Antibody-drug conjugates targeting claudin 18.2

Abstract

An antibody-drug conjugate (ADC) targeting Claudin 18.2, and compositions containing the molecule, and therapeutic and diagnostic uses thereof.

Description

[0001] This invention relates to antibody-drug conjugates (ADCs) targeting Claudin18.2 (CLDN18.2) and compositions containing said molecules. The invention also relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments. Background of the Invention

[0003] Claudins are a family of proteins that are essential components of tight junctions in cells. They establish intercellular barriers that control the flow of molecules between cells. Claudins have four transmembrane domains, with their N-terminus and C-terminus both located in the cytoplasm. Different claudins are expressed in different tissues, and alterations in their function are associated with cancer development in various tissues. For example, Claudin-1 is expressed in colon cancer and has prognostic value, Claudin-18 is highly expressed in gastric and pancreatic cancers, and Claudin-10 is highly expressed in hepatocellular carcinoma. As cell membrane surface proteins, claudins are useful targets for various therapeutic strategies.

[0004] Claudin-18 isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker, whose expression in normal tissues is strictly limited to epithelial cells differentiated from the gastric mucosa, but not in gastric stem cell regions. CLDN18.2 is expressed in a significant proportion of primary gastric cancers and retains its expression level in metastatic gastric cancer tissues. In addition to gastric cancer, CLDN18.2 expression has also been found in pancreatic cancer, making it an ideal target molecule for the treatment of 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).

[0005] There is a large unmet clinical need for treatment of gastric cancer, making it essential to develop drugs that target Claudin18.2.

[0006] Despite the clinical success of therapeutic antibodies, naked MAbs targeting cell surface tumor antigens rarely provide sufficient efficacy on their own. To increase the low activity of MAbs, new strategies focus on binding them to toxic molecules. Plant and bacterial toxins, as well as small chemotherapeutic molecules, are promising candidates because they are highly effective and active in very small amounts.

[0007] Thanks to technological advancements over the past few years, the field of antibody-drug conjugates (ADCs) for cancer treatment has recently experienced a surge in development activity by pharmaceutical companies aimed at addressing the initial issues they presented regarding immunogenicity, affinity, specificity, undesirable toxicity, yield, half-life, and other aspects.

[0008] Despite some progress, there remains a need for other therapeutic strategies for treating cancer, and for components used in such strategies, particularly for antibodies against Claudin18.2 with high affinity, good specificity and / or low risk of immunogenicity, and for ADC molecules with high activity, low toxicity, long half-life, good specificity or affinity, and / or good half-life or druggability. Invention Overview

[0010] This invention provides an antibody-drug conjugate (ADC) targeting Claudin18.2, which has the following advantages:

[0011] (1) It binds to target cells expressing human CLDN18.2 with high affinity;

[0012] (2) It can enter cells through endocytosis and kill target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency.

[0013] (3) It has a significant bystander effect;

[0014] (4) It has high anti-tumor efficacy;

[0015] (5) It has low toxicity;

[0016] (6) It has good stability;

[0017] (7) It has good drug-like properties.

[0018] In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface; in some embodiments, the target cells are CHO cells or 293 cells expressing CLDN18.2, such as CHO-S cells or HEK293 cells; in some embodiments, the target cells are cancer cells expressing CLDN18.2, such as cells naturally expressing CLDN18.2 or cells artificially transfected with CLDN18.2 or cells artificially transfected with increased levels of CLDN18.2, such as gastric cancer cells, pancreatic cancer cells, colon cancer cells, or colorectal cancer cells expressing CLDN18.2. In some embodiments, the target cells are cell lines with intermediate hCLDN18.2 expression levels, such as NUGC-4 and SNU620. In some embodiments, the target cells are cell lines with high expression levels, such as DAN-G cells overexpressing hCLDN18.2. Attached image description:

[0019] Figure 1 The HB37A6 antibody was shown to specifically bind to CLDN18.2 on the cell surface.

[0020] Figure 2 The results showed that the HB37A6 antibody did not bind to CLDN18.1 on the cell surface.

[0021] Figure 3 The binding of HB37A6 antibody to gastric cancer cell lines NUGC-4, KATO III-hCLDN18.2, and DAN-G-hCLDN18.2 was demonstrated.

[0022] Figure 4 The study demonstrated the antitumor effect of HB37A6 antibody in a mouse model of pancreatic cancer.

[0023] Figure 5 The antitumor effect of HB37A6 antibody in a mouse model of gastric cancer was demonstrated.

[0024] Figure 6 The cell-binding activity of the IEX019 molecule was demonstrated.

[0025] Figure 7 shows The endocytosis of IEX019 molecules in DANG-hCLDN18.2 cells was demonstrated.

[0026] Figure 8 shows the killing effect of IEX019 molecule in cell lines with low expression of hCLDN18.2. Figure 8A) The killing effect of IEX019 molecule in cell lines with moderate expression levels of hCLDNA8.2 ( Figure 8B) The killing effect of IEX019 molecules in the hCLDN18.2 cell line with high expression ( Figure 8C ).

[0027] Figure 9 shows This demonstrates the bystander effect of the IEX019 molecule.

[0028] Figure 10 shows the tumor-suppressive efficacy of IEX019 molecules in mice. Figure 10A ) and weight change ( Figure 10B ).

[0029] Figure 11 shows the tumor-suppressive efficacy of IEX019 molecule in mice. Figure 11A) and weight change ( Figure 11B ).

[0030] Figure 12 shows the tumor-suppressive efficacy of IEX019 molecule in mice. Figure 12A ) and weight change ( Figure 12B ). Invention Details

[0032] I. Definition

[0033] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0035] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0036] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0037] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0038] The term "CLAUDIN" or "CLDN" used in this article refers to the most important skeletal protein determining the structure of tight junctions between cells. Claudin proteins participate in adhesion and play a crucial role in tumor cell metastasis and invasion. Claudin proteins are widely distributed in mammalian epithelial and endothelial cells, primarily on the lateral surfaces of epithelial cells and the plasma membrane of basal cells. Different Claudin proteins exhibit specific expression in different tissues. The Claudin18 (CLDN18) gene, located at 3q22.3, has a molecular weight of 24 kDa, contains 261 amino acid residues, and belongs to the Claudins superfamily. Its protein structure includes two extracellular loops and four transmembrane regions. The two isoforms of human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1) and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2). In their primary structural sequences, they differ only in certain amino acid residues at positions from the N-terminal signal peptide to the extracellular loop 1 structure, particularly at the extracellular loop 1, where CLDN18.1 and CLDN18.2 differ by only 8 amino acids. The two isoforms of CLDN18 also exhibit very high interspecies sequence homology. The extracellular loop 1 of CLDN18.2 is completely sequence-identical in different species, including humans, mice, and macaques, with 84% homology between human and mouse CLDN18.2 proteins, indicating extreme conservation of the CLDN18.2 protein sequence (O. Tureci. et al., Gene 481:83-92, 2011). CLDN18.2 or any variants and isotypes thereof can be isolated from cells or tissues that naturally express them, or recombinantly generated using techniques well known in the art and / or those described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0039] As used herein, the terms "anti-CLDN18.2 antibody," "anti-CLDN18.2," "CLDN18.2 antibody," "CLDN18.2-binding antibody," or "antibody specifically binding to CLDN18.2" refer to antibodies that bind with sufficient affinity to (human) CLDN18.2 such that the antibody can be used as a therapeutic agent targeting (human) CLDN18.2. In one embodiment, the (human) CLDN18.2 antibody binds to (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, the (human) CLDN18.2 antibody does not bind to CLDN18.1. In one embodiment, the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), thin-layer biofilm interferometry (BLI), MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0040] The expression of CLDN18.2 in cells can be determined in various ways, such as by using anti-CLDN18.2 antibodies. For example, the binding strength of anti-CLDN18.2 antibodies to cells that "highly express CLDN18.2" (e.g., as determined by FACS) can be 500, 600, 700, 800, 900, or preferably more than 1000 times, such as 1100, 1200, 1300, 1400 times or more, that of anti-CLDN18.2 antibodies to cells that do not express CLDN18.2. For example, the binding strength of cells that "moderately express CLDN18.2" to anti-CLDN18.2 antibodies (e.g., as determined by FACS) can be 5 to 500 times stronger than the binding strength of anti-CLDN18.2 antibodies to cells that do not express CLDN18.2, such as 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or higher, but not exceeding 500 times.

[0041] The terms "complete antibody," "full antibody," or "full-length antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that only has heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds.

[0042] For conventional four-chain IgG antibodies, the full-length antibody heavy chain typically consists of a heavy chain variable region (abbreviated as VH in this article) and a heavy chain constant region, wherein 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 in this article) and a light chain constant region, wherein the light chain constant region consists of one domain: CL. Each heavy chain variable region (VH) and each light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0043] The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0044] "Antigen-binding fragment" refers to a molecule that is distinct from the intact antibody, contains a portion of the intact antibody, and binds the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; bivalent antibodies or fragments thereof; or camelid antibodies.

[0045] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0046] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: 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)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)).

[0047] The following are the area ranges of CDRs defined using the kabat, AbM, Chothia, Contact, and IMGT schemes.

[0048]

[0049] CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0050] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0051] Unless otherwise stated, in this invention, when referring to the position of a residue in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position numbered according to 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)).

[0052] In one embodiment, the heavy chain variable region (CDR) of the antibody of the present invention is determined according to the following rules:

[0053] VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule.

[0054] In one embodiment, the light chain variable region (CDR) of the antibody of the present invention is determined according to the Kabat rule.

[0055] In one embodiment, the heavy chain variable region CDR of the antibody of the present invention is determined according to the following rules: VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule; and the light chain variable region CDR is determined according to the Kabat rule.

[0056] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined by the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0057] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs (within the same assignment system). However, although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0058] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this document, the term "Fc region" excludes the heavy chain variable region VH and light chain variable region VL of immunoglobulins, as well as the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region.

[0059] "IgG form 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 form antibody means its heavy chain constant region comes from IgG4, or an IgG1 form antibody means its heavy chain constant region comes from IgG1.

[0060] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0061] As used in this article, "antibody-drug conjugate (ADC)" refers to a structure obtained by linking an antibody to a drug.

[0062] As used herein, the general term "sugar" refers to monosaccharides such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). The term "sugar derivative" as used herein refers to a derivative of a monosaccharide, i.e., a monosaccharide 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), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Examples of sugar derivatives also include compounds denoted herein as E(A)x, where E is a sugar or a sugar derivative, and where E includes x functional groups A.

[0063] The core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as GlcNAc bonded to the antibody via a C1 bond, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of the antibody's asparagine amino acid. The core-GlcNAc substituent may be present at the antibody's native glycosylation site, but it may also be introduced at different sites on the antibody. In this context, 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 referred to as GlcNAc(Fuc).

[0064] "Glycosylation modification" refers to the process of altering the glycan chains of an antibody through glycosylation engineering. Antibody glycosylation can be further modified for various purposes to obtain antibodies with novel glycosylations. For example, glycosylation can be removed to eliminate FcγR affinity and complement binding / effective function; fucose and sialic acid groups can be reduced and dimeric N-acetylglucosamine, galactose, and mannose can be added to enhance Fc-mediated ADCC and CDC effects. Glycosylation modification methods are known in the art, such as increasing or decreasing the glycan chains on the antibody surface by altering the glycosylation sites, modifying the glycan chains in vitro via chemical or enzymatic methods, 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 influencing cell culture conditions. In some embodiments, the "glycosylation modification" of this invention is performed by in vitro enzymatic modification of the glycan chains. Preferably, the glycosylation modification of the present invention is carried out by modifying the glycan chain with glycosidases (e.g., glycosidases or glycosyltransferases).

[0065] The modified glycosylated antibody of the present invention refers to an antibody whose glycosylation pattern has been modified compared to an antibody with a natural glycosylation pattern. Preferably, the modified glycosylated antibody refers to an antibody obtained by in vitro enzymatic modification of the glycan chain (e.g., by modification of the glycan chain by a glycosidase (e.g., an endonuclease or glycosyltransferase)) after expression in an expression system (e.g., mammalian cells). More preferably, the modified glycosylated antibody of the present invention refers to an antibody comprising a core -GlcNAc and a sugar derivative E(A)x linked thereto, wherein GlcNAc is bonded to the antibody via a C1 bond, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of the asparagine amino acid of the antibody. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucose-substituted, typically fucose is linked to the C6 of the -GlcNAc substituent via α-1,6. The -GlcNAc substituent in fucosylation refers to the core -GlcNAc(Fuc), and the GlcNAc-E(A)x substituent in fucosylation refers to GlcNAc(Fuc)-E(A)x.

[0066] The term "site-specific conjugation" as used in this article refers to the conjugation that specifically links a drug / active substance to a specific site on an antibody via a linker.

[0067] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1-24 carbon atoms, more preferably 1-16 carbon atoms, 1-12 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0068] The term "aryl" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6-20, for example, 6-12 carbon atoms in the ring moiety. Preferably, the aryl group is (C6-C6) 10 Aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may optionally be substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamide, heterocyclic, etc., wherein R is independently hydrogen, alkyl, aryl, heteroaryl, aryl-alkyl-, heteroaryl-alkyl-, etc.

[0069] The term "cycloalkyl" refers to a cyclic alkyl group, that is, 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 3-18, 3-10, or 3-8, and typically, according to the invention, contains 3 to 6 atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0070] As used herein, the term "heteroaryl" refers to a 5- to 20-membered (e.g., 5-14, 5-8, 5-6) monocyclic, bicyclic, or fused polycyclic system containing 1-8 heteroatoms selected from N, O, or S. Preferably, the heteroaryl group is a 5- to 10-membered ring system. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furanyl, 2- or 3-pyrroleyl, 2-, 4- or 5-imidazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isooxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridinyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl.

[0071] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention, and that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugates of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0072] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0073] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0074] The term "drug:antibody ratio" or "DAR" refers to the ratio of the small molecule drug portion (D) conjugated to the Ab portion described herein to the Ab portion. In some embodiments described herein, the DAR may be determined by p and r in Formula I, for example, the DAR may be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the small molecule drug portion (D) conjugated to the Ab portion described herein to the Ab portion in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 20, for example 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example 1.0-8.0, 2.0-6.0, 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, 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, a range with two of these values ​​as endpoints.

[0075] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants).

[0076] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0077] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0078] The term "small molecule drug" refers to low-molecular-weight organic compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0079] As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. An immune response can be a humoral or cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0080] The term "effective amount" refers to such an amount or dose of the antibody, fragment, composition, or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention.

[0081] "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody, antibody fragment, composition, or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, "therapeutic effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, and more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0082] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0083] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in their nucleic acid content and may contain mutations. This document includes mutant progeny with the same function or biological activity screened or selected from the initially transformed cells.

[0084] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0085] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0086] "Isolated" antibodies or other molecules (e.g., ADC molecules) are antibodies or molecules that have been separated from components of their natural environment or the environment in which they are expressed. In some embodiments, the antibody or ADC molecule is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0087] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0088] The terms “tumor” and “cancer” are used interchangeably in this article to cover both solid tumors and hematologic malignancies.

[0089] The terms "cancer" and "cancerous" 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, including metastatic forms of those cancers.

[0090] The term "tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when used in this article.

[0091] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0092] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0093] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) the ADC molecule of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. In some embodiments, the ADC molecule of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0094] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients 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 prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0095] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0096] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0097] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0098] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0099] II. Antibody-Drug Conjugates

[0100] This invention provides an antibody-drug conjugate having formula (I):

[0101] Ab-(L-(D) r ) p (I)

[0102] Or its pharmaceutically acceptable salts or solvates,

[0103] in:

[0104] Ab is an antibody or fragment thereof that binds to CLDN18.2 (e.g., human CLDN18.2);

[0105] L is the connector;

[0106] D represents a drug, including prodrugs, preferably antitumor compounds; and

[0107] p is 1 to 10, for example 1-9, 2-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0108] r is 1-5, for example 1, 2, 3, 4 or 5, preferably 1 or 2.

[0109] 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); linear antibody; single-chain antibody (e.g., scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.

[0110] In some embodiments, Ab in formula (I) of the present invention is a bispecific antibody or a multispecific antibody.

[0111] In a preferred embodiment of the invention, Ab contains three complementary determinant regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3.

[0112] In a preferred embodiment of the invention, Ab comprises three complementary determinant regions (LCDRs) from the light chain variable region, namely LCDR1, LCDR2 and LCDR3.

[0113] In some implementations, Ab includes three complementary determinant regions (HCDRs) from the heavy chain variable region and three complementary determinant regions (LCDRs) from the light chain variable region.

[0114] In some aspects, Ab includes a heavy chain variable region (VH). In some aspects, Ab includes a light chain variable region (VL). In some aspects, Ab includes both a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region includes three complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementary determinant regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0115] In some implementations, VH

[0116] (i) comprising or consisting of 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

[0117] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:4; or

[0118] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:4, preferably, the amino acid changes do not occur in the CDR region.

[0119] In some implementations, VL

[0120] (i) comprising or consisting of 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

[0121] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:9; or

[0122] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:9, preferably, the amino acid changes do not occur in the CDR region.

[0123] In some embodiments, the three complementary determination regions (HCDRs) from VH in this invention, HCDR1, HCDR2, and HCDR3, are

[0124] (i) the three complementary determinant regions HCDR1, HCDR2 and -HCDR3 contained in VH as shown in SEQ ID NO:4, or

[0125] (ii) A sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions.

[0126] In some embodiments, the three complementary determination regions (LCDRs) from VL in this invention, LCDR1, LCDR2, and LCDR3, are

[0127] (i) The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:9,

[0128] (ii) The sequence relative to (i) contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions.

[0129] In some embodiments, HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:1, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:1.

[0130] In some embodiments, HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:2, or HCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:2.

[0131] In some embodiments, HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:3, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3.

[0132] In some embodiments, LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:6, or LCDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6.

[0133] In some embodiments, LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:7, or LCDR2 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7.

[0134] In some embodiments, LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:8, or LCDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8.

[0135] In some embodiments, Ab in formula (I) of the present invention comprises a heavy chain constant region. In some embodiments, Ab in formula (I) of the present invention comprises a light chain constant region. In some embodiments, Ab in formula (I) of the present invention further comprises both a heavy chain constant region and a light chain constant region.

[0136] 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, such as the wild-type IgG1 heavy chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a lambda or kappa light chain constant region.

[0137] In some preferred embodiments, the heavy chain constant region HC of the present invention

[0138] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:5;

[0139] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:5; or

[0140] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:5.

[0141] In some embodiments, the antibody light chain constant region LC of the present invention

[0142] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:10;

[0143] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:10; or

[0144] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:10.

[0145] In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain. In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises a light chain. In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises both a heavy chain and a light chain.

[0146] In some specific embodiments of the present invention, the heavy chain of the present invention includes a heavy chain variable region and a heavy chain constant region, or is composed of the heavy chain variable region and the heavy chain constant region. In some specific embodiments of the present invention, the light chain of the present invention includes a light chain variable region and a light chain constant region, or is composed of the light chain variable region and the light chain constant region.

[0147] In some specific embodiments, Ab in formula (I) of the present invention specifically binds to CLDN18.2 and includes the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:4, and / or the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:9.

[0148] In some specific embodiments of the present invention, Ab in formula (I) of the present invention comprises HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, and / or LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7 and 8, respectively.

[0149] In some specific embodiments of the present invention, Ab in formula (I) of the present invention includes

[0150] VH, comprising 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 with it, and / or composed of said amino acid sequence, and / or

[0151] VL, which contains the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 90% identity with it or is composed of said amino acid sequence.

[0152] In some embodiments of the present invention, Ab in formula (I) of the present invention comprises VH and VL, wherein 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.

[0153] In some specific embodiments of the present invention, the Ab in formula (I) is an IgG antibody, i.e., it comprises a heavy chain and a light chain that bind to CLDN18.2. In some embodiments, the Ab in formula (I) is a complete antibody.

[0154] In some implementations, the heavy chain of Ab in formula (I)

[0155] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:11;

[0156] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:11; or

[0157] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:11.

[0158] In some implementations, the light chain of Ab in formula (I)

[0159] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:12;

[0160] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:12; or

[0161] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:12.

[0162] In some embodiments of the present invention, Ab in formula (I) of the present invention comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:11 and the amino acid sequence of the light chain is shown in SEQ ID NO:12.

[0163] In one embodiment of the invention, the amino acid alteration described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid alteration described herein is an amino acid substitution, preferably a conservative substitution. In a preferred embodiment, the amino acid alteration described herein occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described herein occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid alteration described herein occurs in the Fc region of the antibody heavy chain constant region.

[0164] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. In some embodiments, the substitution occurs in the CDR region of the antibody. Typically, the resulting variant has modifications (e.g., improvements) and / or will have substantially retained certain biological properties of the parent antibody relative to certain biological characteristics (e.g., increased affinity). An exemplary substitution variant is an affinity-matured antibody.

[0165] The antibody Ab in formula (I) of the present invention can be an antibody with modified glycosylation. In some embodiments, the antibody is an antibody obtained by in vitro enzymatic modification of the glycan chain (e.g., by modification of the glycan chain by a glycosidase (e.g., an endonuclease or a glycosyltransferase)). In some embodiments, the antibody with modified glycosylation refers to an antibody in which the glycan chain at the antibody glycosylation site is modified from a heterogeneous N-glycan chain to a single-structure N-glycan chain with a reactive group (e.g., any reactive group capable of reacting with the linker moiety, such as an azide, ketone, or alkynyl group). In a preferred embodiment, the N-glycosylation site of the antibody is a conserved N-glycosylation site on the antibody Fc domain, such as Asn297.

[0166] Methods suitable for modifying antibody glycosylation according to the present invention can be found, for example, in PCT / NL2013 / 050744, PCT / EP2016 / 059194 or PCT / EP2017 / 052792, the entire contents of which are incorporated herein by reference.

[0167] In a preferred embodiment, the modified glycosylated antibody of the present invention is a GlcNAc-E(A) antibody. x An antibody with substituents, wherein GlcNAc is N-acetylglucosamine, and E(A)x is a sugar derivative comprising x functional groups A, wherein A is independently selected from azido, keto, and alkynyl groups and x is 1, 2, 3, or 4; wherein the GlcNAc-E(A)x substituent is bonded to the antibody via a C1 bond of the N-acetylglucosamine with the GlcNAc-E(A)x substituent, wherein the N-acetylglucosamine is optionally fucosylated. In the case of fucosylation of N-acetylglucosamine, it is bonded to fucose via a C6 bond.

[0168] In one embodiment, the modified glycosylated antibody of the present invention is an antibody of formula (III), wherein Ab represents an antibody, GlcNAc is N-acetylglucosamine, Fuc is fuc, b is 0 or 1, y is 1 to 20, and E(A)x is a sugar derivative comprising x functional groups A, wherein A is an azide, keto, or alkynyl group, and x is 1, 2, 3, or 4. In a 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.

[0169]

[0170] The sugar derivative E(A)x in the GlcNAc-E(A)x substituent of the modified glycosylated antibody can be linked to C4 of the GlcNAc via a β(1,4)-glycosidic bond or to C3 of the GlcNAc via an α(1,3)-glycosidic bond, preferably to C4 of the GlcNAc via a β(1,4)-glycosidic bond. The N-acetylglucosamine of the GlcNAc-E(A)x substituent is linked to the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom (GlcNAcβ1-Asn) in the side chain of the asparagine of the antibody. The GlcNAc on the GlcNAc-E(A)x substituent is optionally fucosylated. Accordingly, GlcNAc-E in the antibody-drug conjugate can also be linked as described above when present.

[0171] In a 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 may be bonded to C2, C3, C4, or C6 of the sugar or sugar derivative E, replacing the hydroxyl group (OH). It should be understood that the bonding position of functional group A corresponds to the position where the sugar containing A is connected to the linker.

[0172] In a preferred embodiment, the sugar derivative E(A)x is derived from a sugar or sugar derivative E. In a 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, with GalNAc being the most preferred.

[0173] In another preferred embodiment, E(A)x is GalNAc-N3, preferably, E(A)x is 6-azido-6-deoxy-2-acetamidogalactose.

[0174] It should be understood that, without contradiction, the above description and explanation of sugars (including but not limited to the glycosidic linkage of GlcNAc-E(A)x) also applies to the corresponding sugars in the conjugate of formula (II) below, such as the sugar linkage therein. Those skilled in the art will understand that, although in some embodiments a reactive group (e.g., functional group A) is linked to the antibody's sugar chain during antibody glycosylation modification, and therefore "antibody with modified glycosylation" is defined as an antibody containing that reactive group, during antibody-drug conjugate formation, this reactive group may react with the linker portion to form a new group, which is then considered part of the linker in the antibody-drug conjugate.

[0175] 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.

[0176] When the modified antibody is a complete antibody, it preferably contains two or more, more preferably two, GlcNAc-E(A)x substituents, which are optionally fucosylated. However, if the modified antibody is an antibody fragment, such as a Fab or Fc fragment, it 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, provided that the substituent does not impede 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.

[0177] As described above, the modified glycosylated antibody of the present invention contains one or more GlcNAc-E(A)x substituents, for example, two GlcNAc-E(A)x substituents.

[0178] In a preferred embodiment, the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (e.g., a natively conserved N-glycosylation site) of the antibody, such as a glycosylation site in the Fc region (more preferably the CH2 domain). In yet another preferred embodiment, the antibody is an IgG antibody and the GlcNAc-E(A)x substituent is present at a native N-glycosylation site (a natively conserved N-glycosylation site) of the IgG antibody. In yet another preferred embodiment, the native site is an 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 a preferred embodiment, the GlcNAc-E(A)x substituent is present at the Asn297-glycosylation sites of both heavy chains of the antibody.

[0179] In some embodiments, L in formula (I) of the present invention is a linker. Any linker known in the art can be used to connect with the anti-human CLDN18.2 of the present invention; preferably, the linker enables site-specific coupling of the ADC.

[0180] In some embodiments, the linker suitable for use in this invention can be any linker capable of conjugating an antibody to a drug. In some embodiments, the linker can be a linker used in techniques capable of site-specific conjugation.

[0181] In a preferred embodiment, the linker of the present invention is a linker linked to an antibody oligosaccharide. As defined herein, a "linker linked to an antibody oligosaccharide" refers to any linker that connects to a reactive group on the glycan chain at the antibody glycosylation site to conjugate an antibody to a drug. The glycan chain at the antibody glycosylation site is generally an N-glycan chain and is often modified to transform a heterogeneous N-glycan chain into a single-structure N-glycan chain with a reactive group. This reactive group is then used to connect to the "linker," achieving site-specific conjugation of the drug to the antibody, resulting in an antibody-drug conjugate. In a preferred embodiment, the N-glycosylation site of the antibody is the antibody Fc domain, preferably a conserved N-glycosylation site on the CH2 domain, such as Asn297. Therefore, in one embodiment, the “linker linked to the antibody oligosaccharide” of the present invention is particularly any linker capable of site-specific coupling with a reactive group on the N-glycan chain at a conserved N-glycosylation site (e.g., Asn297) on the Fc domain of the antibody, such as the linker described in PCT / NL2013 / 050744 or PCT / EP2021 / 075401, which are incorporated herein in their entirety. In one embodiment, the reactive group of the present invention is an azide, keto, or 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, keto, or alkynyl group, preferably an azide group, and the linker of the present invention is a linker containing an alkynyl group. When referring to such linkers in the present invention, since the reactive group reacts with the group of the linker to form a new group, the group formed after the reactive group in the antibody-drug conjugate reacts may also be defined as part of the “linker,” for example as shown in formula (II) of the present invention.

[0182] Linkers suitable for use in this invention also include, for example, cathepsin-degradable linkers, such as Val-Cit linkers (e.g., vc-PAB), cBu-Cit linkers, and CX linkers; non-breakable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared sensitive linkers, and ultraviolet sensitive linkers such as PC4AP (Antibody–drug conjugates: Recent advances in linker chemistry, Su, Z., Xiao, D., Xie, F., Liu, L., Wang, Y., Fan, S., … Li, S. (2021). Antibody–drug conjugates: Recent advances in linker chemistry. Acta Pharmaceutica Sinica B.). The linkers suitable for use in this invention can also be combinations of one or more linkers. For example, a linker degraded by cathepsin can be combined with other types of linkers to form a new linker. Therefore, the term "linker" in this invention encompasses a single type of linker or a combination of different types of linkers, as long as it can conjugate the antibody and drug of this invention. Thus, in one embodiment, the linkers suitable for use in this invention are MC-VC-PAB, vc-PAB, SMCC, or MC-GGFG.

[0183] In this invention, D in formula (I) can be any antitumor compound, as long as it has antitumor effects and contains substituents or partial structures that can be attached to the linker structure; there are no particular limitations. For example, the antitumor compound can be a pharmaceutically active compound that acts on tumors. For antitumor compounds, preferably, part or all of the linker can be cleaved within tumor cells, releasing the antitumor compound portion and thus exhibiting an antitumor effect. When the linker is cleaved at the linker portion with the drug, the antitumor compound is released in its unmodified structure, allowing it to exert its original antitumor effect.

[0184] In some embodiments, the antitumor compound can be, for example, a cytotoxic agent or chemotherapeutic agent, such as camptothecin compounds like eczetidine (a topoisomerase I inhibitor, Exatecan), Dxd (a novel topoisomerase I inhibitor derivative of Exatecan), auristatin compounds such as monomethylaurestatin E (MMAE), or maytansine compounds such as the small molecule microtubule inhibitor DM1. The structures in the embodiments of this application show the structures of representative compounds of these antitumor compounds.

[0185] In one embodiment, the present invention provides (II) an antibody-drug conjugate or a pharmaceutically acceptable salt or solvation thereof.

[0186]

[0187] Ab represents the antibody as defined in the text.

[0188] L1 is the connector;

[0189] E is a sugar or sugar derivative, such as the sugar or sugar derivative defined above;

[0190] Furthermore, GlcNAc is N-acetylglucosamine, and Fuc is fucose;

[0191] D and r are defined as in equation I above;

[0192] b is either 0 or 1, for example, 0;

[0193] x is 1, 2, 3, or 4; preferably 1 or 2;

[0194] y is 1-20; 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.

[0195] 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.

[0196] 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, with the most preferred GalNAc, such as 6-deoxy-2-acetamidogalactose, which is preferably linked to L1 via a C atom at the 6-position.

[0197] In a preferred embodiment, the GlcNAc linked to the Ab is located at a native N-glycosylation site (e.g., a natively conserved N-glycosylation site) of the antibody, such as a glycosylation site in the Fc region. In yet another preferred embodiment, the antibody is IgG and the GlcNAc is located at a native N-glycosylation site (e.g., a natively conserved N-glycosylation site) of IgG, such as a glycosylation site in the Fc region. In yet another preferred embodiment, the native site is an Asn297-glycosylation site of IgG. The Asn297-glycosylation site is located in the Fc region of the heavy chain of the IgG antibody. In a preferred embodiment, the GlcNAc group is located at the Asn297-glycosylation sites of both heavy chains of the antibody.

[0198] In one embodiment, L1 in formula (II) has the following structure

[0199]

[0200] Q is -N(H)C(O)CH2- or -CH2-;

[0201] R1 is independently selected from hydrogen, halogens, -OR2, -NO2, -CN, -S(O)2R2, Cl-C 12 Alkyl, aryl, heteroaryl, C1-C 12 Alkyl aryl, C1-C 12 Alkyl heteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 Alkyl, wherein the alkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, arylalkyl, and heteroarylalkyl are optionally substituted, wherein two substituents R1 may be linked together to form a fused, bridged, or helical cycloalkyl or a fused or helical aromatic or heteroaromatic substituent, and wherein R2 is independently selected from hydrogen, halogen, C1-C 24 Alkyl, aryl, heteroaryl, C1-C 12 Alkyl aryl, C1-C 12 Alkyl heteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 alkyl;

[0202] R3 and R4 are each independently selected from hydrogen, halogens, and C1-C atoms. 24 Alkyl, aryl, heteroaryl, C1-C 12 Alkyl aryl, C1-C 12 Alkyl heteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 alkyl;

[0203] c can be 0, 1, 2, 3, or 4;

[0204] m is 0 or 1;

[0205] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0206] Y is O, S, or NR1;

[0207] L2 and L3 are each independently C1-C 12 An alkyl group, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are optionally replaced by heteroatoms selected from O, N and S, provided that the O, N and / or S are not directly connected to each other;

[0208] Each L4 is an independently divisible linker.

[0209] It should be understood that when m is 0, it means that Q does not exist, and thus the relevant nitrogen atom of the triazole is directly connected to the E part through a covalent single bond.

[0210] In one implementation scheme, L4 is independently...

[0211] R5 and R6 are each independently selected from hydrogen and C1-C. 12 alkyl;

[0212] Ar is selected from aryl, preferably phenyl;

[0213] In one implementation scheme, L4 is independently for

[0214] In one implementation, -L1- has the following structure

[0215]

[0216] In one embodiment, the antibody-drug conjugate has an average DAR of 1-15, such as 1-10, 2-8, 2-6, or 3-5.

[0217] In one embodiment, the antibody-drug conjugate of the present invention is selected from:

[0218]

[0219]

[0220] Where Ab is HB37A6;

[0221] Where q represents the average DAR.

[0222] In IEX019-02, IEX019-04, and IEX019-05, q is 2-5, for example, 3-5, 3-4, or 3.5-4.5. In IEX019-03, q is 5-11, for example, 6-10, 7-9, or 7.5-8.5.

[0223] III. Preparation of the ADC molecule of the present invention

[0224] This invention provides a method for preparing antibodies with modified glycosylation, the method comprising:

[0225] (1) Preparation of glycosylated antibody: Under conditions suitable for antibody expression, host cells containing nucleic acids encoding said antibody (e.g., any one and / or multiple polypeptide chains) or expression vectors containing said nucleic acids, as provided above, are cultured, and the antibody is optionally recovered from said host cells (or host cell culture medium) to obtain an antibody containing a core N-acetylglucosamine substituent (core-GlcNAc substituent), wherein said core N-acetylglucosamine and said core N-acetylglucosamine substituent are optionally fucosylated;

[0226] (2) Preparation of trimmed antibody: The antibody prepared in step (1) is deglycosylated in the presence of a glycosidase to obtain an antibody containing a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated.

[0227] (3) The trimmed antibody obtained in (2) is contacted 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 attached to the antibody via a C1 bond of 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).

[0228] To perform step (1), nucleic acids encoding antibodies (such as those described above, such as any one or more polypeptide chains) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0229] The glycosidase in step (2) can be selected based on the properties of the glycosylation antibody, for example, from Endo S, Endo A, Endo F, Endo M, Endo D and Endo H enzymes and / or combinations thereof, and also, for example, from Endo S, Endo S49, Endo F or combinations thereof. In a preferred embodiment, the glycosidase is the glycosidase described in PCT / EP2017 / 052792, most preferably Endo SH from PCT / EP2017 / 052792.

[0230] The glycosyltransferase in step (3) is preferably a glycosyltransferase derived from or derived from β-(1,4)-N-acetylgalactosamine transferase, more preferably any β-(1,4)-GalNAcT enzyme described in PCT / EP2016 / 059194. In some embodiments, the β-(1,4)-GalNAcT enzyme is or is derived from an invertebrate β-(1,4)-GalNAcT enzyme. The β-(1,4)-GalNAcT enzyme can be or can be derived from any invertebrate β-(1,4)-GalNAcT enzyme known to those skilled in the art. Preferably, the β-(1,4)-GalNAcT enzyme is or is derived from a β-(1,4)-GalNAcT enzyme derived from Nematoda, preferably Chromadorea or Seminentea, or from Arthropoda, preferably Insecta. More preferably, β-(1,4)-GalNAcT enzymes are found in *C. elegans*, *Ascaris lumbricoides*, *Spodoptera litura*, *Drosophila melanogaster*, *Caenopsis rhabditis briggsae*, *Caenopsis wuce*, *Loa filariasis*, *P. piscis spp.*, *Termitomyces hygrophorus*, *Caryota flos*, *Oyster oyster*, and *Monarchus davidii*. Preferably, the glycosyltransferase suitable for step (3) is the *Spodoptera litura* β-(1,4)-GalNAcT enzyme (e.g., His-TnGalNacT) disclosed in PCT / EP2016 / 059194 and named TnGalNAcT.

[0231] Another aspect of the present invention provides a method for preparing an antibody-adjuvant antibody (ADC) using the antibody of the present invention. In this invention, "ADC" is defined as an antibody coupled to an active substance (D) having biological and / or pharmaceutical activity via a linker (L). The method comprises coupling the antibody of the present invention to one or more active substances D via one or more linkers (L) defined in the present invention. Preferably, the linker-active substance site is specifically coupled to the antibody.

[0232] In one embodiment, a method for preparing the ADC of the present invention is provided, the method comprising glycosylation of an antibody of formula III.

[0233]

[0234] The meanings of each variable or symbol are as defined above;

[0235] Reaction with a linker-payload containing an alkynyl group and one or more (e.g., 1, 2, 3, or 4) drug molecules.

[0236] Generate antibody-drug conjugate of formula (II)

[0237]

[0238] The meanings of each variable or symbol are as defined above.

[0239] In one embodiment, the linker-drug compound has the following structure:

[0240]

[0241] The variables are defined as described above.

[0242] IV. Pharmaceutical Compositions

[0243] In some embodiments, the present invention provides compositions comprising any ADC molecule described herein or a pharmaceutically acceptable salt thereof, preferably pharmaceutical compositions or pharmaceutical formulations. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example a pharmaceutical composition, comprises an ADC molecule of the present invention, and a combination of one or more other therapeutic agents.

[0244] The present invention also includes compositions comprising the ADC molecule of the present invention or a pharmaceutically acceptable salt thereof (including pharmaceutical compositions). These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0245] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0246] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0247] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0248] A drug comprising the ADC described herein can be prepared by mixing the ADC molecules of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0249] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including 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 suitably combined in amounts effective for the intended use.

[0250] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0251] VII. Drug combinations and medicine boxes

[0252] In some embodiments, the present invention also 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)).

[0253] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0254] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0255] - A first container containing a pharmaceutical composition comprising the ADC molecule of the present invention;

[0256] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0257] VIII. Applications and Methods

[0258] In one aspect, the present invention provides a method for preventing or treating tumors (e.g., cancer) in a subject, comprising administering to the subject an effective amount of the ADC molecule, pharmaceutical composition, pharmaceutical combination, or cassette of the present invention.

[0259] In some embodiments, the tumor (e.g., cancer) patient has CLDN18.2 (e.g., elevated levels, such as nucleic acid or protein levels). In some embodiments, the patient's tumor cells express CLDN18.2, for example, moderately expressed CLDN18.2, preferably highly expressed CLDN18.2.

[0260] In some embodiments, the tumor, such as cancer, includes solid tumors, hematologic malignancies, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be in the early, intermediate, or late stage, or metastatic stage.

[0261] In one specific embodiment, the ADC molecule of the present invention is capable of killing tumor cells and / or inhibiting tumor cell proliferation, such as tumor cells expressing CLDN18.2, such as gastrointestinal tumor cells, such as gastric cancer cells, pancreatic cancer cells, colon cancer cells, or colorectal cancer cells.

[0262] In some implementations, the tumor is a tumor immune escape.

[0263] In some implementations, the tumor is cancer, such as an epithelial tumor, a gastrointestinal tumor, such as epithelial cancer or gastroesophageal cancer, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.

[0264] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., cancer). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0265] In other respects, the present invention provides the use of ADC molecules or pharmaceutical compositions or combinations or kits in the manufacture or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0266] In some embodiments, the ADC molecules or pharmaceutical compositions or kits of the present invention may delay the onset of symptoms and / or symptoms associated with the disease.

[0267] In some embodiments, the ADC molecules or pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as treatments and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0268] In some implementation schemes, treatment methods include surgery; radiotherapy, local irradiation, or focused irradiation.

[0269] In some implementations, 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).

[0270] Other exemplary antibodies include those that specifically bind to immune checkpoints.

[0271] The combination therapy of the present invention covers combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which the administration of the ADC molecule of the present invention may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceuticals.

[0272] The drug composition can be administered via known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (internal parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; via a continuous release system or via an implantable device. In some embodiments, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0273] The composition can also be applied topically via an implantable membrane, a sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In some embodiments, when an implantable device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecules via diffusion, timed release of a large pellet, or continuous administration.

[0274] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0275] Example 1.1 Construction of a stable expression cell line

[0276] Preparation of human CLDN18.2 overexpression cell line

[0277] Use according to the manufacturer's instructions. The kit (Invitrogen, A1369601) was used to construct a cell line stably expressing human Claudin18.2 (hereinafter referred to as CLDN18.2). First, the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pCHO1.0 to construct plasmids. The constructed plasmids were then transfected into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) using chemical transfection and electrotransfection methods, respectively. The transfected cells underwent two rounds of pressure selection to obtain cell pools expressing CLDN18.2. Then, the cells that highly expressed CLDN18.2 were sorted out using a flow cytometer (MoFlo XDP, Beckman Coulter), and the monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2 that stably expressed CLDN18.2 were obtained by dilution.

[0278] Preparation of human CLDN18.1 overexpressing cell lines

[0279] Use according to the manufacturer's instructions. A cell line stably expressing human Claudin18.1 (hereinafter referred to as CLDN18.1) was constructed using a kit (Invitrogen, A1369601). First, the full-length human CLDN18.1 (UniProt ID: P56856-1) gene was constructed into the vector pCHO1.0 (Invitrogen, A1369601) to form a plasmid. The constructed plasmid was then chemically transfected into CHO-S cells (Invitrogen, A1369601). The transfected cells underwent two rounds of pressure selection to obtain cell pools expressing CLDN18.1. Then, cells highly expressing CLDN18.1 were sorted using a flow cytometry system (MoFlo XDP, Beckman Coulter), and a dilution method was used to obtain the stable CLDN18.1 monoclonal cell line CHO-hCLDN18.1.

[0280] Construction of tumor cell lines overexpressing CLDN18.2

[0281] The full-length human CLDN18.2 (UniProt ID: P56856-2) gene was constructed into the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence. This vector, along with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), was co-transfected into HEK293T (ATCC, CRL-3216) cells for viral packaging. Culture supernatants were collected after 48 and 72 hours of culture, and the lentivirus was concentrated using PEG8000. Pancreatic cancer DAN-G cells (CLS Cell Lines Service GmbH, 300162) and gastric cancer KATO III cells (ATCC, HTB-103) were transfected with concentrated virus. Then, cells expressing CLDN18.2 were sorted using a flow cytometry system (MoFlo XDP, Beckman Coulter) to obtain stable CLDN18.2-transfected tumor cell lines DAN-G-hCLDN18.2 and KATOIII-hCLDN18.2.

[0282] Example 1.2, Generation of CLDN18.2 Monoclonal Cells

[0283] This invention employs hybridoma technology, using cells (CHO-hCLDN18.2) obtained in Example 1 to immunize H2L2 fully human antibody-transgenic mice (purchased from Harbour BioMed). Then, spleen cells from these mice were electrofused with myeloma cells. The supernatant was then collected and screened using flow cytometry (FACS) to identify hybridoma cells specifically expressing anti-CLDN18.2 antibodies, whose secreted antibodies did not bind to CLDN18.1. The test cells (HEK293-hCLDN18.2) obtained in Example 1 were counted and diluted to 1×10⁻⁶. 6 Cells / ml, add 100 μl / well to a U-bottom 96-well plate. Centrifuge at 500g for 5 min, remove cell culture medium. Add the supernatant from the hybridoma 96-well plate to the U-bottom plate and resuspend the cells, 100 μl per well, incubate on ice for 30 min. Centrifuge at 500g for 5 min, remove supernatant, wash cells once with PBS. Add 100 μl of FITC-labeled anti-mouse Fab secondary antibody (1:500 diluted in PBS) to each well, and add 100 μl of FITC-labeled anti-human Fab secondary antibody to the positive control antibody. Incubate on ice in the dark for 30 min. Centrifuge at 500g for 5 min, remove supernatant, wash cells once with PBS. Resuspend cells in 50 μl of 1×PBS, and analyze by FACS. Positive clones are then screened again for CHO-hCLDN18.1 using the same method. After two rounds of screening, the fully human antibody clone HB37A6 was obtained.

[0284] Example 1.3: Preparation of recombinant CLDN18.2 monoclonal antibody

[0285] The anti-CLDN18.2 monoclonal antibody HB37A6 (see CN202010570517.X) and the control antibody zolbetuximab (abbreviated as Zmab, sequence derived from INN117) were expressed as full-length monoclonal antibodies in HEK293 cells (Invitrogen, A14527).

[0286] First, expression vectors were constructed by placing the heavy chain variable region and light chain variable region (see sequence listing information) of HB37A6 and the control antibody at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 5) and light chain kappa constant region (SEQ ID NO: 10), respectively. These were then incorporated into the pcDNA3.1 expression vector containing the N-terminal signal peptide to obtain the light and heavy chain expression vectors. The obtained light and heavy chain expression vectors were co-transfected into HEK293 cells using PEI (Polysciences Inc, 23966). After 7 days of culture, the supernatant was collected. The supernatant was purified using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), then ultrafiltered and the medium was changed to PBS (Gibco, 70011-044). The concentration was detected using the A280 method, and the purity was determined by SEC-HPLC. An antibody solution with a purity greater than 95% was obtained, yielding the recombinant CLDN18.2 monoclonal antibody HB37A6.

[0287] The specific transfection and purification process is as follows:

[0288] Expi293 cells (Invitrogen, A14527) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 × 10⁻⁶ cells / day before transfection. 6 Cells / ml. The cell density on the day of transfection was approximately 3 × 10⁻⁶ cells / ml. 6 Cells / ml. Take 1 / 10 of the final volume of Opti-MEM medium (Gibco, 31985-070) as transfection buffer, add an appropriate amount of plasmid at 1.0 μg / ml for cell transfection, and mix well. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the plasmid to PEI ratio is 1:3 in 293F cells), mix well, and incubate at room temperature for 20 min to obtain a DNA / PEI mixture. Slowly add the DNA / PEI mixture to the cells while gently shaking the flask, then incubate at 36.5℃ in an 8% CO2 incubator. After seven days, obtain the cell culture medium, collect the cell supernatant, and purify it.

[0289] The Protein A column (Hitrap Mabselect Sure, GE, 11-0034-95) used for purification was treated with 0.1M NaOH for 2 hours. Glass bottles and other equipment were washed with distilled water and then dried at 180°C for 4 hours. Before purification, the collected cell material 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). The filtered supernatant was added to the purification column and equilibrated with 10 column volumes of binding buffer. 5 ml of elution buffer (0.1 M citric acid + sodium citrate, pH 3.5) was added, and the eluent was collected. 80 μl of 2M Tris-HCl was added to every 1 ml of eluent. The collected antibody was ultrafiltered and concentrated, then exchanged for PBS (Gibco, 70011-044), and the concentration was determined.

[0290] Example 1.4: Determination of CLDN18.2 antibody affinity by SPR method

[0291] The equilibrium dissociation constant (K0) of HB37A6 bound to human CLDN18.2 was determined using surface plasmon resonance (SPR). D Following the manufacturer's instructions, human Claudin 18.2 (GenScrip, P50251802) was conjugated onto the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino-conjugation kit (GE Healthcare, BR-1006-33). After conjugation, 1M ethanolamine was injected to block the remaining activation sites. Affinity and kinetic constants were obtained by detecting the binding and dissociation of the chip surface antigen with the antibody in the mobile phase using a Biacore (GE Healthcare, T200) analyzer. Serially diluted antibodies (0-100 nM) were flowed sequentially from low to high concentration onto the chip surface, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). Data were analyzed using Biacore T200 software with a 1:1 binding model for kinetic analysis. As shown in Table 1, HB37A6 has a better affinity than the control antibody Zmab.

[0292] Table 1. Affinity constants (equilibrium dissociation constants) for SPR assay of CLDN18.2 antibody

[0293]

[0294] Example 1.5: Binding specificity of CLDN18.2 antibody to CLDN18 cells

[0295] The binding of the above-mentioned anti-CLDN18.2 monoclonal antibody HB37A6 and control antibody Zmab to the CHO-S cell lines (i.e., CHO-hCLDN18.2 and CHO-hCLDN18.1 prepared as described in Example 1) stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1 was determined by flow cytometry (FACS).

[0296] Specifically, the cells to be tested (CHO-hCLDN18.2 and CHO-hCLDN18.1) obtained in Example 1 were counted and diluted to 1×10⁻⁶. 6 Cells / ml, add 100 μl / well to a 96-well U-bottom plate. Centrifuge at 500g for 5 min, remove cell culture medium. Add anti-CLDN18.2 monoclonal antibody HB37A6 and control antibody Zmab to the U-bottom plate and resuspend cells, 100 μl per well. The initial antibody concentration is 900 nM, then 3-fold diluted sequentially for a total of 10 concentration points. Incubate on ice for 30 min. Remove supernatant at 500g for 5 min, wash cells once with PBS. Add 100 μl of PE-labeled goat anti-human Fc secondary antibody (SouthernBiotech, J2815-5H87B) to each well. Incubate on ice in the dark for 30 min. Remove supernatant at 500g for 5 min, wash cells once with PBS. Resuspend cells in 50 μl of 1×PBS, and analyze using FACS. Analyze experimental data using GraphPad Prism software. Figure 1 and Figure 2 .like Figure 1 and Figure 2 As shown, all of the antibodies specifically bind to human CLDN18.2 ( Figure 1 ), and not combined with human CLDN18.1 ( Figure 2 ).

[0297] Example 1.6: Binding of CLDN18.2 antibody to tumor cell lines

[0298] Referring to Example 1.5, the binding of HB37A6 to the gastric cancer cell lines NUGC-4 (JCRB, JCRB0834), KATO III-hCLDN18.2, and DAN-G-hCLDN18.2 was determined by FACS. Figure 3 The results showed that the fully human antibody HB37A6 had good tumor cell-specific binding, which was superior to the control antibody Zmab.

[0299] Example 1.7: In vivo antitumor effect of CLDN18.2 antibody

[0300] 1. Antibody activity against the DAN-G-CLDN18.2 tumor-bearing mouse model

[0301] The antitumor effect of HB37A6 antibody in NOD-SCID mice with human pancreatic cancer (female NOD-SCID mice (15-18g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was tested. The human pancreatic cancer cells DAN-G-hCLDN18.2 constructed in Example 1 were routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) to obtain a cell suspension with a cell density of 12×10^5 cells / ml. The cell suspension was mixed with Matrigel gel 1:1 to prepare a cell suspension with a cell concentration of 6×10^5 cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously injected into the right abdominal region of NOD-SCID mice to establish a DAN-G-CLDN18.2 tumor-bearing mouse model.

[0302] Five days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of 43.36 mm were selected. 3 ~89.47mm 3 Mice within the range were grouped in a serpentine pattern according to tumor size (8 mice per group).

[0303] Each mouse was administered hIgG (Equitech-Bio, batch number 160308-02), HB37A6, and the control antibody Zmab at a dose of 10 mg / kg per administration, on days 5, 9, 12, and 16 post-inoculation. Tumor volume was monitored 2-3 times per week. Tumor volume was measured using calipers to determine the maximum long axis (L) and maximum wide axis (W) of the tumor. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0304] 2. Antibody activity against NUCG-4 tumor-bearing mouse model

[0305] The antitumor effect of HB37A6 antibody in NOG mice with human gastric cancer (female NOG mice (15-18g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was tested. PBMC cells (Allcells) were resuscitated, centrifuged to collect cells, and dispersed in PBS (1×) at a cell density of 2.5×10^6 cells / ml. On day 0, 0.2ml of the cell suspension was injected intravenously into the eye vein of NOG mice to establish a NOG humanized mouse model.

[0306] NUGC-4 cells were routinely resuscitated and passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) at a density of 12×10^6 cells / ml. The PBS was then mixed 1:1 with Matrigel gel to prepare a cell suspension with a concentration of 6×10^6 cells / ml. On day 5, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOG humanized mice to establish a NUGC-4 tumor-bearing mouse model.

[0307] On day 1 after tumor cell inoculation, mice were randomly divided into groups of 7 mice each. Each mouse was administered hIgG (Equitech-Bio, batch number 160308-02, control group), HB37A6 (treatment group), and positive control antibody Zmab (treatment group) at a dose of 10 mg / kg per administration. Administration was performed on days 1, 5, 8, and 12 post-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 wide axis (W) of the tumor. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance. The relative tumor inhibition rate (TGI%) was calculated on day 26 after vaccination using the following formula:

[0308] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in control group before drug administration).

[0309] 3. Results

[0310] The results are as follows Figure 4 As shown, both HB37A6 and the control antibody Zmab inhibited tumor growth in a human pancreatic cancer DANG-CLDN18.2 mouse model, with a TGI of 28% for HB37A6 and 24% for Zmab. Figure 5 As shown, HB37A6 exhibited better anti-tumor effects than the control antibody Zmab in the NUGC-4 mouse model of human gastric cancer, with a TGI of 31% for HB37A6 and 0% for Zmab.

[0311] Example 2.1 Synthesis of IEX019ADC molecule

[0312] IEX019

[0313] Based on HB37A6, ADCs coupled with different small molecule compounds were further designed and synthesized. The specific process is as follows:

[0314] Example 2.1.1 Preparation of IEX019-02

[0315] 1). Preparation of compound 6

[0316]

[0317] Under a nitrogen 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 150 mL of DCM. The mixture was stirred for 10 minutes and quenched by adding saturated NH4Cl aqueous solution (150 mL). After separation, the aqueous layer was extracted with DCM (150 mL). The combined organic layers were dried (Na2SO4) and concentrated. The residue was purified by column chromatography. Product 6 (2.06 g, 5.72 mmol, 57%) was given as a pale yellow, viscous oil. 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).

[0318] 2 Preparation of compound 7

[0319]

[0320] CSI (11 μL, 18 mg, 0.13 mmol) was added to a stirred solution of 6 (47 mg, 0.13 mmol) in DCM (10 mL). After 30 minutes, a solution of Et3N (91 μL, 66 mg, 0.65 mmol) and diethanolamine (16 mg, 0.16 mmol) in DMF (0.5 mL) was added. After another 30 minutes, p-nitrophenyl chloroformate (52 mg, 0.26 mmol) and Et3N (54 μL, 39 mg, 0.39 mmol) were added. After another 4.5 hours, the reaction mixture was concentrated and the residue was purified by gradient column chromatography (33→66% EtOAc / heptane (1% AcOH)) to give 7 (88 mg, 0.098 mmol, 75%) as a colorless oil. 1H 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).

[0321] 3) Preparation of compound 9

[0322]

[0323] Compound 8 (163 mg, 240 μmol) was added to a mixture of ethanotecan 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 to 9 mL of DCM and purified by gradient column chromatography (0→40% MeOH / DCM) to give 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.

[0324] 4) Preparation of Compound 1

[0325] To a solution of compound 9 (155 mg, 159 μmol) in DMF (1.6 mL), a solution 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 give compound 1 (94 mg, 44 μmol, 28%) as a pale yellow solid. LCMS (ESI+)C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + Calculated value: 1066.88; Measured value: 1067.12.

[0326] 5) Enzymatic reconstitution of HB37A6 into HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2

[0327] HB37A6 was expressed and purified in HEK293 cells according to Example 1.3. The obtained HB37A6 (16.4 mg / mL) was incubated with EndoSH (1% w / w) as described in PCT / EP2017 / 052792 to obtain pruned HB37A6 with -GlcNAc or GlcNAc(Fuc) at the Asn297 position. The pruned HB37A6 was then incubated with the enzyme His-TnGalNAcT (4.5% w / w) disclosed in PCT / EP2016 / 059194 and 6-azido-GalNAc-UDP (25 eq (equivalent to antibody)) disclosed in PCT / EP2016 / 059194 (in a solution containing 6 mM MnCl2, histidine (20 mM) + NaCl (150 mM)) at 30°C for 16 h.

[0328] Next, the incubation mixture obtained above was purified using a 50 mL protA column (Hitrap Mabselect Sure, GE, 11-0034-95). The obtained incubation mixture was loaded onto the column and washed with TBS + 0.2% Triton and TBS. The column was then eluted with 0.1 M acetate buffer (pH 2.9) and neutralized with 2.5 M Tris-HCl (pH 7.2). After dialyzing three times with PBS, the obtained (modified) glycosylated antibody was concentrated to 32.6 mg / mL using a Vivaspin Turbo 15 ultrafiltration device (Sartorius).

[0329] The obtained glycosylated antibody was analyzed by mass spectrometry. The main steps are as follows: Before mass spectrometry analysis, the glycosylated antibody was treated with IdeS to enable analysis of the Fc / 2 fragment. 20 μg of the (modified) glycosylated antibody solution was mixed with IdeS (Fabricator). TM (1.25 U / μL) was incubated in PBS pH 6.6 at 37°C for 1 hour with a total volume of 10 μL. The sample was diluted to 80 μL and then analyzed on a JEOL AccuTOF (ESI-TOF) spectrometer. The deconvolution spectrum was obtained using Magtran software. Mass spectrometry analysis of the IdeS digested sample showed that the major product corresponded to the obtained HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc) 2，， The observed mass is 24330.4.

[0330] Therefore, the above results demonstrate that HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 was obtained, in which GlcNAc at Asn297 of both heavy chains is replaced by 6-azido-GalNAc (4-position substitution).

[0331] 6) Preparation of HB37A6-SYNtecan E conjugate (IEX019-02)

[0332] The bioconjugate IEX019-02 according to the present invention is prepared by coupling compound 1 (linker-payload) as a linker-conjugate to HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 as an azide-modified biomolecule. Therefore, 808 μL of PBS pH 7.4, 11.3 mL of 1,2-propanediol, and 350 μL of 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 pH 7.4). The reaction was incubated overnight at room temperature, then filtered and dialyzed to PBS pH 7.4. Residual free payload was removed by adding charcoal (350 mg) and then rotating overnight at room temperature. Charcoal was removed by centrifugation and filtration, and ADC was purified on an AKTA Purifier-10 (GE Healthcare) column equipped with a Superdex200 26 / 600SEC (GE Healthcare) column.

[0333] Mass spectrometry analysis of the IdeS digested sample showed that both major products corresponded to the obtained ADC, namely the HB37A6-SYNtecan E conjugate. The first peak, with an observed mass of 26469 Da (calculated mass 26465 Da), corresponds to the conjugated Fc / 2 fragment (the 2x blocked lactone form of the payload). The second peak, with an observed mass of 26499 Da (calculated mass 26501 Da), corresponds to the bound Fc / 2 fragment (the 2x open carboxylate form of the payload).

[0334] The obtained structure is shown below. The concentration, DAR value, and SEC purity of the ADC product were determined by UV, SEC, RP-HPLC, and LC-MS. The monomer purity detected by SE-HPLC was >99%, and the concentration was 6.12 mg / ml.

[0335]

[0336] Where q represents the average DAR, for example, 3-5, 3.2-4.8, or 3.5-4.5, such as 3.52 as measured in Table 2.

[0337] Where Ab is HB37A6.

[0338] Example 2.1.2 Preparation of IEX019-03

[0339]

[0340] Where q represents the average DAR, for example, 5-11, 6-10, 7-9 or 7.5-8.5, such as 7.9 as measured in Table 2.

[0341] Where Ab is HB37A6.

[0342] (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 place the reaction mixture in a shaker for 2-4 hours.

[0343] (i) The optimal concentration of HB37A6 is 5-10 mg / mL.

[0344] (ii) The optimal molar ratio of TCEP / mAb is 4.5 to 6.5.

[0345] (iii) The optimal temperature for the reaction is 20-37℃.

[0346] (iv) The optimal pH for the reaction is generally between 6.5 and 8.0.

[0347] (b) An excess linker-toxin MC-GGFG-DXD (purchased from Levena Biopharma, SET0218, structure shown below) was dissolved in DMSO and reacted with the thiol groups of the antibody reduced in step (a). The reaction mixture was placed in a shaker for 1–2 h, wherein…

[0348] (i) The optimal molar ratio of DXD / mAb is 10.0–12.0.

[0349] (ii) The optimal temperature for the reaction is 20-37℃.

[0350] Obtain the crude ADC product.

[0351] (c) The crude ADC product obtained was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IEX019-03.

[0352] (d) The ADC products were analyzed by HIC, LC-MS and SEC HPLC to determine the average DAR value and SEC purity.

[0353]

[0354] Example 2.1.3 Preparation of IEX019-04

[0355]

[0356] Where q represents the average DAR, for example, 3-5, 3.2-4.8 or 3.0-4.0, such as 3.5 as measured in Table 2.

[0357] Where Ab is HB37A6.

[0358] This molecule was prepared according to the following method.

[0359] (a) Add the reducing agent solution (TCEP (Sigma, C4706), dissolved in water) to the antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer). After the addition is complete, place the reaction mixture in a shaker for 2-4 hours.

[0360] (i) The optimal concentration of antibody HB37A6 is 5-10 mg / mL

[0361] (ii) The optimal molar ratio of TCEP / mAb is 1.9 to 2.7.

[0362] (iii) The optimal temperature for the reduction reaction is 20-37℃.

[0363] (iv) The optimal pH for the reaction is generally between 6.5 and 8.0.

[0364] (b) An excess of the linker-payload MC-VC-PAB-MMAE (purchased from Levena Biopharma, SET0201) with a reactive group (maleimide-linked drug) was dissolved in the organic solvent DMSO and reacted with the reduced thiol group generated in step (a). The reaction mixture was placed in a shaker for 1–2 h.

[0365] (i) The optimal molar ratio of MC-VC-PAB-MMAE / mAb is 8.0 to 10.0.

[0366] (ii) The optimal temperature for the combination reaction is 20–37 °C.

[0367] (c) After the binding reaction is complete, add acetylcysteine ​​solution to terminate the reaction in step (b). After mixing, incubate at 20-25°C for 5-15 minutes.

[0368] (d) The crude ADC product obtained was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IEX019-04.

[0369] (e) The ADC products were analyzed using HIC and SEC-HPLC to determine the average DAR value and SEC purity.

[0370]

[0371] Example 2.1.4 Preparation of IEX019-05

[0372]

[0373] Where q represents the average DAR, for example, 3-5, 3.2-4.8, or 3.0-4.5, such as 3.3 as measured in Table 2.

[0374] Where Ab is HB37A6.

[0375] This molecule was prepared using the following method.

[0376] (a) Add the linker-payload SMCC-DM1 solution (purchased from Levena Biopharma, SET0101, dissolved in organic solvents such as DMSO) to the antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer). After addition, place the reaction mixture in a shaker for 2-5 hours.

[0377] (i) The optimal concentration of antibody HB37A6 is 5–10 mg / mL;

[0378] (ii) The optimal molar ratio of connector-payload / mAb is 5.5 to 6.5;

[0379] (iii) The optimal pH for the reaction is generally between 6.5 and 8.0;

[0380] (iv) The optimal temperature for the reaction is 20-37℃;

[0381] Obtain the crude ADC product.

[0382] (b) The crude ADC product obtained was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IEX019-05.

[0383] (c) The average DAR value and SEC purity of the ADC products were determined by ultraviolet spectrophotometry and SEC high-performance liquid chromatography.

[0384]

[0385] Example 2.1.5 Preparation of IEX019-06

[0386] The preparation process is similar to that of IEX019-02, except that the HB37A6 monoclonal antibody is replaced with the control antibody IgG.

[0387] Example 2.1.6 Preparation of IEX019-07

[0388] The preparation process is similar to that of IEX019-04, except that the HB37A6 monoclonal antibody is replaced with the control antibody IgG.

[0389] Information on all monoclonal antibodies and ADCs is summarized in Table 2.

[0390]

[0391] *The heavy chain of IgG is SEQ ID NO:21, and the light chain is SEQ ID NO:22.

[0392] Example 2.2 Cell binding assay of IEX019 molecule

[0393] To investigate whether small molecule conjugation alters the binding properties of IEX019-01 monoclonal antibody to target cells, we used the DANS-G cell line (hCLDN18.2 negative) and the DANS-G-hCLDN18.2 cell line prepared in Example 1 (overexpressing hCLDN18.2) to detect the affinity of IEX019-01 and IEX019-02 for the target site by flow cytometry. The experimental method was the same as in Example 1.5.

[0394] Neither IEX019-01 nor IEX019-02 bound to non-target cells (DAN-G), but showed strong affinity for DANG-hCLDN18.2, indicating that antibody binding depends on target expression specificity, and that conjugation with Exatecan does not affect antibody binding. Meanwhile, the control molecule IEX019-06 (a negative control of IgG, which was conjugated to Exatecan toxin using the same technique) did not bind to target cells (…). Figure 6 ).

[0395] Example 2.3 Internalization experiment of IEX019 molecules

[0396] Strong endocytosis is one of the key characteristics of ADC drugs. When an ADC binds to an antigen on the cell membrane, the ADC-antigen complex enters the cell through endocytosis, killing the target cell. Therefore, the endocytosis efficiency of an ADC is one of the important indicators determining its tumor-suppressive effect.

[0397] To assess the endocytosis efficiency of antibodies after conjugation with small molecule compounds, we used flow cytometry to detect the endocytosis of different IEX019 molecules on DANG-hCLDNA18.2 cells. After digestion of DANG-hCLDNA18.2 cells, the cell density was adjusted to 1*102. 5 Seed cells / well into a 96-well plate. Centrifuge at 500g for 3 min and discard the supernatant. Resuspend the cells in 100 μl of the target molecule (50 nM). Set up 5 replicates for each sample (i.e., endocytosis time: 0 h, 1 h, 2 h, 3 h, 4 h). Incubate on ice for 1 h. After 1 h, centrifuge at 500g for 3 min and discard the supernatant. Add 200 μl of FACS buffer (1% FBS, 1XPBS) to each well and wash twice. Transfer one set of samples to a new 96-well plate and incubate at 37°C for 4 h. Continue incubating the remaining samples on ice. Repeat the previous steps, incubating samples at 37°C for 3 h, 2 h, 1 h, and 0 h respectively. After each specific incubation time, centrifuge at 500g for 3 min and discard the supernatant. Add 100 μl of 1:400 diluted anti-hFC-PE antibody (SouthernBiotech) to each well and incubate on ice in the dark for 30 min. After secondary antibody incubation, centrifuge at 500g for 3 minutes and discard the supernatant. Add 200μl of FACS buffer to each well and wash twice. Finally, resuspend the cells in 100μl of PBS and perform analysis.

[0398] Experimental results are as follows Figure 7 The results showed that, with 0 h of incubation at 37°C as the zero point for endocytosis, after 2 h of incubation, all molecules reached the maximum endocytosis limit, approximately 60%, indicating that the ADC molecules designed and synthesized based on IEX019-01 maintained a strong endocytosis capacity consistent with that of monoclonal antibodies after binding to tumor cells.

[0399] Example 2.4: In vitro cell-killing effect of IEX019 molecule

[0400] The effects of ADCs on cell viability in various cell lines expressing different levels of hCLDNA8.2 were detected using the CellTiter-Glo (Promega, G9242) assay kit (Table 3).

[0401] Table 3. Expression levels of hCLDN18.2 in the cell lines used.

[0402]

[0403] Cells were digested with EDTA / Trypsin, adjusted in density, and evenly seeded into 96-well plates (Table 4). Specific concentrations of diluted IEX019 molecules (IEX019-02, IEX019-03, IEX019-04, initial dilution 100 nM, dilution factor 3) were added. Wells without added IEX019 molecules served as controls. The plates were incubated at 37°C for 5 days. After 5 days, 100 μl of CellTiter-Glo assay reagent was added to each well, and the plates were incubated at room temperature for 30 min. Cells were then analyzed using a microplate reader. Relative cell viability was calculated (relative cell viability = sample / control * 100%), and a curve was fitted using Graph Pad Prism 8.0.

[0404] As shown in Figure 8, the killing effect of ADC molecules on cell lines depends on the expression level of surface hCLDNA8.2. In hCLDNA8.2-negative DANG cells, IEX019 molecules had no significant effect on cell viability. Figure 8A) In cell lines expressing IEX019 at certain levels (NUGC-4, SNU620), the IEX019 molecule exhibited a certain degree of cell killing effect. Figure 8B) In the high-expression cell line DN-G-hCLDN18.2, the IEX019 molecule showed significant killing effects. Figure 8C This indicates that the IEX019 molecule has good selectivity and effectiveness.

[0405] Table 4. Cell seeding density

[0406]

[0407]

[0408] Example 2.5: Bystander killing effect

[0409] In the synthesis of ADC drugs, small molecule compounds can be linked to antibodies via cleavable linkers. After being endocytosed into the cell membrane, the linkers are cleaved, releasing the small molecule and killing the target cell. Following target cell death, the small molecule compound is released into the intercellular space, further killing non-target cells within a certain range; this effect is known as the bystander effect. Because tumor cells exhibit significant differences in target expression levels (tumor heterogeneity), the bystander effect is crucial for effectively killing tumor cells and inhibiting tumor growth.

[0410] This invention utilizes non-target cells DAN-G and target cells DANG-hCLDN18.2 to detect the bystander effect of the IEX019 molecule.

[0411] After digesting the cells with EDTA / Trypsin, adjusting the density, and preparing a six-well cell culture plate, 7.5 × 10⁶ DANG cells and 7.5 × 10⁶ DANG-hCLDN18.2 were added. 4 Two cell types were co-cultured. 200 μl of the test sample (IgG (SEQ ID NO:21, SEQ ID NO:22), IEX019-02, IEX019-05, IEX019-06, Exatecan (Macklin, E881532)) was added to each well, with a final concentration of 50 nM. Three replicates were set for each sample. The cell culture plates were placed in a 37°C incubator and cultured for 5 days. After 5 days, the culture supernatant was discarded, and the cells were washed with PBS and then digested with Trypsin-EDTA. All digested cells were collected and transferred to 96-well plates. Following the antibody incubation protocol for flow cytometry, the cells were incubated with the primary antibody (IEX019-01, 100 nM) and the secondary antibody (anti-hFc-PE, Southern Biotech) at 4°C for 1 h and 0.5 h, respectively. After washing with PBS, the cells were incubated with live / dead antibodies. Violet dye (Thermo, L34964) was diluted 1:1000, and 100 μl was added to each well. Cells were incubated at 4°C for 20 min. After washing with PBS, cells were resuspended in 100 μl of PBS and analyzed. Live / dead dye was used to separate the cell populations in each sample. IEX019-01 negative (i.e., hCLDN18.2 negative) cells were designated as DAN-G cells, and IEX019-01 positive (i.e., hCLDN18.2 positive) cells were designated as DAN-G-hCLDN18.2 cells. The number of each cell type in each sample was counted, and the relative cell viability of each cell type was calculated using the following equation. A curve was fitted using Graph Pad Prism 8.0.

[0412] DANG cell relative viability = (Number of DNAG cells in the sample group / Number of DNAG cells in the IgG group) * 100%

[0413] DANG-hCLDN18.2 cell relative viability = (Number of DNAG-18.2 cells in the sample group / Number of DNAG-18.2 cells in the IgG group) * 100%

[0414] Cell viability was set to 100% for the IgG group.

[0415] like Figure 9The results showed that the negative control sample IEX019-06 had no killing effect on either type of cell. The IEX019-05 molecule, by linking the DM1 toxin to the IEX019-01 monoclonal antibody via an insoluble linker, lacked the ability to kill surrounding non-target cells; therefore, it only killed DANG-hCLDNA8.2 cells and had no effect on DANG cells. Only IEX019-02 exhibited a significant bystander effect, capable of killing both target and non-target cells simultaneously.

[0416] Example 2.6: Antitumor efficacy of IEX019 molecule in DANN-G-hCLDN18.2 mouse xenograft model

[0417] To demonstrate the in vivo efficacy of the IEX019 molecule, we used DANG-hCLDN18.2 cells to inoculate CB-17-SCID mice and determined the antitumor efficacy of the antibody molecule of this invention. SPF-grade female CB-17-SCID mice (14-17g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), certificate number NO.110011201108225246, were used in the experiment.

[0418] DANG-hCLDN18.2 cells were passaged using standard methods for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) to prepare a cell concentration of 3×10⁻⁶ cells / mL. 6 Cell suspension of 1 cell / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB-17SCID mice to establish a DANG-hCLDN18.2 tumor-bearing mouse model.

[0419] On day 5 after tumor cell inoculation, the tumor volume of all mice ranged from 50.16 to 136.68 mm. 3 Mice were divided into serpentine groups (6 mice per group). The dosage and administration method are shown in Table 5. Administration was performed on day 5 post-inoculation. Tumor volume and body weight were monitored twice weekly. Figure 10A , 10B As shown, monitoring ended after 92 days.

[0420] The relative tumor inhibition rate (TGI%) was calculated on day 50 post-vaccination using the following formula:

[0421] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in control group before drug administration).

[0422] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers, and the tumor volume was calculated using the following formula:

[0423] V = L * W2 / 2. Weight was measured using an electronic balance.

[0424] Table 5. Experimental Design Table (Note: Single dose, 1 dose in total)

[0425] IEX019-01 (Control Group) 10mg / kg Single dose Intraperitoneal injection IEX019-02 (Treatment Group) 10mg / kg Single dose Intraperitoneal injection IEX019-03 (Treatment Group) 10mg / kg Single dose Intraperitoneal injection IEX019-04 (Treatment Group) 10mg / kg Single dose Intraperitoneal injection

[0426] The tumor inhibition rate results are shown in Table 6 and Figure 10A As shown: On day 50 post-vaccination, compared to IEX019-01 monoclonal antibody, IEX019-02 achieved a tumor inhibition rate of 103.60% after a single dose, significantly superior to IEX019-03 and IEX019-04, with tumor inhibition rates of 93.70% and 35.20%, respectively. After 82 days post-vaccination, 100% of the tumors in the IEX019-02 group showed complete regression. Simultaneously, mouse body weight was measured, and the results are as follows... Figure 10B The mice shown did not show a significant difference in body weight.

[0427] Table 6. Tumor suppression rate on day 50

[0428]

[0429] Example 2.7: Antitumor efficacy of IEX019 molecule in NUGC-4 mouse xenograft model

[0430] To demonstrate the in vivo efficacy of the IEX019 molecule, we used NUGC-4 cells to inoculate CB-17-SCID mice to determine the antitumor efficacy of the antibody molecule of this invention.

[0431] The experiment used SPF-grade female CB-17-SCID mice (14-17g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), certificate number NO.110011201109348141.

[0432] NUGC-4 cells were passaged using standard methods for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) to prepare a cell concentration of 3×10⁶ cells / year. 7 Cell suspension of 1 cell / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB-17SCID mice to establish the NUGC-4 tumor-bearing mouse model.

[0433] On day 5 after tumor cell inoculation, the tumor volume of all mice ranged from 72.25 to 140.50 mm. 3 The mice were divided into serpentine groups (6 mice per group). The dosage and administration method are shown in Table 7. The mice were given the drug on the 5th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 11A and 11B. Monitoring continued for 40 days.

[0434] The relative tumor inhibition rate (TGI%) was calculated on day 33 post-vaccination using the following formula:

[0435] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in control group before drug administration).

[0436] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers, and the tumor volume was calculated using the following formula:

[0437] V = L * W 2 / 2. Weight was measured using an electronic balance.

[0438] Table 7. Experimental Design Table (Note: Single dose, 1 dose in total)

[0439]

[0440] The tumor inhibition rate results are shown in Tables 8 and 11A: On day 33 post-inoculation, compared to the negative control IEX019-06, the single-dose tumor inhibition rates of IEX019-02 and IEX019-03 were 80.04% and 54.31%, respectively. Simultaneously, we measured the mouse body weight, and the results are as follows... Figure 11B The mice shown did not show a significant difference in body weight.

[0441] Table 8. Tumor suppression rate on day 33

[0442]

[0443] Example 2.8: Antitumor efficacy of IEX019 molecule in SNU620 mouse xenograft model

[0444] To demonstrate the in vivo efficacy of the IEX019 molecule, we used SNU620 cells to inoculate CB-17-SCID mice and determined the antitumor efficacy of the molecule (IEX019-02) of this invention. SPF-grade female CB-17-SCID mice (18-20g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), certificate number NO.110011211102179364, were used in the experiment.

[0445] SNU620 cells were passaged using standard methods for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) to prepare a cell concentration of 3×10⁶ cells / cells. 7 Cell suspension of 1 cell / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB-17SCID mice to establish the SNU620 tumor-bearing mouse model.

[0446] On day 7 after tumor cell inoculation, the tumor volume of all mice ranged from 58.1 to 117.3 mm. 3 Mice were divided into serpentine groups (6 mice per group). The dosage and administration method are shown in Table 9. Administration was performed on day 7 post-inoculation. Tumor volume and body weight were monitored twice weekly. Figure 12A , 12B As shown, monitoring ended after 39 days.

[0447] The relative tumor inhibition rate (TGI%) was calculated on day 39 post-vaccination using the following formula:

[0448] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in control group before drug administration).

[0449] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers, and the tumor volume was calculated using the following formula:

[0450] V = L * W 2 / 2. Weight was measured using an electronic balance.

[0451] Table 9. Experimental Design Table (Note: Single dose, 1 dose in total)

[0452]

[0453] The tumor inhibition rate results are shown in Table 10 and Figure 12A As shown: On day 39 post-inoculation, compared with hIgG, a single dose of IEX019-0210 mg / kg resulted in a tumor inhibition rate of 143.77%, with 100% complete tumor regression in mice. There was no significant difference in mouse body weight between the treatment groups and the hIgG control group. Figure 12B ).

[0454] Table 10. Tumor suppression rate on day 39

[0455]

[0456] Sequence information:

[0457]

[0458]

[0459]

Claims

1. An antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof, Equation (II) in Ab is an antibody or its antigen-binding fragment that binds to CLDN18.

2. L1 is ; E is 6-deoxy-2-acetamidogalactose, and it is linked to L1 through the C atom at position 6; Furthermore, GlcNAc is N-acetylglucosamine, and Fuc is fucose; D stands for Exatecan; r is 2; b is 0 or 1; x is 1; y is 1 or 2; Ab contains a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2, and 3, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7, and 8, respectively.

2. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4; and the light chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

9.

3. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

9.

4. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

9.

5. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the Ab further comprises a heavy chain constant region and / or a light chain constant region.

6. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein Ab is an IgG antibody.

7. The antibody-drug conjugate of any one of claims 1-6 or a pharmaceutically acceptable salt thereof, wherein the heavy chain constant region of the Ab is derived from IgG1, IgG2, IgG3 or IgG4.

8. The antibody-drug conjugate of claim 7 or a pharmaceutically acceptable salt thereof, wherein the heavy chain constant region of the Ab is derived from IgG1.

9. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the Ab comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:11; and the light chain comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

12.

10. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the Ab comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:11; and the light chain comprises the amino acid sequence of SEQ ID NO:

12.

11. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the Ab comprises a heavy chain and a light chain, wherein the heavy chain consists of the amino acid sequence of SEQ ID NO:11; and the light chain consists of the amino acid sequence of SEQ ID NO:

12.

12. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein Ab is a human antibody.

13. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein Ab is an antigen-binding fragment.

14. The antibody-drug conjugate of claim 13 or a pharmaceutically acceptable salt thereof, wherein the antigen-binding fragment is selected from Fv, Fab, Fab', Fab'-SH, F(ab')2 and scFv.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 1-6 and 8-14, wherein the GlcNAc linked to the Ab is present at the Asn297-glycosylation site of both heavy chains of the antibody.

16. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is selected from: IEX019-02 Wherein Ab is as defined in claim 10 or 11, Where q represents the average DAR, and q is 2-5.

17. The antibody-drug conjugate of claim 16 or a pharmaceutically acceptable salt thereof, wherein q is 3-5.

18. The antibody-drug conjugate of claim 17 or a pharmaceutically acceptable salt thereof, wherein q is 3.5-4.

5.

19. A pharmaceutical composition comprising an antibody-drug conjugate of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, and optionally one or more other therapeutic agents, and optionally pharmaceutical excipients.

20. The pharmaceutical composition of claim 19, wherein the other therapeutic agent is a chemotherapeutic agent, angiogenesis inhibitor, cytokine, cytotoxic agent, small molecule drug, or immunomodulator.

21. A pharmaceutical composition comprising an antibody-drug conjugate of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

22. Use of the antibody-drug conjugate of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 19 to 21, in the preparation of a medicament for treating a subject with cancer, wherein said cancer is gastric cancer, gastroesophageal junction cancer, or colorectal cancer.

23. The use of claim 22, wherein the cancer is colon cancer.

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