Antibody-drug conjugate targeting Claudin 18.2

Abstract

The present invention provides an antibody-drug conjugate (ADC) targeting Claudin 18.2 and a composition containing the same. [Solution] An antibody-drug conjugate comprising the following formula. JPEG2026053502000038.jpg62150 (Ab: Anti-Claudin 18.2 antibody. q: Drug-antibody ratio of 3-5.)

Description

[Technical Field]

[0001] The present invention relates to antibody-drug conjugates (ADCs) targeting Claudin 18.2 (CLDN 18.2) and compositions comprising the above molecule. The present invention further relates to the therapeutic and diagnostic use of these antibodies or antibody fragments. [Background technology]

[0002] Claudins are a family of proteins that are essential components of tight junctions in cells. They establish intercellular barriers that control molecular movement between cells. Claudins family proteins have four transmembrane domains, with both their N-terminus and C-terminus located in the cytoplasm. Different Claudins proteins are expressed in different tissues, and changes in their function are associated with cancer development in each tissue. For example, Claudin-1 is expressed in colon cancer and has shown 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. Its expression in normal tissue is strictly limited to differentiated epithelial cells of the gastric mucosa, but not in the gastric stem cell region. CLDN18.2 is expressed in a considerable number of primary gastric cancers and maintains its expression level in cancerous tissue of gastric metastases. In addition to gastric cancer, CLDN18.2 expression is also found 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] Regarding gastric cancer, in 2014 there were approximately 410,000 new cases and 290,000 deaths nationwide, accounting for nearly half of the world's total cases and deaths, and the trend continues to increase. However, there is a large demand for unsatisfactory clinical oncology treatments, making the development of drugs targeting Claudin 18.2 extremely necessary.

[0005] Despite the clinical success of therapeutic antibodies, naked MAbs targeting cell surface tumor antigens have rarely provided sufficient efficacy on their own. To increase the low activity of MAbs, a novel strategy focuses on binding to toxic molecules. Plant and bacterial toxins, as well as small chemotherapeutic molecules, may be good candidates because they are highly effective and active even in very small amounts.

[0006] Technological advancements over the past few years have led to increasing development activity by pharmaceutical companies in the field of antibody-drug conjugates (ADCs) for cancer treatment, with the aim of solving the initial problems related to immunogenicity, affinity, specificity, undesirable toxicity, productivity, and half-life.

[0007] Although some progress has been achieved, there is a need for other treatment strategies for treating tumors, as well as components used in such treatment strategies, particularly antibodies against Claudin18.2 that have high affinity, high specificity, and / or low immunogenic risk, and ADC molecules that have relatively high activity, low toxicity, long half-life, high specificity, or high affinity, and / or good half-life or pharmacophore properties.

Summary of the Invention

[0008] The present invention provides an antibody-drug conjugate (ADC) targeting Claudin18.2, and the conjugate has the following advantages.

[0009] (1) It binds to target cells expressing human CLDN18.2 and has a high affinity for it. (2) It can enter cells by endocytosis and kill target cells. In some embodiments, the ADC of the present invention has a high endocytosis efficiency. (3) It has a significant bystander killing effect. (4) It has a high anti-tumor drug efficacy. (5) It has low toxicity. (6) It has good stability. (7) It has good drug development potential.

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

[0011] [Figure 1] This demonstrates that the HB37A6 antibody specifically binds to CLDN18.2 on the cell surface. [Figure 2] This indicates that the HB37A6 antibody does not bind to CLDN18.1 on the cell surface. [Figure 3] This shows the binding of the HB37A6 antibody to the gastric cancer cell line NUGC-4, the gastric cancer cell line KATO III-hCLDN18.2, and the pancreatic cancer cell line DAN-G-hCLDN18.2. [Figure 4] This study demonstrates the antitumor effect of the HB37A6 antibody in a mouse model of pancreatic cancer. [Figure 5] This study demonstrates the antitumor effect of the HB37A6 antibody in a mouse model of gastric cancer. [Figure 6] The IEX019 molecule exhibits cell-binding activity. [Figure 7] This demonstrates endocytosis of the IEX019 molecule in DANG-hCLDN18.2 cells. [Figure 8A]Figure 8A shows the killing effect of the IEX019 molecule in cell lines with low expression of hCLDN18.2, Figure 8B shows the killing effect of the IEX019 molecule in cell lines with moderate expression levels of hCLDNA8.2, and Figure 8C shows the killing effect of the IEX019 molecule in cell lines with high expression of hCLDN18.2. [Figure 8B] Figure 8A shows the killing effect of the IEX019 molecule in cell lines with low expression of hCLDN18.2, Figure 8B shows the killing effect of the IEX019 molecule in cell lines with moderate expression levels of hCLDNA8.2, and Figure 8C shows the killing effect of the IEX019 molecule in cell lines with high expression of hCLDN18.2. [Figure 8C] Figure 8A shows the killing effect of the IEX019 molecule in cell lines with low expression of hCLDN18.2, Figure 8B shows the killing effect of the IEX019 molecule in cell lines with moderate expression levels of hCLDNA8.2, and Figure 8C shows the killing effect of the IEX019 molecule in cell lines with high expression of hCLDN18.2. [Figure 9] The IEX019 molecule demonstrates bystander-killing effects. [Figure 10A] The tumor-suppressing effect (Figure 10A) and body weight change (Figure 10B) of the IEX019 molecule in mice are shown. [Figure 10B] The tumor-suppressing effect (Figure 10A) and body weight change (Figure 10B) of the IEX019 molecule in mice are shown. [Figure 11A] The tumor-suppressing effect (Figure 11A) and body weight change (Figure 11B) of the IEX019 molecule in mice are shown. [Figure 11B] The tumor-suppressing effect (Figure 11A) and body weight change (Figure 11B) of the IEX019 molecule in mice are shown. [Figure 12A] The tumor-suppressing effect (Figure 12A) and body weight change (Figure 12B) of the IEX019 molecule in mice are shown. [Figure 12B] The tumor-suppressing effect (Figure 12A) and body weight change (Figure 12B) of the IEX019 molecule in mice are shown. [Modes for carrying out the invention]

[0012] I. Definition

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

[0014] The following definitions are used to interpret this specification, and where appropriate, a singular term may include a plural form, and vice versa. It should be understood that the terms used herein are merely descriptive and not restrictive to specific embodiments.

[0015] When used with a number or figure, the term "approximately" means covering a range of numbers or figures that is 5% smaller than the lower limit and 5% larger than the upper limit.

[0016] As used herein, the terms "and / or" refer to one or more of the selectable options.

[0017] As used herein, the terms “contains” or “includes” mean including the elements, integers, or steps described above, but not excluding any other elements, integers, or steps. Where the terms “contains” or “includes” are used herein, unless otherwise specified, they also include any other combinations of the elements, integers, or steps described above. For example, when an antibody variable region “contains” a particular sequence is referred to, it is also intended to include the antibody variable region consisting of that particular sequence.

[0018] As used herein, the terms "CLAUDIN" or "CLDN" refer to the most important skeletal proteins that determine the tight junction structure between cells, participating in adhesion junctions and playing a crucial role in tumor cell metastasis and invasion. Claudin proteins are widely present in mammalian epithelial and endothelial cells, primarily distributed on the lateral surfaces of epithelial cells and the basal cytoplasmic membrane. 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, contains 261 amino acid residues, belongs to the Claudins superfamily, and its protein structure consists of two extracellular loops and four membrane-permeable regions. The two subtypes of the human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1) and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2), respectively. In the primary structural sequences of both proteins, they differ only in amino acid residues at a certain position from the N-terminal signal peptide to the extracellular loop 1 structure. Specifically, on the extracellular loop 1, CLDN18.1 and CLDN18.2 differ by only eight amino acids. The interspecies sequence homology between the two subtype proteins of CLDN18 is also very high. Of these, the extracellular loop 1 of CLDN18.2 has a perfect sequence match in different organisms such as humans, mice, and rhesus monkeys, and the homology between human and mouse CLDN18.2 proteins reaches 84%, revealing that the CLDN18.2 protein sequence is extremely conserved (O. Tureci et al., Gene 481:83-92, 2011). CLDN18.2 or any of its variants and isotypes can be isolated from cells or tissues that express them naturally, or produced by recombinant methods well known in the art and / or the methods described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0019] As used herein, the terms “anti-CLDN18.2 antibody,” “anti-CLDN18.2,” “CLDN18.2 antibody,” “antibody that binds to CLDN18.2,” or “antibody that specifically binds to CLDN18.2” refer to an antibody that can be used as a therapeutic agent targeting (human) CLDN18.2 by binding to (human) CLDN18.2 with sufficient affinity. 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 does not bind to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), biolayer interferometry (BLI), MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0020] The expression of CLDN18.2 in cells can be determined by various means, such as anti-CLDN18.2 antibodies. For example, the binding strength (measured, for example, by FACS) between cells that "highly express CLDN18.2" and anti-CLDN18.2 antibodies may be 500, 600, 700, 800, 900, or preferably 1000 times or more than the binding strength between anti-CLDN18.2 antibodies and cells that do not express CLDN18.2, for example, 1100, 1200, 1300, 1400 times or more. For example, the binding strength (e.g., 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 times or greater, but not exceeding 500 times.

[0021] The terms “complete antibody,” “whole antibody,” or “full-length antibody” are interchangeable herein and refer to antibody molecules that have a natural immunoglobulin molecular structure. In the case of a typical four-chain IgG antibody, a full-length antibody contains 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 contains 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 as VH herein) and a heavy chain constant region, where the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The full-length antibody light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region, where the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus.

[0023] The term "antibody fragment" refers to a portion of a complete antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

[0024] An "antigen-binding fragment" refers to a molecule that contains part of a complete antibody and binds to the antigen that the complete antibody binds to, but is a molecule distinct from the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies (e.g., VHH), bivalent antibodies or their fragments, or camelid antibodies.

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

[0026] A "complementarity-determining region," "CDR region," or "CDR" is a region in the antibody variable domain that is hypervariable in sequence and structurally formed and determined ("hypervariable loop"), and / or contains antigen contact residues ("antigen contact sites"). CDRs primarily play a role in binding to antigen epitopes. Heavy chain and light chain CDRs are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. CDRs in the heavy chain variable domain of an antibody are called HCDR1, HCDR2, and HCDR3, and CDRs in the light chain variable domain of an antibody are called LCDR1, LCDR2, and LCDR3.In the amino acid sequence of a given light chain variable region or heavy chain variable region, the precise amino acid sequence boundary of each CDR can be determined by one or a combination of many known antibody CDR assignment systems, such as Chothia (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)), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USD Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and International ImMunoGeneTics. This includes the database (IMGT) (available at imgt.cines.fr / on the World Wide Web) and the North CDR definition based on affinity propagation clustering that utilizes 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] The following are the CDR domain boundaries as defined in the kabat, AbM, Chothia, Contact, and IMGT schemes.

[0028] [Table 1]

[0029] The CDR may be determined by having the same Kabat numbering position as the sequence of reference CDRs (e.g., any one of the exemplary CDRs of the present invention).

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

[0031] Unless otherwise specified, in this invention, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the 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 heavy chain variable region (CDR) of the antibody according to the present invention is determined by the following rules.

[0033] VH CDR1 is determined by the AbM rule, while VH CDR2 and VH CDR3 are both determined by the Kabat rule.

[0034] In one embodiment, the light chain variable region (CDR) of the antibody according to the present invention is determined by the Kabat rule.

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

[0036] It should be noted that the CDR boundaries of the variable region of the same antibody obtained by different assignment systems may differ. That is, there are differences in the CDR sequences of the variable region of the same antibody as defined by different assignment systems. Therefore, when an antibody is limited by a specific CDR sequence as defined in the present invention, the range of antibodies may include antibodies whose variable region sequence includes the specific CDR sequence, but whose CDR boundary differs from the specific CDR boundary defined in the present invention because a different approach (e.g., different rules or combinations of assignment systems) is used.

[0037] Antibodies with different specificities (i.e., targeting different binding sites of different antigens) have different CDRs (Cellular Derived Sequences) (within the same assignment system). However, despite the differences in CDRs among antibodies, the number of amino acid positions directly involved in antigen binding is limited within the CDR. At least two of the Kabat, Chothia, AbM, Contact, and North methods can be used to determine the minimum overlapping region and provide a "minimal binding unit" for antigen binding. The minimal binding unit may be a subpart of the CDR. As is known to those skilled in the art, the structure of the antibody and protein folding can determine the remaining residues of the CDR sequence. Accordingly, the present invention also considers variants of any CDR provided herein. For example, in a variant of a single CDR, the amino acid residues of the minimal binding unit may be retained unchanged, while the remaining CDR residues, defined based on Kabat or Chothia, may be replaced with conserved amino acid residues.

[0038] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least some constant regions. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin “Fc domain” includes two or three constant domains, namely a CH2 domain, a CH3 domain, and a selective CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain includes second and third constant domains (CH2 and CH3 domains) derived from two heavy chains of IgG, IgA, and IgD antibodies, or second, third, and fourth constant domains (CH2, CH3, and CH4 domains) derived from two heavy chains of IgM and IgE 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 known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this specification, the term “Fc region” does not include the heavy chain variable region VH and light chain variable region VL, or the heavy chain constant region CH1 and light chain constant region CL of immunoglobulins, but may, in some cases, include the hinge region at the N-terminus of the heavy chain constant region.

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

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

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

[0042] As used herein, the general term “sugar” refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). As used herein, the term “sugar derivative” refers to a derivative of a monosaccharide, i.e., a monosaccharide containing substituents and / or functional groups. Examples of sugar derivatives include amino sugars and sugar acids such as glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc), N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Examples of sugar derivatives further include compounds represented herein as E(A)x, where E is a sugar or sugar derivative, and E contains x functional groups A.

[0043] The core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as a GlcNAc that binds to the antibody via C1, preferably via an N-glycosidic bond to an amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. The core-GlcNAc substituent may be present at the natural glycosylation site of the antibody, but may also be introduced at a different site of the antibody. In this specification, the core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fucα1-6)GlcNAc substituent, also known as GlcNAc(Fuc).

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

[0045] The modified glycosylation antibody of the present invention refers to an antibody whose glycosylation mode is modified compared to an antibody having a natural glycosylation mode. Preferably, the modified glycosylation antibody refers to an antibody obtained after expression in an expression system (e.g., mammalian cells) followed by enzymatic modification of the glycans in vitro (e.g., modification of the glycans by glycosidase (endoglycosidase or glycosyltransferase, etc.)). More preferably, the modified glycosylation antibody of the present invention refers to an antibody comprising a core-GlcNAc and a linked sugar derivative E(A)x, where GlcNAc is bound to the antibody via C1, preferably via an N-glycosidic bond of an amide nitrogen atom on the side chain of the asparagine amino acid of the antibody. When the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, fucose is generally linked to C6 of the -GlcNAc substituent via α-1,6. The fucosylated -GlcNAc substituent refers to the core -GlcNAc(Fuc), and the fucosylated GlcNAc-E(A)x substituent refers to GlcNAc(Fuc)-E(A)x.

[0046] As used herein, the term "site-specific coupling" refers to a coupling that specifically links a drug / active substance to a particular site on an antibody via a linker.

[0047] As used herein, the term “alkyl group” refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 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. Typical 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, and n-decyl groups.

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

[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. A cycloalkyl group may contain three or more carbon atoms in the ring, for example, 3 to 18, 3 to 10, or 3 to 8 carbon atoms, and generally, according to the present invention, it contains 3 to 6 atoms. 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. Typical heteroaryl groups include 2-thienyl or 3-thienyl group, 2-furyl or 3-furyl group, 2-pyrrolyl or 3-pyrrolyl group, 2-imidazolyl group, 4-imidazolyl or 5-imidazolyl group, 3-pyrazolyl group, 4-pyrazolyl or 5-pyrazolyl group, 2-thiazolyl group, 4-thiazolyl or 5-thiazolyl group, 3-isothiazolyl group, 4-isothiazolyl or 5-isothiazolyl group, 2-oxazolyl group, 4-oxazolyl or 5-oxazolyl group , containing a 3-isoxazolyl group, a 4-isoxazolyl group or a 5-isoxazolyl group, a 3-triazolyl group or a 5-1,2,4-triazolyl group, a 4-triazolyl group or a 5-1,2,3-triazolyl group, a tetrazolyl group, a 2-pyridyl group, a 3-pyridyl group or a 4-pyridyl group, a 3-pyridazinyl group or a 4-pyridazinyl group, a 3-pyrazineyl group, a 4-pyridazinyl group or a 5-pyridazinyl group, a 2-pyridinyl group, a 2-pyrimidinyl group, a 4-pyrimidinyl group or a 5-pyrimidinyl group.

[0051] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and performance of the ADC complex of the present invention, and that is biologically or otherwise undesirable. The ADC complex of the present invention may exist in the form of pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt means a salt formed by combining the ADC complex of the present invention with an organic or inorganic acid, and the organic or inorganic acid includes, but is not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. A pharmaceutically acceptable, non-toxic base addition salt means a salt formed with 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-group-containing organic base.

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

[0053] Unless otherwise specified by context, the terms "pharmaceutically acceptable" and "medicinal" are used interchangeably in this specification.

[0054] The term “drug:antibody ratio” or “DAR” refers to the ratio of the small molecule drug moiety (D) coupled to the Ab moiety as described herein. In some embodiments described herein, the DAR can be determined by p and r in formula I, for example, the DAR may be 1 to 20, for example 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, 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 DAR mean of the molecular population in the product, i.e., the overall ratio of the small molecule drug moiety (D) coupled to the Ab moiety as described herein in the product, as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC), and this DAR is referred to herein as the DAR mean. In some embodiments, the average DAR values ​​of the composite according to the present invention are 1 to 20, for example 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, for example 1.0 to 8.0, 2.0 to 6.0, for example 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 This range has two endpoints from the following values: 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0.

[0055] As used herein, the term “therapeutic agent” includes any substance effective in treating or preventing tumors such as cancer, and includes chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0056] In this invention, the term "cytotoxic agent" refers to a substance that inhibits or blocks cellular function and / or causes cell death or destruction.

[0057] "Chemotherapy agents" include chemical compounds useful for treating cancer or immune system disorders.

[0058] The term "small molecule drug" refers to low molecular weight organic compounds that can modulate 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 mimetic compounds, and antibody mimetic compounds. As therapeutic agents, small molecules have higher cell permeability, are more susceptible to degradation, and are less likely to induce an immune response than large molecules.

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

[0060] The term "effective dose" refers to the amount or dosage of the antibody, fragment, composition, or combination of the present invention that, after being administered to a patient, produces the desired effect in the patient requiring treatment or prevention.

[0061] The "therapeutic dose" refers to the amount necessary to effectively achieve the desired therapeutic outcome over the required period of time. The therapeutic dose is also the amount at which the toxicity or adverse effects of any antibody, antibody fragment, composition, or combination do not outweigh the beneficial effects of the treatment. Compared to an untreated subject, the "therapeutic dose" preferably suppresses a measurable parameter (e.g., tumor volume) by at least about 30%, and more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and even 100%.

[0062] The "preventive effective dose" refers to the amount of medication needed to effectively achieve the desired preventive outcome over the required period at the required dosage. Generally, the preventive dose is less than the therapeutic effective dose because preventive doses are applied before or during the early stages of the disease in the target population.

[0063] The terms “host cell,” “host cell line,” and “host cell culture” may be used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including their offspring. Host cells include “transformed organisms” and “transformed cells,” including primary transformed cells and their offspring regardless of passage number. Offspring may not be exactly the same as the parent cells in terms of nucleic acid content and may contain mutations. This specification includes mutant offspring having the same function or biological activity, screened or selected from primary transformed cells.

[0064] As used herein, the term “labeling” refers to a compound or composition that is directly or indirectly bound or fused to a reagent (e.g., a polynucleotide probe or antibody) and that facilitates detection by the reagent to which it is bound or fused. Labeling can be detectable itself (e.g., radioisotope labeling or fluorescent labeling) or, when labeled by an enzyme catalyst, can catalytically alter the chemical properties of a detectable substrate compound or composition. The term is intended to include direct labeling of a probe or antibody by coupling (i.e., physical linking) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reaction with another directly labeled reagent.

[0065] "Individual" or "Subject" includes mammals. Mammals include, but are not limited to, livestock (e.g., cattle, 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 isolated from its natural environment or a component of its expression environment. In some embodiments, the antibody or ADC molecule is purified to a purity of 95% or more than 99%, 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 "antitumor effect" refers to a biological effect that can be expressed by various means, including, but is not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival rate.

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

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

[0070] The term “tumor” refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive as used herein.

[0071] The term "medicinal adjuvants" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete or incomplete)), excipients, vectors, and stabilizers that are administered together with the active substance.

[0072] The term "pharmaceutical composition" refers to a composition in which the active ingredient contained herein exists in a form that enables its biological activity, and which does not contain any other ingredient that is toxic to the 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. “Non-fixed combination” means that the active ingredients (e.g., (i) the ADC molecule of the present invention, and (ii) other therapeutic agents) are administered to a patient sequentially in separate entities simultaneously, without specific time constraints, or at the same or different time intervals, where such administration provides the patient with two or more active ingredients at levels that are prophylactically or therapeutically effective in vivo. In some embodiments, the ADC molecule of the present invention and other therapeutic agents used in a pharmaceutical combination are administered at levels not exceeding those used individually. “Fixed combination” means that two or more active ingredients are 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 ingredients, the combined use of each ingredient can achieve a better effect in treating a disease or condition than using any one ingredient alone. Each ingredient may be in a single formulation form, and these formulation forms may be the same or different.

[0074] The term “combination therapy” refers to the treatment of a disease described herein by administering two or more therapeutic agents or forms of treatment (e.g., radiation therapy or surgery). Such administration includes co-administration of these therapeutic agents nearly simultaneously, for example, in a single capsule having a fixed proportion of the active ingredient. Alternatively, such administration includes co-administration of each active ingredient in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose before administration. Such administration also includes using each type of therapeutic agent sequentially at substantially the same time or at different times. In any case, the treatment program provides the beneficial effect of the combination of pharmaceuticals in the treatment of the disease or condition described herein.

[0075] As used herein, “treatment” means reducing, interrupting, delaying, relieving, stopping, decreasing, or reversing the progression or severity of any existing symptoms, symptoms, conditions, disease, or illness.

[0076] As used herein, “prevention” includes inhibiting the onset or progression of a disease, condition, or symptoms associated with a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a prevention program. Generally, in the context of cancer, the term “prevention” means the administration of a drug before the onset of any signs or symptoms of cancer, particularly before cancer develops in subjects at risk of developing cancer.

[0077] As used herein, the term “vector” refers to a nucleic acid molecule capable of replicating another nucleic acid ligated to it. The term includes vectors that are self-replicating nucleic acid structures, and vectors that are bound to the genome of a host cell into which they are introduced. Some vectors can guide the expression of a nucleic acid manipulably ligated to them. 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. Tissue or cell samples may originate from solid tissues such as fresh, frozen, and / or preserved organs or tissue samples, biopsy samples, or puncture samples; blood or any blood component; cerebrospinal fluid; amniotic fluid; peritoneal fluid; interstitial fluid; or cells derived from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that do not naturally mix with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics.

[0079] II. Antibody-drug conjugates

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

[0081] In some embodiments, Ab in formula (I) of the present invention is a human antibody or a humanized antibody, preferably a human antibody. In some embodiments, 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, dAb (domain antibody), a linear antibody, a single-chain antibody (e.g., scFv), a single-domain antibody (e.g., VHH), a bivalent antibody or a fragment thereof, or a camelid antibody.

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

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

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

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

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

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

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

[0089] In some embodiments, the three complementarity determination regions (HCDRs) from the VH according to the present invention, HCDR1, HCDR2, and HCDR3, are (i) The three complementarity determination regions included in VH shown in Sequence ID No. 4, HCDR1, HCDR2 and HCDR3, or (ii) The sequence of (i) contains at least one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in total across the three HCDR regions, and which is 5, 4, 3, 2, or 1 or less.

[0090] In some embodiments, the three complementarity determination regions (LCDRs) from the VL according to the present invention, LCDR1, LCDR2, and LCDR3, are (i) LCDR1, LCDR2 and LCDR3, which are the three complementarity determination regions included in the VL shown in Sequence ID No. 9, or (ii) The sequence of (i) contains at least one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in total across the three LCDR regions, and which is 5, 4, 3, 2, or 1 or less.

[0091] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or 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 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, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, or 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, Ab in formula (I) of the present invention includes a heavy chain steady region. In some embodiments, Ab in formula (I) of the present invention includes a light chain steady region. In some embodiments, Ab in formula (I) of the present invention further includes a heavy chain steady region and a light chain steady region.

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

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

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

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

[0102] In some specific embodiments of the present invention, the heavy chain of the present invention includes or comprises a heavy chain variable region and a heavy chain steady region. In some specific embodiments of the present invention, the light chain of the present invention includes or comprises a light chain variable region and a light chain steady region.

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

[0104] In some specific embodiments of the present invention, 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, 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 VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and / or It contains the amino acid sequence shown in Sequence ID No. 9 or an amino acid sequence having at least 90% identity thereto, or contains a VL consisting of such sequences.

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

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

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

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

[0110] In some embodiments of the present invention, Ab in formula (I) comprises a heavy chain and a light chain, where the amino acid sequence of the heavy chain is shown in SEQ ID NO: 11 and the amino acid sequence of the light chain is shown 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, and preferably conservative substitutions. In a preferred embodiment, the amino acid changes described herein occur in a region outside the CDR (e.g., FR). More preferably, the amino acid changes described herein occur in a region 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 substitutions are conservative substitutions. A conservative substitution means that one amino acid is replaced by another amino acid of the same type, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is replaced by another neutral amino acid. In some embodiments, the substitution occurs in the CDR region of the antibody. Generally, the resulting variant has modifications (e.g., improvements) to a certain biological property (e.g., improved affinity) compared to the parent antibody and / or retains some substantially preserved biological properties of the parent antibody. An exemplary substitution variant is an affinity-mature antibody.

[0113] The antibody Ab in formula (I) of the present invention may be an antibody having a modified glycosylation. In some embodiments, the antibody is obtained after enzymatic modification of the glycan in vitro (e.g., modification of the glycan by a glycosidase (endoglycosidase or glycosyltransferase, etc.)). In some embodiments, the antibody having the modified glycosylation refers to an antibody in which the glycan of the glycosylation site of the antibody has been modified from a heterogeneous N-glycan to a single-structure N-glycan having a reactive group (e.g., any reactive group that can react with the 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 of the antibody Fc domain, e.g., Asn297.

[0114] For modifications of 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 by reference in their entirety.

[0115] In one preferred embodiment, the modified glycosylated antibody of the present invention is GlcNAc-E(A) x An antibody containing a substituent, wherein GlcNAc is N-acetylglucosamine, and E(A)x is a sugar derivative containing 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 bound to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent, and the N-acetylglucosamine is optionally fucosylated. When N-acetylglucosamine is fucosylated, it is bound to fucose (Fuc) via C6.

[0116] In one embodiment, the modified glycosylated antibody of the present invention is an antibody of formula (III), where Ab represents an antibody, GlcNAc is N-acetylglucosamine, Fuc is fucose, b is 0 or 1, and y is 1 to 20, where E(A)x is a sugar derivative containing x functional groups A, where A is an azide group, a ketone group, or an alkynyl group, and x is 1, 2, 3, or 4. In one preferred embodiment, y is 1 to 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 modified glycosylated antibody can, for example, bind to C4 of the GlcNAc via a β(1,4)-glycosidic bond, or to C3 of the GlcNAc via an α(1,3)-glycosidic bond, preferably to C4 of the GlcNAc via a β(1,4)-glycosidic bond. The N-acetylglucosamine in the GlcNAc-E(A)x substituent binds to the antibody via C1, preferably to the amide nitrogen atom (GlcNAcβ1-Asn) in the asparagine side chain of the antibody via an N-glycosidic bond. The GlcNAc in the GlcNAc-E(A)x substituent is optionally fucosylated. Correspondingly, if GlcNAc-E is present in the antibody-drug conjugate, it can also be linked as described above.

[0118] In one preferred embodiment, the functional group A is an azide group. When A is an azide group, preferably A is bonded to C2, C3, C4, or C6. As described above, one or more azide substituents in E(A)x can bond to C2, C3, C4, or C6 of the sugar or sugar derivative E and substitute for a hydroxyl group (OH). It should be understood that the bond position of the functional group A corresponds to the position where the sugar containing A and the linker are linked.

[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 selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc), and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose, and GalNAc, and most preferably GalNAc.

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

[0121] To the extent that they do not contradict each other, the above descriptions and explanations regarding sugars (including, but not limited to, the linking schemes of the glycosidic bond of GlcNAc-E(A)x) should be understood to apply similarly to sugars corresponding to the complex of formula (II) below, for example, the linking schemes of sugars. In some embodiments, a reactive group (e.g., functional group A) is linked to the sugar chain of the antibody in the glycosylation modification of the antibody, so an "antibody having modified glycosylation" is defined as an antibody containing such reactive group. However, it will be understood by those skilled in the art that when an antibody-drug complex is formed, the reactive group reacts with the linker moiety to form a new group with the linker moiety, so that in the antibody-drug complex, the new group is considered part of the linker.

[0122] Preferably, the antibody of the present invention is a monoclonal antibody, more preferably an IgG antibody (e.g., a tetrachain IgG antibody), and most preferably an IgG1 antibody. In one embodiment, the antibody is a complete antibody.

[0123] If the modified antibody is a complete antibody, it preferably contains two or more, more preferably two, GlcNAc-E(A)x substituents, and the GlcNAc-E(A)x substituents are optionally fucosylated. However, if the modified antibody is an antibody fragment, for example, a Fab or Fc fragment, the antibody may have only one GlcNAc-E(A)x substituent, which is optionally fucosylated. The GlcNAc-E(A)x substituent can be located at any position on the antibody, as long as the substituent does not interfere with the binding of the 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 described above, the modified glycosylated antibody of the present invention comprises one or more GlcNAc-E(A)x substituents, for example, two GlcNAc-E(A)x substituents.

[0125] In one preferred embodiment, the GlcNAc-E(A)x substituent is located at the native N-glycosylation site of the antibody (e.g., a naturally conserved N-glycosylation site), for example, the Fc region (more preferably the CH2 domain). In a further preferred embodiment, the antibody is an IgG antibody, and the GlcNAc-E(A)x substituent is located at the native N-glycosylation site (naturally conserved N-glycosylation site) of the IgG antibody. In a further preferred embodiment, the native site is the Asn297-glycosylation site of the IgG antibody. The Asn297-glycosylation site is located in the Fc region of the heavy chain of the IgG antibody. In one preferred embodiment, the GlcNAc-E(A)x substituent is located at the Asn297-glycosylation sites 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 the anti-human CLDN18.2 of the present invention, preferably the linker can achieve site-specific coupling of the ADC.

[0127] In some embodiments, the linker applied to 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 technique that can achieve site-directed coupling.

[0128] In one preferred embodiment, the linker of the present invention is a linker that links to an oligosaccharide of an antibody. As defined herein, “a linker that links to an oligosaccharide of an antibody” refers to any linker that links to a reactive group of a glycan at the glycosylation site of an antibody to couple the antibody to a drug. The glycan at the glycosylation site of an antibody is generally an N-glycan, and is usually modified from a heterogeneous N-glycan to a single-structure N-glycan with a reactive group, and further links to a “linker” using the reactive group of the glycan to achieve site-specific coupling between the drug and the antibody, thereby obtaining an antibody-drug conjugate. In one preferred embodiment, the N-glycosylation site of the antibody is the antibody Fc domain, and preferably the conserved N-glycosylation site of the CH2 domain, for example, Asn297. Accordingly, in one embodiment, the “linker 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 of the antibody Fc domain (e.g., Asn297), such as the linker described in PCT / NL2013 / 050744 or the linker described in PCT / EP2021 / 075401, the whole of which is incorporated herein. 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 containing 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 containing an alkynyl group. When such a linker is referred to in the present invention, the reactive group reacts with the linker group to form a new group, so the group formed after the reaction of the reactive group in the antibody-drug conjugate can also be defined as part of the "linker," for example, as shown in formula (II) of the present invention.

[0129] The linkers applicable to the present invention include, for example, cathepsin-degrading linkers such as Val-Cit linkers (vc-PAB, etc.), cBu-Cit linkers and CX linkers, non-cleavable linkers such as SMCC linkers or MD linkers, acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, and ultraviolet-sensitive linkers, further comprising PC4AP (Antibody-drug conjugates: Recent advances in linker chemistry, Su, Z., Xiao, D., Xie, F., Liu, L., Wang, Y., Fan, S., Li, S. (2021). Antibody-drug conjugates: Recent advances in linker chemistry. Acta Pharmaceutica Sinica B.). The linker applied to the present invention may be a combination of one or more linkers, for example, a cathepsin-degrading linker can be combined with other types of linkers to form a new linker. Accordingly, the “linker” as described in the present invention encompasses 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. Accordingly, in one embodiment, the linker applied to the present invention is MC-VC-PAB, vc-PAB, SMCC, or MC-GGFG.

[0130] In formula (I) of the present invention, D may be any antitumor compound, and is not particularly limited, as long as it has an antitumor effect and a substituent or substructure that can be linked to the linker structure. For example, the antitumor compound may be a pharmaceutically active compound that acts on tumors. In the case of an antitumor compound, preferably part or all of the linker is cleaved within the tumor cell, 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 its unmodified structure, thereby exhibiting its inherent antitumor effect.

[0131] In some embodiments, the antitumor compound may be, for example, a cytotoxic agent or a chemotherapeutic agent, and may be a camptothecin-based compound such as ixitecan (the topoisomerase I inhibitor exatecan) or Dxd (a novel topoisomerase I inhibitor exatecan derivative), an auristatin-based compound such as monomethyl auristatin E (MMAE), or a mytansin-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 as described in (II) or a pharmaceutically acceptable salt or solvate thereof, [ka] Ab represents the antibody as defined in the specification. L1 is the linker, E is a sugar or sugar derivative, for example, a sugar or sugar derivative as defined above. GlcNAc is N-acetylglucosamine, and Fuc is fucose. D and r are as defined in the above formula I, b is either 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 pharmaceutically 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, and is preferably linked to L1 via the C atom at the 6-position.

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

[0136] In one embodiment, in formula (II), L1 has the following structure

Chemical formula

[0137] It should be understood that when m is 0, Q does not exist, which means that the associated nitrogen atom of the triazole is directly bonded to the E portion by a covalent single bond.

[0138] In one embodiment, each L4 is independently [ka] And, Here, R5 and R6 are independently hydrogen and C1-C, respectively. 12 Selected from alkyl groups, Ar is selected from aryl groups, preferably phenyl groups. In one embodiment, each L4 is independently [ka] That is the case.

[0139] In one embodiment, -L1- has the following structure in the formula. [ka]

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

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

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

[0143] III. Production of the ADC molecule of the present invention

[0144] One aspect of the present invention is a method for preparing an antibody having a modified glycosylation,

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

[0146] (2) Preparation of trimmed antibodies: The antibodies prepared in step (1) are deglycosylated in the presence of endoglycosidase to obtain antibodies containing core N-acetylglucosamine substituents, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituents are optionally fucosylated.

[0147] (3) A method is provided comprising 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 containing a GlcNAc-E(A)x substituent, wherein the GlcNAc-E(A)x substituent is conjugated to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent, wherein the catalyst is a glycosyltransferase, and P is selected from uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP).

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

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

[0150] The glycosyltransferase in step (3) is preferably a glycosyltransferase of β-(1,4)-N-acetylgalactosaminetransferase, or derived therefrom, and more preferably any β-(1,4)-GalNAcT enzyme described in PCT / EP2016 / 059194. In some embodiments, the β-(1,4)-GalNAcT enzyme is an invertebrate β-(1,4)-GalNAcT enzyme, or derived therefrom. The β-(1,4)-GalNAcT enzyme may be an invertebrate β-(1,4)-GalNAcT enzyme known to those skilled in the art, or derived therefrom. Preferably, the β-(1,4)-GalNAcT enzyme is derived from or is derived from the phylum Nematoda, preferably from the class Chromadorea or Seernentea, or from the phylum Arthropoda, preferably from the class Insecta. More preferably, the β-(1,4)-GalNAcT enzyme is derived from or is derived from the nematode Mylestinellosa, the roundworm, the nettle caterpillar, the fruit fly, the saprophytic fruit nematode, Caeno rhabditis briggsae, the nematode Wu, the Robaria beetle, the Piseorhiza, the Japanese termite, the Japanese horned fly, the Biki and the Hamster butterfly. Preferably, the glycosyltransferase suitable for step (3) is the nettle-leaved nettle β-(1,4)-GalNAcT enzyme (e.g., His-TnGalNacT) disclosed in PCT / EP2016 / 059194 and named TnGalNAcT.

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

[0152] In one embodiment, a method for preparing the ADC of the present invention, wherein a glycosylated antibody of formula (III) is used. [ka] In the formula, the meaning of each variable or symbol is as defined above. A linker-drug compound (linker-payload) containing an alkynyl group and one or more (e.g., 1, 2, 3, or 4) drug molecules is reacted with the drug molecule. The process involves generating an antibody-drug conjugate (Formula II), [ka] The method is provided, in which the meaning of each variable or symbol in the formula is as defined above.

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

[0154] IV. Pharmaceutical Compositions

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

[0156] The present invention further comprises compositions (including pharmaceutical compositions) comprising the ADC molecule of the present invention or a pharmaceutically acceptable salt thereof. These compositions may also comprise suitable pharmaceutically acceptable materials, such as pharmaceutically acceptable excipients including pharmaceutically acceptable vectors known in the art and buffers.

[0157] As used herein, “medicinal vector” includes any or all of the following: a physiologically compatible solvent, dispersion medium, isotonic agent, absorption retarder, etc.

[0158] For information on the use of medicinal excipients and their applications, see also the Handbook of Pharmaceutical Excipients, 8th edition, RCRowe, PJSeskey, and SCOwen, Pharmaceutical Press, London, Chicago.

[0159] The compositions of the present invention may be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., solutions for injection and solutions for infusion), dispersants or suspensions, liposomes, and suppositories. The preferred form is determined by the desired mode of administration and therapeutic use.

[0160] A drug of the ADC molecule described herein can be produced by mixing the ADC molecule of the present invention having a desired purity with one or more optionally selected medicinal adjuvants, preferably in the form of a lyophilized formulation or an aqueous solution.

[0161] The pharmaceutical composition or formulation of the present invention may further contain one or more active ingredients, which are necessary for the specific indication being treated and preferably have complementary activities that do not adversely affect each other. It is also desirable to provide other therapeutic agents, for example, which include 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 appropriate combinations in amounts effective for the intended use.

[0162] Sustained-release formulations can be manufactured. A suitable example of a sustained-release formulation comprises a semipermeable matrix of a hydrophobic solid polymer containing an antibody, the matrix being a molded article such as a film or microcapsule.

[0163] VII. Combinations of pharmaceuticals and drug kits

[0164] In some embodiments, the present invention further provides a pharmaceutical combination or pharmaceutical combination product comprising the ADC molecule of the present 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 immunomodulators (e.g., immune checkpoint inhibitors or agonists)).

[0165] Another object of the present invention is to provide a drug kit comprising the pharmaceutical combination of the present invention, preferably the drug kit in the form of pharmaceutical dose units. This allows the dose units to be provided by an administration program or pharmaceutical administration interval.

[0166] In one embodiment, the drug kit of the present invention is in the same package, - A first container containing a pharmaceutical composition comprising the ADC molecule of the present invention, -Includes a second container containing a pharmaceutical composition including other therapeutic agents.

[0167] VIII. Use and Method

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

[0169] In some embodiments, the patient with the tumor (e.g., cancer) has CLDN18.2 (e.g., elevated levels of nucleic acids or proteins). In some embodiments, the tumor cells of the patient express CLDN18.2, for example, moderately expressing CLDN18.2, and preferably highly expressing CLDN18.2.

[0170] In some embodiments, the tumor, for example, cancer, includes solid tumors, hematological malignancies, and metastatic lesions. In one embodiment, an example of a solid tumor includes a malignant tumor. Cancer may be early, intermediate, or late-stage cancer or metastatic cancer.

[0171] In one specific embodiment, the ADC molecule of the present invention can kill tumor cells and / or suppress the proliferation of tumor cells that express, for example, CLDN18.2, such as gastrointestinal tumor cells, such as gastric cancer cells, pancreatic cancer cells, colon cancer cells, or colorectal cancer cells.

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

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

[0174] The subjects may be mammals such as primates, preferably higher primates, such as humans (e.g., individuals suffering from or at risk of suffering from one of the diseases described herein). In one embodiment, the subjects are suffering from or at risk of suffering from one of the diseases described herein (e.g., cancer). In some embodiments, the subjects are receiving or have already received other treatments such as chemotherapy and / or radiation therapy. In some embodiments, the subjects have previously received or are receiving immunotherapy.

[0175] In other embodiments, the present invention provides the use of ADC molecules, pharmaceutical compositions, pharmaceutical combinations, or drug kits in the production or manufacture of a drug, wherein the drug is used for the use described herein, for example, to prevent or treat a disease or condition related herein.

[0176] In some embodiments, the ADC molecule, pharmaceutical composition, pharmaceutical combination, or drug kit of the present invention delays the onset of a disease and / or symptoms associated with the disease.

[0177] In some embodiments, the ADC molecule or pharmaceutical composition of the present invention may be administered in combination with one or more other therapies, such as therapeutic schemes and / or other therapeutic agents, and used for the purposes described herein, for example, to prevent and / or treat the diseases or conditions relating to the present invention.

[0178] In some embodiments, the treatment method includes surgical procedures, radiation therapy, localized or intensive irradiation, etc.

[0179] In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

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

[0181] The combination of therapies of the present invention includes combination administration (for example, two or more therapeutic agents contained in the same or separate formulations), and individual administration, in the latter case, administration of the ADC molecule of the present invention may be performed before, simultaneously with, and / or after the administration of other therapeutic agents and / or drugs.

[0182] The administration route of the pharmaceutical composition may be by known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intrafocal routes, by a continuous release system, or by an implantable device. In some embodiments, the composition may be administered by bolus injection, continuous infusion, or by an implantable device.

[0183] The composition may be administered topically via an implantable membrane, sponge, or another suitable material that absorbs or encapsulates the desired molecule. In one embodiment, if an implantable device is used, the device can be implanted in any suitable tissue or organ and can deliver the desired molecule by diffusion, sustained-release bolus, or continuous administration.

[0184] 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 each embodiment of the present invention. The present invention will be further described using the following examples, which are described by description and not by limitation, and it should be understood that various modifications can be made. [Examples]

[0185] Example 1.1: Construction of a stable expression cell line

[0186] Preparation of a human CLDN18.2 overexpressing cell line

[0187] Following the manufacturer's instructions, cell lines stably expressing human Claudin18.2 (abbreviated as CLDN18.2, hereafter the same) were constructed using the Freedom® CHO-S® reagent kit (Invitrogen, A1369601). First, the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed in the vector pCHO1.0 to create a plasmid. The constructed plasmids were then introduced into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) using chemical and electrical transfection methods, respectively. The transfected cells underwent two rounds of press screening to obtain cell pools expressing CLDN18.2. Next, cells highly expressing CLDN18.2 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter), and monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2, which stably express CLDN18.2, were obtained by dilution.

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

[0189] Following the manufacturer's instructions, a cell line stably expressing human Claudin 18.1 (referred to as CLDN18.1; the same applies hereafter) was constructed using the Freedom® CHO-S® reagent kit (Invitrogen, A1369601). 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. The plasmid constructed by chemical transfection was introduced into CHO-S cells (Invitrogen, A1369601), and the transfected cells underwent two press screenings to obtain a cell pool expressing CLDN18.1. Next, cells highly expressing CLDN18.1 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter), and a monoclonal cell line CHO-hCLDN18.1 stably expressing CLDN18.1 was obtained by dilution.

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

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

[0192] Example 1.2, Generation of CLDN18.2 Monoclonal

[0193] This invention utilizes hybridoma technology to immunize genetically modified mice (purchased from Harbour BioMed) with fully human H2L2 antibody cells (CHO-hCLDN18.2) obtained in Example 1. Next, mouse spleen cells were obtained and electrofused with myeloma cells. Subsequently, the supernatant was collected and screened for hybridoma cells specifically expressing anti-CLDN18.2 antibody by flow cytometry (FACS). The secreted antibody did not bind to CLDN18.1. The cells to be detected (HEK293-hCLDN18.2) obtained in Example 1 were counted, and 1 × 10⁶ cells were identified. 6The cells were diluted to individual cells / mL and added to a 96-well plate with a U-shaped bottom at a rate of 100 μL / well. Centrifuged at 500 g for 5 min and the cell medium was removed. The supernatant from the hybridoma culture in the 96-well plate was added to the U-shaped plate, the cells were resuspended, 100 μL was added per well, and the mixture was incubated on ice for 30 min. The supernatant was removed and the cells were washed once with PBS. 100 μL of FITC-labeled anti-mouse Fab secondary antibody (diluted 1:500 in PBS) was added per well, and 100 μL of FITC-labeled anti-human Fab secondary antibody was added to the positive control antibody. The mixture was incubated on ice for 30 min in the dark. The supernatant was removed and the cells were washed once with PBS. The cells were resuspended in 50 μL of 1× PBS and detected by FACS. Positive clones were re-screened for CHO-hCLDN18.1 using the same method as described above. After two rounds of screening, the 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, sequence derived from INN117) were expressed in full-length monoclonal antibody form in HEK293 cells (Invitrogen, A14527).

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

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

[0198] Depending on the required transfection volume, Expi293 cells (Invitrogen, A14527) are passaged, and the cell density is increased to 1.5 × 10⁶ the day before transfection. 6 The concentration was adjusted to 10 cells / mL. On the day of transfection, the cell density was approximately 3 × 10⁶. 6 The cell count was cell / mL. 1 / 10 of the final volume of Opti-MEM medium (Gibco, 31985-070) was taken as transfection buffer, and an appropriate plasmid was added to achieve a transfection cell count of 1.0 μg / mL, and the mixture was homogeneously mixed. An appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the plasmid-to-PEI ratio is 1:3 for 293F cells), and after homogeneous mixing, the mixture was incubated at room temperature for 20 minutes to obtain the DNA / PEI mixture. The DNA / PEI mixture was gradually added to the cells while gently shaking the flask, and then the cells were cultured in a 36.5°C, 8% CO2 incubator. After 7 days, the cell saturation 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 hours, and after washing the glass bottles with distilled water, they were dried at 180°C for 4 hours. Before purification, the collected cell saturation was centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered through a 0.22 μm filter. The Protein A column was equilibrated with 10 column volumes of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). After adding the filtered supernatant to the purification column, it was equilibrated with 10 column volumes of binding buffer. 5 mL of eluent buffer (0.1 M citrate + sodium citrate, pH 3.5) was added to collect the eluent, and 80 μL of 2 M Tris-HCl was added per 1 mL of eluent. The collected antibodies were concentrated by ultrafiltration, replaced with PBS (Gibco, 70011-044), and their concentrations were detected.

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

[0201] Using surface plasmon resonance (SPR), the equilibrium dissociation constant (K) of HB37A6 bound to human CLDN18.2 was determined. DThe following measurements were taken. Following the manufacturer's instructions, human Claudin 18.2 (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). After coupling, 1M ethanolamine was injected to seal the remaining active site. Following the manufacturer's instructions, the binding and dissociation of the chip surface antigen and antibody in the mobile phase were detected using Biacore (GE Healthcare, T200) to obtain affinity and kinetic constants. Gradient-diluted antibodies (0nM to 100nM) were flowed across the chip surface from low to high concentration, with a binding time of 180s and a dissociation time of 600s. Finally, the chip was regenerated using 10mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). The data results were analyzed using Biacore T200 analysis software in a 1:1 binding model for dynamic analysis. 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] Flow cytometry (FACS) was used to measure the binding of the anti-CLDN18.2 monoclonal antibody HB37A6 and the control antibody Zmab to CHO-S cell lines stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1 (i.e., CHO-hCLDN18.2 and CHO-hCLDN18.1 prepared as described in Example 1).

[0205] Specifically, count the cells to be detected (CHO-hCLDN18.2 and CHO-hCLDN18.1) obtained in Example 1, and then 1 × 10⁻⁶ 6The antibody was diluted to individual cells / mL and added to a 96-well plate with a U-shaped bottom at a rate of 100 μL / well. Centrifuged at 500 g 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 a rate of 100 μL per well to resuspend the cells, setting the initial antibody concentration to 900 nM, and then sequentially diluting it 3-fold to obtain a total of 10 concentration points. The cells were incubated on ice for 30 min. The supernatant was removed and the cells were washed once with PBS after incubation at 500 g for 5 min. 100 μL of PE-labeled secondary antibody against goat anti-human Fc (SouthernBiotech, J2815-5H87B) was added per well. The cells were incubated on ice in the dark for 30 min. The supernatant was removed and the cells were washed once with PBS after incubation at 500 g for 5 min. The cells were resuspended in 50 μL of 1× PBS and detected by FACS. Experimental data was analyzed using GraphPad Prism software to obtain Figures 1 and 2. As shown in Figures 1 and 2, both antibodies specifically bound to human CLDN18.2 (Figure 1), but did not bind to human CLDN18.1 (Figure 2).

[0206] Example 1.6: Conjugation of CLDN18.2 antibody to tumor cell line

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

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

[0209] 1. Activity of antibodies against the DAN-G-CLDN18.2 tumor-carrying mouse model.

[0210] The antitumor effect of the HB37A6 antibody was tested in NOD-SCID mice (female NOD-SCID mice (15g-18g), purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) with human pancreatic cancer. Human pancreatic cancer cells DAN-G-hCLDN18.2 constructed in Example 1 were subjected to conventional subculture and used in subsequent in vivo experiments. The cells were collected by centrifugation, and DAN-G-hCLDN18.2 was dispersed in PBS (1×) and divided into 12×10⁶ cells. 5 A cell suspension with a cell density of cells / mL was obtained. The cell suspension and Matrigel gel were mixed in a 1:1 ratio, and 6 × 10⁶ cells were collected. 5 A cell suspension with a cell concentration of cells / mL was prepared. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right flank region of NOD-SCID mice to establish a DAN-G-CLDN18.2 tumor-bearing mouse model.

[0211] After inoculating each mouse with tumor cells for 5 days, the tumor volume was detected, and the tumor volume was 43.36 mm². 3 ~89.47mm 3 Mice within the specified range were divided into S-type groups based on tumor volume (8 mice per group).

[0212] Each mouse was administered hIgG (Equitech-Bio, lot number 160308-02), HB37A6, and the control antibody Zmab at a dose of 10 mg / kg per dose, administered on days 5, 9, 12, and 16 after inoculation. Tumor volume was monitored 2-3 times per week. Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using calipers, and the tumor volume was calculated as V = L × W. 2 The calculation was performed using the formula / 2. Weight was measured using an electronic balance.

[0213] 2. Activity of antibodies against NUCG-4 tumor-bearing mouse models

[0214] We selected the HB37A6 antibody and tested its antitumor effect in NOG mice (female NOG mice (15g-18g), purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) that had human gastric cancer. PBMC cells (Allcells) were resuscitated, collected by centrifugation, and measured 2.5 × 10⁶ cells. 6 PBMC cells were dispersed in PBS (1×) to create a cell suspension with a cell density of cells / mL. On day 0, 0.2 mL of the cell suspension was taken and used for intravenous injection into the eyes of NOG mice to establish a humanized NOG mouse model.

[0215] NUGC-4 cells were subjected to standard resuscitation and subculturing and used in subsequent in vivo experiments. Cells were collected by centrifugation and measured in 12 × 10⁶ cells. 6 Disperse NUGC-4 cells in PBS (1×) to a cell density of cells / mL, mix with Matrigel gel in a 1:1 ratio, and then add 6×10⁶ cells. 6 A cell suspension with a cell concentration of cells / mL was prepared. On day 5, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right flank region of NOG-humanized mice to establish a NUCG-4 tumor-bearing mouse model.

[0216] On day 1 after inoculation with tumor cells, mice were randomly divided into groups of 7. Each group received either hIgG (Equitech-Bio, lot number 160308-02, control group), HB37A6, or the positive control antibody Zmab (treatment group). The dosage was 10 mg / kg per dose, administered on days 1, 5, 8, and 12 after inoculation. Tumor volume and body weight were monitored 2-3 times per week. Tumor volume was measured using calipers to determine the maximum long axis (L) and maximum width axis (W) of the tumor, and the tumor volume was calculated as V = L × W. 2 The formula used was / 2. Body weight was measured using an electronic balance. On the 26th day after vaccination, the relative tumor suppression rate (TGI%) was calculated using the formula:

[0217] TGI% was calculated as 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group tumor volume before administration).

[0218] 3, results

[0219] As shown in Figure 4, both HB37A6 and the control antibody Zmab were able to suppress 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 a better antitumor effect than the control antibody Zmab in the human gastric cancer NUGC-4 mouse model, with a TGI of 31% for HB37A6 and 0% for Zmab.

[0220] Example 2.1 Synthesis of IEX019 ADC molecule

[0221] IEX019

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

[0223] Example 2.1.1 Preparation of IEX019-02

[0224] 1). Preparation of compound 6

Chemical formula

[0225] 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 to a solution of BCN-OH (5, 1.5 g, 10 mmol) in DCM (150 mL). The mixture was stirred for 10 minutes and quenched by adding saturated aqueous NH4Cl solution (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), CSI (11 μL, 18 mg, 0.13 mmol) was added. After 30 minutes, Et3N (91 μL, 66 mg, 0.65 mmol) and diethanolamine (16 mg, 0.16 mmol) in DMF (0.5 mL) were added. After 30 minutes, p-nitrophenyl chloroformate (52 mg, 0.26 mmol) and Et3N (54 μL, 39 mg, 0.39 mmol) were added. After a further 4.5 hours, the reaction mixture was concentrated, and the residue was purified by gradient column chromatography (33 → 66% Â / heptane (1% AcOH)) to obtain a colorless oily substance 7 (88 mg, 0.098 mmol, 75%).

[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 hours, the reaction mixture was diluted in 9 mL of DCM and purified by gradient column chromatography (0 → 40% MeOH / DCM) to obtain compound 9 (155 mg, 159 μmol, 68%). LCMS(ESI+)C 55 H 54 FN6O 10 + (M+H) + Calculated value: 977.39, measured value: 977.80.

[0232] 4) Preparation of Compound 1

[0233] To a solution of compound 9 (155 mg, 159 μmol) in DMF (1.6 mL), solutions of Et3N (73 mg, 101 μL, 0.72 mmol) and compound 7 (65 mg, 72 μmol) in DMF (1.4 mL) were added. The reaction mixture was stirred for 24 hours, diluted with DCM (20 mL), and purified by gradient column chromatography (0 → 40% MeOH / DCM) to obtain 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, measured value: 1067.12.

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

[0235] HB37A6 was expressed in HEK293 cells according to Example 1.3 and purified. The obtained HB37A6 (16.4 mg / mL) was incubated with EndoSH (1% w / w) as described in PCT / EP2017 / 052792 to obtain trimmed HB37A6 having -GlcNAc or GlcNAc(Fuc) at the Asn297 position. 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 (equivalents) relative to the antibody) (in a 6 mM solution containing histidine (20 mM) + NaCl (150 mM) of MnCl2) disclosed in PCT / EP2016 / 059194 for 16 hours at 30°C.

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

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

[0238] Therefore, the above results demonstrated that two heavy chains both had GlcNAc located at Asn297 substituted with 6-azido-GalNAc (4-substitution) to obtain HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2.

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

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

[0241] Mass spectrometry of the IdeS digestion sample showed that both of the two main products corresponded to the obtained ADC, namely the HB37A6-SYNtecan E complex. The first peak: The observed mass was 26469 Da (calculated mass: 26465 Da), corresponding to the coupled Fc / 2 fragment (2x closed lactone form of the payload). The second peak: The observed mass was 26499 Da (calculated mass: 26501 Da), corresponding to the bound 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 exceeded 99%, and the concentration was 6.12 mg / mL.

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

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

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

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

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

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

[0249] (b) An excess of the linker-toxin MC-GGFG-DXD (purchased from Levena Biopharma, SET0218, structure as shown below) was dissolved in DMSO and reacted with the thiol group of the antibody reduced in step (a). The reaction mixture was left in a shaker for 1 to 2 hours, and then (i) The optimal molar ratio of DXD / mAb was 10.0 to 12.0. (ii) The optimal temperature for the reaction is 20°C to 37°C. Crude ADC product was obtained.

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

[0251] (d) ADC products were detected using HIC, LC-MS, and SEC HPLC, and the average DAR value and SEC purity were determined.

[0252] [ka]

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

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

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

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

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

[0258] (c) After the binding reaction was complete, 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 obtained crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain the final ADC product IEX019-04.

[0260] (e) ADC products were detected using HIC and SEC-HPLC, and the average DAR value 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, 3-5, 3.2-4.8, or 3.0-4.5, and was 3.3 as measured in Table 2.

[0264] In the formula, Ab was HB37A6.

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

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

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

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

[0269] [ka]

[0270] Example 2.1.5 Preparation of IEX019-06

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

[0272] Example 2.1.6 Preparation of IEX019-07

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

[0274] Table 2 summarizes the information on all monoclonal antibodies and ADCs.

[0275] [Table 3]

[0276] Example 2.2 Cell binding experiment of IEX019 molecule

[0277] To detect whether the binding properties of the IEX019-01 monoclonal antibody to target cells change due to small molecule coupling, the inventors used a DAN-G cell line (hCLDN18.2 negative) and a 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 using flow cytometry. The experimental procedure was the same as in Example 1.5.

[0278] Both IEX019-01 and IEX019-02 did not bind to non-target cell DAN-G, but showed very high affinity for DANG-hCLDN18.2, indicating that antibody binding is dependent on the target expression specificity and that exatecan coupling does not affect antibody binding. Simultaneously, the control molecule IEX019-06 (a monoclonal antibody that is a negative control of IgG coupled to exatecan toxin using the same technique) did not bind to target cells (Figure 6).

[0279] Example 2.3 Endocytosis experiment of IEX019 molecule

[0280] Strong endocytosis is one of the key properties of ADC drugs. After ADCs bind to antigens on the cell membrane surface, endocytosis allows the ADC-antigen complex to enter the cell and kill the target cell. Therefore, the endocytotic efficiency of ADCs is one of the important indicators determining the tumor suppressor effect.

[0281] To detect the endocytosis efficiency of antibodies coupled to small molecule compounds, the inventors detected endocytosis of different IEX019 molecules in DANG-hCLDNA18.2 cells using flow cytometry techniques. After digesting the DANG-hCLDNA18.2 cells, the cell density was adjusted to 1 × 10⁶ 5Cells were seeded individually per well in a 96-well plate. Centrifuged at 500g for 3 minutes, and the supernatant was discarded. 100 μL of the molecule to be detected was taken and the cells were resuspended (molecule concentration 50 nM), and five repeats were set up for each sample (i.e., endocytosis times: 0h, 1h, 2h, 3h, 4h). The samples were incubated on ice for 1 hour. After 1 hour, the samples were centrifuged at 500g for 3 minutes, and the supernatant was discarded. 200 μL of FACS buffer (1% FBS, 1×PBS) was added to each well, and the plates were washed twice. One set of samples was taken and transferred to a new 96-well plate, and incubated at 37°C for 4 hours. The remaining samples were continued to be incubated on ice. The previous procedure was repeated, with each sample incubated at 37°C for 3h, 2h, 1h, and 0h. After the specified incubation time was completed, the samples were centrifuged at 500g for 3 minutes, 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 in the dark for 30 minutes. After incubation of the secondary antibody was complete, the cells were centrifuged at 500 g for 3 minutes and the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. Finally, the cells were resuspended in 100 μL of PBS and detected in-vector.

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

[0283] Example 2.4: In vitro cell-killing effect of the IEX019 molecule

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

[0285] [Table 4]

[0286] After digesting the cells with EDTA / Trypsin, the density was adjusted and the cells were uniformly seeded in a 96-well plate (Table 4). Sample IEX019 molecules at specific diluted concentrations (IEX019-02, IEX019-03, IEX019-04, starting dilution concentration 100 nM, dilution factor 3) were added, and wells without IEX019 molecules were used as controls. The cells were incubated in a 37°C incubator for 5 days. After 5 days, 100 μL of CellTiter-Glo detection reagent was added to each well, incubated at room temperature for 30 minutes, and detected using a microplate reader. Relative cell viability (relative cell viability = sample / control × 100%) was calculated, and the curve was fitted using GraphPad Prism 8.0.

[0287] As shown in Figure 8, the killing effect of the ADC molecule on cell lines depended on the expression level of surface hCLDN18.2. In hCLDN8.2-negative DANG cells, the IEX019 molecule did not significantly affect cell viability (Figure 8A). In cell lines with moderate expression levels (NUGC-4, SNU620), the IEX019 molecule showed a certain degree of cell killing (Figure 8B). In high-expression cell lines DAN-G-hCLDN18.2, the IEX019 molecule showed a significant killing effect in all cases (Figure 8C). This demonstrated that the IEX019 molecule has 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 a cleavable linker and, after endocytosis to the cell membrane, the linker is cleaved, releasing the small molecule and killing the target cells. After the target cells are killed, the small molecule compounds are released from the target cells into the intercellular spaces, further killing non-target cells within a certain range; this effect is called bystander killing. Because cells within tumors differ greatly in target expression levels (tumor heterogeneity), bystander killing was crucial for effective killing of tumor cells and suppression of tumor growth.

[0291] This invention detects 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, adjust the density and prepare a 6-well cell culture plate, then fill each well with 7.5 × 10⁶ DANG cells and DANG-hCLDN18.2 cells. 4Two types of cells were co-cultured. 200 μL of the sample to be measured (IgG (SEQ ID NO: 21, SEQ ID NO: 22), IEX019-02, IEX019-05, IEX019-06, Exatecan (Macklin, E881532)) was added to each well to a final concentration of 50 nM, with 3 replicates set for each sample. The cell culture plates were left in an incubator at 37°C and cultured for 5 days. After 5 days, the culture supernatant was discarded, washed with PBS, and Trypsin-EDTA was added to digest the cells. All digested cells were collected and transferred to a 96-well plate. Following the antibody incubation process of flow cytometry technology, the cells were incubated at 4°C with primary antibody (IEX019-01, 100 nM) and secondary antibody (anti-hFc-PE, SouthernBiotech) for 1 hour and 0.5 hours, respectively. After washing with PBS, live / dead cells were incubated. Violet dye (Thermo, L34964) was diluted 1:1000, 100 μL was added to each well, and incubated at 4°C for 20 minutes. After washing with PBS, the cells were resuspended in 100 μL of PBS and detected in-instrumentally. Each sample was divided into populations using live / dead dyes, where the IEX019-01 negative (i.e., hCLDN18.2 negative) population consisted of DAN-G cells, and the IEX019-01 positive (i.e., hCLDN18.2 positive) cells consisted of DAN-G-hCLDN18.2 cells. The number of each of the two cell types in each sample was statistically determined, and the relative cell viability of each cell type was calculated according to the following formula, and the curves were fitted using GraphPad Prism 8.0.

[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 of the IgG group was set to 100%.

[0296] As shown in Figure 9, the negative control sample IEX019-06 had no killing effect on either of the two cell types. The IEX019-05 molecule linked the DM1 toxin to the IEX019-01 monoclonal antibody via an uncleavable linker and lacked the ability to kill surrounding non-target cells, thus only killing DANG-hCLDNA8.2 cells and not affecting DANG cells. Only IEX019-02 exhibited a significant bystander killing effect, capable of simultaneously killing both target and non-target cells.

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

[0298] To demonstrate the in vivo efficacy of the IEX019 molecule, the inventors inoculated DANG-hCLDN18.2 cells into CB-17-SCID mice and measured the antitumor efficacy of the antibody molecule of the present invention. SPF-grade female CB-17-SCID mice (14g-17g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used in the experiment, with certificate number NO.110011201108225246.

[0299] DANG-hCLDN18.2 cells were regularly subcultured and used in subsequent in vivo experiments. Cells were collected by centrifugation, and the DANG-hCLDN18.2 cells were dispersed in PBS (1×) and divided into 3×10⁶ cells. 6 A cell suspension with a cell concentration of cells / mL was prepared. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right ventral region of CB-17 SCID mice to establish a DANG-hCLDN18.2 tumor-bearing mouse model.

[0300] Five days after inoculation of tumor cells, the tumor volume was 50.16 mm². 3 ~136.68mm 3 All mice were divided into snake-like groups (6 mice each), and administered on the 5th day after inoculation according to the dosage and method shown in Table 5. Tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 10a and 10b, and monitoring was discontinued after 92 days.

[0301] The relative tumor suppression rate (TGI%) was calculated 50 days after vaccination, using the following formula: TGI% was calculated as 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group tumor volume before administration).

[0302] Tumor volume measurement: Measure the longest axis (L) and widest axis (W) of the tumor using calipers. The tumor volume is calculated as V = L × W. 2 The calculation was performed using the formula / 2. Weight was measured using an electronic balance.

[0303] [Table 6]

[0304] As shown in Table 6 and Figure 10A, the tumor suppression rate after a single dose of IEX019-02 reached 103.60% at 50 days post-inoculation compared to IEX019-01 monoclonal antibody, which was clearly superior to IEX019-03 and IEX019-04, which had tumor suppression rates of 93.70% and 35.20%, respectively. 82 days after inoculation, 100% of the mouse tumors in the IEX019-02 group had completely regressed. Simultaneously, the inventors detected the body weight of the mice, and the results are shown in Figure 10B; there was no significant difference in mouse body weight.

[0305] [Table 7]

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

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

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

[0309] NUGC-4 cells were regularly subcultured and used in subsequent in vivo experiments. Cells were collected by centrifugation, and the NUGC-4 cells were dispersed in PBS (1×) and divided into 3×10⁶ cells. 7 A cell suspension with a cell concentration of 1 cell / mL was prepared. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right ventral region of CB-17 SCID mice to establish a NUGC-4 tumor-bearing mouse model.

[0310] Five days after inoculation of tumor cells, the tumor volume was 72.25 mm². 3 ~140.50mm 3 All mice were divided into snake-like groups (6 mice each), and administered on the 5th day after inoculation using the dosage and method shown in Table 7. Tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 11A and 11B, and monitoring was discontinued after 40 days.

[0311] On the 33rd day after vaccination, the relative tumor suppression rate (TGI%) was calculated using the following formula: TGI% was calculated as 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group tumor volume before administration).

[0312] Tumor volume measurement: Measure the longest axis (L) and widest axis (W) of the tumor using calipers. The tumor volume is calculated as V = L × W. 2 The calculation was performed using the formula / 2. Weight was measured using an electronic balance.

[0313] [Table 8]

[0314] As shown in Tables 8 and 11A, the tumor suppression rates 33 days after inoculation were 80.04% and 54.31% compared to single doses of the negative controls IEX019-06, IEX019-02, and IEX019-03, respectively. Simultaneously, mouse body weight was detected, and as shown in Figure 11B, there was no significant difference in mouse body weight.

[0315] [Table 9]

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

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

[0318] SNU620 cells were regularly subcultured and used in subsequent in vivo experiments. Cells were collected by centrifugation, dispersed in PBS (1×), and then divided into 3×10⁶ cells. 7 A cell suspension with a cell concentration of cells / mL was prepared. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right ventral region of CB-17 SCID mice to establish a SNU620 tumor-bearing mouse model.

[0319] Seven days after inoculation of tumor cells, the tumor volume was 58.1 mm². 3 ~117.3mm 3 All mice were divided into snake-like groups (6 mice each), and administered on the 7th day after inoculation using the dosage and method shown in Table 9. Tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 12a and 12b, and monitoring was discontinued after 39 days.

[0320] On the 39th day after vaccination, the relative tumor suppression rate (TGI%) was calculated using the following formula: TGI% was calculated as 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group tumor volume before administration).

[0321] Tumor volume measurement: Measure the longest axis (L) and widest axis (W) of the tumor using calipers. The tumor volume is calculated as V = L × W. 2 The calculation was performed using the formula / 2. Weight was measured using an electronic balance.

[0322] [Table 10]

[0323] As shown in Table 10 and Figure 12A, the tumor suppression rate after a single dose of IEX019-02 10 mg / kg on day 39 after inoculation was 143.77% compared to hIgG, with 100% of mouse tumors completely regressing. There were no significant differences in mouse body weight in any of the administration groups compared to 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, Ab is an anti-Claudin 18.2 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, In the formula, q is the drug-antibody ratio (DAR) of 3 to 5. The antibody-drug conjugate.

2. The antibody-drug conjugate according to claim 1, wherein Ab is an anti-Claudin 18 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 aforementioned Ab includes the heavy chain constant region from IgG1, The antibody-drug conjugate according to claim 2.

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

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

8.

7. The antibody-drug conjugate according to claim 4, wherein the coupling to Ab is performed at the position of Asn297.

8. The following formula: 【Chemistry 2】 The antibody-drug conjugate, In the formula, Ab is an anti-Claudin 18.2 antibody comprising 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; The coupling to Ab is performed at the position of Asn297; and, In the formula, q is the DAR of 3 to 4. Antibody-drug conjugate.

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 according to claim 8 for treating Claudin18-expressing cancer in a subject.

18. The pharmaceutical composition according to claim 16 for treating Claudin 18-expressing cancer in a subject.

19. The antibody-drug conjugate according to claim 8 for treating gastric cancer in a subject.

20. The antibody-drug conjugate according to claim 8 for treating pancreatic cancer in a subject.

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