co-cultured the two types of cells. 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 at a final concentration of 50 nM, and three replicates were set up for each sample. The cell culture plate was left in a 37°C incubator and cultured for 5 days. After 5 days, the culture supernatant was discarded and washed with PBS, and then Trypsin-EDTA was added to digest the cells, and all the digested cells were collected and transferred to a 96-well plate. According to the antibody incubation process of the flow cytometry technique, the cells were incubated with a primary antibody (IEX019-01, 100 nM), a secondary antibody (anti-hFc-PE, SouthernBiotech) at 4°C for 1 h and 0.5 h, respectively. After washing with PBS, the live / dead Violet dye (Thermo, L34964) was diluted 1:1000, 100μL was added to each well, and incubated at 4℃ for 20min. After washing with PBS, cells were resuspended in 100μL PBS and detected on-board. Live / dead dye was used to separate each sample into populations, where IEX019-01 negative (i.e., hCLDN18.2 negative) population was DAN-G cells, and IEX019-01 positive (i.e., hCLDN18.2 positive) cells was DAN-G-hCLDN18.2. The number of the two types of cells in each sample was counted, and the relative cell viability of each type of cell was calculated according to the following equation, and the curve was fitted with Graph Pad Prism8.0.

[0293] Relative viability of DANG 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 in 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 types of cells, and 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, can only kill DANG-hCLDNA8.2 cells and has no effect on DANG cells. Only IEX019-02 has a significant bystander killing effect and can kill target cells and non-target cells simultaneously.

[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 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. In the experiment, SPF grade female CB-17-SCID mice (14g-17g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used, with the qualification certificate number NO.110011201108225246.

[0299] DANG-hCLDN18.2 cells were subcultured periodically and used for subsequent in vivo experiments. Cells were collected by centrifugation, and DANG-hCLDN18.2 cells were dispersed in PBS (1×) at 3×10 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 each) and administered the doses and methods shown in Table 5 on the 5th day after inoculation. The tumor volumes and body weights 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, and the formula was: TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of 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 results of tumor inhibition rate are shown in Table 6 and Figure 10A. On the 50th day after inoculation, the tumor inhibition rate after a single administration of IEX019-02 reached 103.60%, compared with IEX019-01 monoclonal antibody, which is obviously superior to IEX019-03 and IEX019-04, with tumor inhibition rates of 93.70% and 35.20%, respectively. On the 82nd day after inoculation, 100% of the mouse tumors in the IEX019-02 group were completely regressed. At the same time, the inventors detected the weight of the mice, and the results are shown in Figure 10B, and there was no significant difference in the weight of the mice.

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

[0309] NUGC-4 cells were subcultured periodically and used for subsequent in vivo experiments. Cells were collected by centrifugation, and NUGC-4 cells were 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 area 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 each) and administered the doses and regimens shown in Table 7 on the 5th day after inoculation. The tumor volumes and body weights 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, and the formula was: TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of 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 results of tumor inhibition rate are shown in Tables 8 and 11A. On the 33rd day after inoculation, the tumor inhibition rates for single administration of negative control IEX019-06, IEX019-02 and IEX019-03 were 80.04% and 54.31%, respectively. At the same time, the body weight of the mice was detected, and the results shown in Figure 11B showed that there was no significant difference in the body weight of the mice.

[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 IEX019 molecule, the inventors inoculated SNU620 cells into CB-17-SCID mice to measure the antitumor efficacy of the molecule of the present invention (IEX019-02). In the experiment, SPF grade female CB-17-SCID mice (18g-20g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used, with the qualification certificate number NO.110011211102179364.

[0318] SNU620 cells were subcultured periodically and used for subsequent in vivo experiments. Cells were collected by centrifugation, and SNU620 cells were dispersed in PBS (1x) at 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 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 each) and administered the doses and regimens shown in Table 9 on day 7 after inoculation. The tumor volumes and body weights 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, and the formula was: TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of 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, the tumor inhibition rate after a single administration of 10 mg / kg of IEX019-02 was 143.77% compared to hIgG on the 39th day after inoculation, and 100% of the mouse tumors completely regressed. There was no significant difference in the mouse body 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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