Claudin18.2-targeting antibody-drug conjugates

Abstract

The present invention relates to antibody-drug conjugates (ADCs) targeting Claudin18.2, as well as to compositions containing said molecules, and to therapeutic and diagnostic uses thereof.

Description

Antibody-drug conjugates targeting Claudin18.2

[0001] The present invention relates to antibody-drug conjugates (ADCs) targeting Claudin18.2 (CLDN18.2) and compositions containing the same. The present invention also relates to the therapeutic and diagnostic uses of these molecules.

[0002] Background of the Invention

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

[0004] Claudin-18 isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. Its expression in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa, but is absent in the gastric stem cell compartment. CLDN18.2 is expressed in a significant proportion of primary gastric cancers, and its expression levels are retained in gastric metastatic cancer tissues. In addition to gastric cancer, CLDN18.2 expression has also been found in pancreatic cancer, making it an ideal target for the treatment of these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as a new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017). https: / / doi.org / 10.1186 / s13045-017-0473-4).

[0005] Regarding gastric cancer, in 2014, there were approximately 410,000 new cases and 290,000 deaths in China, accounting for nearly half of the global incidence and mortality, and the incidence is still on the rise. However, there is a significant unmet need for clinical cancer treatment, making the development of drugs targeting Claudin18.2 crucial.

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

[0007] Due to technological advances made during the past few years, the field of antibody-drug conjugates (ADCs) for the treatment of cancer has recently experienced an increasing development activity by pharmaceutical companies aimed at solving the problems they initially presented with regard to immunogenicity, affinity, specificity, undesirable toxicity, yield, half-life, etc.

[0008] Despite some progress, there is still a need for other therapeutic strategies for treating tumors, and for components used in such therapeutic strategies, in particular, there is a need for anti-Claudin18.2 antibodies with high affinity, good specificity and / or low immunogenicity risk, and for ADC molecules with higher activity, low toxicity, long half-life, good specificity or affinity, and / or good half-life or druggability.

[0009] SUMMARY OF THE INVENTION

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

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

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

[0013] (3) There is a significant bystander effect;

[0014] (4) High anti-tumor efficacy;

[0015] (5) Low toxicity;

[0016] (6) Good stability;

[0017] (7) It has good drugability.

[0018] In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface; in some embodiments, the target cell is a CHO cell or 293 cell expressing CLDN18.2, such as a CHO-S cell or HEK293 cell; in some embodiments, the target cell is a cancer cell expressing CLDN18.2, such as a cell naturally expressing CLDN18.2 or artificially transfected to express CLDN18.2, or artificially transfected to have increased expression levels of CLDN18.2, such as a gastric cancer cell line or a pancreatic cancer cell line or a colon cancer or colorectal cancer cell line expressing CLDN18.2. In some embodiments, the target cell is a cell line with moderate hCLDN18.2 expression levels, such as NUGC-4, SNU620. In some embodiments, the target cell is a cell line with high expression levels, such as a DAN-G cell overexpressing hCLDN18.2. Description of the drawings:

[0019] FIG1 shows that the HB37A6 antibody specifically binds to CLDN18.2 on the cell surface.

[0020] FIG2 shows that the HB37A6 antibody does not bind to cell surface CLDN18.1.

[0021] FIG3 shows the binding of the HB37A6 antibody to the gastric cancer cell line NUGC-4, the gastric cancer cell line KATO III-hCLDN18.2, and the pancreatic cancer cell line DAN-G-hCLDN18.2.

[0022] FIG4 shows the anti-tumor effect of HB37A6 antibody in a pancreatic cancer mouse model.

[0023] FIG5 shows the anti-tumor effect of HB37A6 antibody in a gastric cancer mouse model.

[0024] FIG6 shows the cell binding effect of IEX019 molecules.

[0025] FIG7 shows the endocytosis of IEX019 molecules in DANG-hCLDN18.2 cells.

[0026] Figure 8 shows the killing effect of IEX019 molecules on cell lines with low hCLDN18.2 expression (Figure 8A), the killing effect of IEX019 molecules on cell lines with medium hCLDNA8.2 expression levels (Figure 8B), and the killing effect of IEX019 molecules on cell lines with high hCLDN18.2 expression (Figure 8C).

[0027] FIG9 shows the bystander effect of the IEX019 molecule.

[0028] FIG10 shows the tumor suppressive efficacy ( FIG10A ) and body weight changes ( FIG10B ) of the IEX019 molecule in mice.

[0029] FIG11 shows the tumor suppressive efficacy ( FIG11A ) and body weight changes ( FIG11B ) of the IEX019 molecule in mice.

[0030] FIG12 shows the tumor suppressive efficacy ( FIG12A ) and body weight changes ( FIG12B ) of the IEX019 molecule in mice.

[0031] Detailed Description of the Invention

[0032] I. Definition

[0033] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0034] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0035] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0036] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0037] As used herein, the terms "comprising" or "including" are intended to include the recited 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 indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0038] The term "claudin" or "cldn" as used herein refers to the most important scaffold protein that determines the structure of tight junctions between cells. It participates in adherens junctions and plays an important role in the metastasis and invasion of tumor cells. Claudin proteins are widely present in mammalian epithelial and endothelial cells, and their distribution is mainly on the lateral surface of epithelial cells and on the basal cell plasma membrane. Different claudin proteins have their own specific expression in different tissues. Among them, the claudin18 (cldn18) gene is located at 3q22.3, has a molecular weight of 24kDa, contains 261 amino acid residues, and is a member of the claudins superfamily. Its protein structure includes two extracellular loops and four transmembrane regions. The two isoforms of human CLDN18, or Claudin18, are Claudin18.1 (UniProt ID: P56856-1) and Claudin18.2 (UniProt ID: P56856-2). Their primary sequences differ only in the amino acid residues at certain positions from the N-terminal signal peptide to the extracellular loop 1 (Loop 1). Specifically, in the extracellular loop 1, CLDN18.1 and CLDN18.2 differ by only eight amino acids. The two CLDN18 isoforms also share very high sequence homology across species. The extracellular loop 1 of CLDN18.2 is completely identical in sequence across species, including humans, mice, and macaques, and the homology between human and mouse CLDN18.2 proteins reaches 84%, indicating extreme sequence conservation of the CLDN18.2 protein (O. Tureci et al., Gene 481:83-92, 2011). CLDN18.2 or any variants and isoforms thereof can be isolated from cells or tissues naturally expressing them, or recombinantly produced using techniques well known in the art and / or those described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0039] As used herein, the terms "anti-CLDN18.2 antibody," "anti-CLDN18.2," "CLDN18.2 antibody," "antibody that binds to CLDN18.2," or "antibody that specifically binds to CLDN18.2" refer to antibodies that are capable of binding to (human) CLDN18.2 with sufficient affinity so that the antibody can be used as a therapeutic agent targeting (human) CLDN18.2. In one embodiment, the (human) CLDN18.2 antibody binds to (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, the (human) CLDN18.2 antibody does not bind to CLDN18.1. In one embodiment, the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 without binding to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), 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 by various means, such as anti-CLDN18.2 antibodies. For example, the binding strength of cells "highly expressing CLDN18.2" to anti-CLDN18.2 antibodies (e.g., as determined by FACS) can be 500, 600, 700, 800, 900, or preferably 1000 times greater, such as 1100 times, 1200 times, 1300 times, 1400 times greater, or even higher, than the binding strength of anti-CLDN18.2 antibodies to cells that do not express CLDN18.2. For example, cells that "moderately express CLDN18.2" can bind to an anti-CLDN18.2 antibody with an intensity (e.g., as determined by FACS) that is 5- to 500-fold, such as 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or more, but no more than 500-fold, as strongly as the anti-CLDN18.2 antibody binds to cells that do not express CLDN18.2.

[0041] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules having the structure of a natural immunoglobulin molecule. In the case of a conventional four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of a heavy chain antibody having only heavy chains and lacking light chains, a full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds.

[0042] For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains: CH1, CH2, and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) 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 from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

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

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

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

[0046] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs 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 one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

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

[0048]

[0049] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0050] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0051] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position 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 CDRs of the antibodies of the present invention are determined according to the following rules:

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

[0054] In one embodiment, the light chain variable region CDRs of an antibody of the invention are determined according to the Kabat rules.

[0055] In one embodiment, the heavy chain variable region CDRs of the antibodies of the invention are 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 CDRs are determined according to the Kabat rule.

[0056] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present 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 for different antigens) have different CDRs (under the same assignment system). However, although CDRs are different between antibodies, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As will be appreciated by those skilled in the art, the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0058] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Herein, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases.

[0059] An "IgG-type antibody" refers to an antibody whose heavy chain constant region belongs to the IgG type. All antibodies of the same type have the same heavy chain constant region, while antibodies of different types differ in their heavy chain constant regions. For example, an antibody of the IgG4 type refers to an antibody whose heavy chain constant region is derived from IgG4, and an antibody of the IgG1 type refers to an antibody whose heavy chain constant region is derived from IgG1.

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

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

[0062] The general term "sugar" used herein refers to monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" used herein refers to a derivative of a monosaccharide, i.e., a monosaccharide comprising a substituent and / or a functional group. Examples of sugar derivatives include amino sugars and sugar acids, such as glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). Examples of sugar derivatives also include compounds represented herein as E(A)x, where E is a sugar or sugar derivative, and where E includes x functional groups A.

[0063] A core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as being bonded to the GlcNAc of the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. The core-GlcNAc substituent may be present at the native glycosylation site of the antibody, but it may also be introduced at a different site of the antibody. Herein, a core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fucα1-6)GlcNAc substituent, also referred to as GlcNAc(Fuc).

[0064] "Glycosylation modification" refers to the process of modifying the sugar chains of an antibody through glycosylation engineering. The glycosylation of an antibody can be further modified for various purposes to obtain an antibody with a new glycosylation, for example, to remove glycosylation in order to eliminate FcγR affinity and complement binding / effector function, to reduce fucose and sialic acid groups and increase bisecting N-acetylglucosamine, galactose and mannose in order to enhance Fc-mediated ADCC and CDC effects. Methods of glycosylation modification are known in the art, for example, by changing the glycosylation sites of an antibody to increase or decrease the sugar chains on the antibody surface, or by modifying the sugar chains in vitro through chemical or enzymatic methods, or by modifying the glycosylation pathway of the expression system (e.g., composed of enzymes such as glycosidases and glycosyltransferases) to catalyze the glycosylation of the antibody, and the glycosylation of the antibody can also be modified by affecting cell culture conditions. In some embodiments, the "glycosylation modification" of the present invention is performed by modifying the sugar chains by enzymatic methods in vitro. Preferably, the glycosylation modification of the present invention is performed by modifying the sugar chain using a glycosidase (such as an endoglycosidase or a glycosyltransferase).

[0065] The antibodies with engineered glycosylation of the present invention refer to antibodies with an engineered glycosylation pattern compared to an antibody with a native glycosylation pattern. Preferably, the antibodies with engineered glycosylation refer to antibodies obtained after enzymatic modification of the sugar chains of an antibody expressed in an expression system (e.g., a mammalian cell) in vitro (e.g., modification of the sugar chains by a glycosidase (e.g., an endoglycosidase or a glycosyltransferase)). More preferably, the antibodies with engineered glycosylation of the present invention refer to antibodies comprising a core -GlcNAc and a sugar derivative E(A)x attached thereto, wherein the GlcNAc is bonded to the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, the fucose is typically attached to C6 of the -GlcNAc substituent via α-1,6. The fucosylated-GlcNAc substituent refers to core-GlcNAc(Fuc), and the fucosylated GlcNAc-E(A)x substituent refers to GlcNAc(Fuc)-E(A)x.

[0066] The term "site-specific conjugation" as used herein refers to conjugation in which a drug / active substance is specifically linked to a specific site of an antibody via a linker.

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

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

[0069] The term "cycloalkyl" is a cyclic alkyl group, that is to say, a monovalent, saturated or unsaturated hydrocarbon group with a ring structure. Cycloalkyl includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups with a ring structure. Cycloalkyl groups can comprise 3 or more, for example 3-18, 3-10 or 3-8 carbon atoms in the ring, and usually according to the present invention, comprise 3 to 6 atoms. The example of cycloalkyl groups includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0070] As used herein, the term "heteroaryl" refers to a 5-20 membered (e.g., 5-14 membered, 5-8 membered, 5-6 membered) monocyclic, bicyclic, or fused polycyclic ring system containing 1-8 heteroatoms selected from N, O, or S. Preferably, the heteroaryl is a 5-10 membered ring system. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 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-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-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 is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed between the ADC conjugates of the present invention and an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. 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 with organic bases containing an N group.

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

[0073] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0074] The term "drug:antibody ratio" or "DAR" refers to the ratio of the small molecule drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety. In some embodiments described herein, the DAR can be determined by p and r in Formula I, for example, the DAR can be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the overall ratio of the small molecule drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), and this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 20, e.g., 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 , 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, and ranges having 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 (e.g., immunosuppressants).

[0076] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0077] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0078] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

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

[0080] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0081] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0082] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0083] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0084] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

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

[0086] An "isolated" antibody or other molecule (e.g., ADC molecule) is one that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody or ADC molecule is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0087] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0088] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and hematological tumors.

[0089] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include gastric cancer, pancreatic cancer, or gastroesophageal junction cancer, including metastatic forms of those cancers.

[0090] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0091] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0092] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0093] The term "pharmaceutical combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an ADC molecule of the present invention, and (ii) another therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or sequentially at equal or different time intervals, as separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient. In some embodiments, the ADC molecule of the present invention and the other therapeutic agent used in the pharmaceutical combination are administered at levels no greater than when they are 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. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the components can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0094] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0095] As used herein, "treat," ...

[0096] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0097] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. 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 a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0099] II. Antibody-Drug Conjugates

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

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

[0102] or a pharmaceutically acceptable salt or solvate thereof,

[0103] in:

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

[0105] L is a linker;

[0106] D is a drug, including a prodrug, preferably an anti-tumor compound; and

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

[0108] r is 1-5, such as 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 (such as 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, the Ab comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3.

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

[0113] In some embodiments, the Ab comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0114] In some aspects, Ab comprises a heavy chain variable region (VH). In some aspects, Ab comprises a light chain variable region (VL). In some aspects, Ab comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0115] In some embodiments, VH

[0116] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4; or

[0117] (ii) comprises or consists 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, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0119] In some embodiments, VL

[0120] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; or

[0121] (ii) comprises or consists 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, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0123] In some embodiments, the three complementarity determining regions (HCDRs) of the present invention, HCDR1, HCDR2 and HCDR3 from VH are

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

[0125] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three HCDR regions relative to the sequence of (i).

[0126] In some embodiments, the three complementarity determining regions (LCDRs) from VL of the present invention, LCDR1, LCDR2 and LCDR3 are

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

[0128] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three LCDR regions relative to the sequence of (i).

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

[0130] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, or HCDR2 comprises an amino acid sequence having one, two or three 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 consists of the amino acid sequence of SEQ ID NO: 3, or HCDR3 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 consists of the amino acid sequence of SEQ ID NO: 6, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6.

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

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

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

[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 light chain constant region LC of the antibody of the present invention is the lambda or kappa light chain constant region.

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

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

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

[0140] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 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: 5, or consists of said amino acid sequence.

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

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

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

[0144] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 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: 10, or consists of said amino acid sequence.

[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 a heavy chain and a light chain.

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

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

[0150] VH comprising or consisting of the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or

[0151] VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% identity thereto.

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

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

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

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

[0157] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 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: 11, or consists of said amino acid sequence.

[0158] In some embodiments, the light chain of Ab in Formula (I)

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

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

[0161] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 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: 12, or consists of said amino acid sequence.

[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 by SEQ ID NO:11, and the amino acid sequence of the light chain is shown by SEQ ID NO:12.

[0163] In one embodiment of the present invention, the amino acid changes described herein include amino acid replacement, insertion or deletion. Preferably, the amino acid changes described herein are amino acid replacements, preferably conservative replacements. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid changes described herein occur in the Fc region of the antibody heavy chain constant region.

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

[0165] The antibody Ab in formula (I) of the present invention may be an antibody with modified glycosylation. In some embodiments, the antibody is an antibody obtained after enzymatic modification of the sugar chain in vitro (e.g., modification of the sugar chain by a glycosidase (e.g., endoglycosidase or glycosyltransferase)). In some embodiments, the antibody with modified glycosylation refers to an antibody in which the sugar chain at the antibody glycosylation site is modified from a non-uniform structure N sugar chain to a single structure N sugar chain with a reactive group (e.g., any reactive group that can react with a linker moiety, such as an azide group, a ketone group, and an alkyne 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 for engineering antibody glycosylation suitable for the present invention are described, for example, in PCT / NL2013 / 050744, PCT / EP2016 / 059194 or PCT / EP2017 / 052792, which are incorporated herein in their entirety.

[0167] In a preferred embodiment, the antibody of the invention having engineered glycosylation is a GlcNAc-E(A) x An antibody comprising a substituent, wherein GlcNAc is N-acetylglucosamine, wherein E(A)x is a sugar derivative comprising x functional groups A, wherein A is independently selected from azide, keto, and alkynyl, and x is 1, 2, 3, or 4; wherein the GlcNAc-E(A)x substituent is bonded to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent, wherein the N-acetylglucosamine is optionally fucosylated. In the case where the N-acetylglucosamine is fucosylated, it is bonded to fucose (Fuc) via C6.

[0168] In one embodiment, the antibody of the present invention with engineered glycosylation is an antibody of formula (III), wherein Ab represents antibody, GlcNAc represents N-acetylglucosamine, Fuc represents fucose, b represents 0 or 1, y represents 1 to 20, and E(A)x represents a sugar derivative comprising x functional groups A, wherein A represents an azide group, a keto group, and an alkyne group, and x represents 1, 2, 3, or 4. In a preferred embodiment, y represents 1 to 10, more preferably, y represents 1, 2, 3, 4, 5, 6, 7, or 8, even more preferably, y represents 1, 2, 3, or 4, and most preferably, y represents 1 or 2.

[0169]

[0170] The sugar derivative E(A)x in the GlcNAc-E(A)x substituent of the antibody with engineered glycosylation can be, for example, bonded 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 bonded to the antibody via C1, preferably to the amide nitrogen atom of the side chain of asparagine of the antibody (GlcNAcβ1-Asn) via an N-glycosidic bond. The GlcNAc on the GlcNAc-E(A)x substituent is optionally fucosylated. Accordingly, the GlcNAc-E in the antibody-drug conjugate, when present, can also be linked as described above.

[0171] In a preferred embodiment, the functional group A is an azido group. When A is an azido 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 the functional group A corresponds to the position at which the sugar containing A is attached to the linker.

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

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

[0174] It should be understood that, unless otherwise stated, the above descriptions and illustrations of sugars (including but not limited to the glycosidic bond linkage of GlcNAc-E(A)x) are also applicable to the corresponding sugars in the conjugate of formula (II) below, such as the sugar linkage therein. Those skilled in the art will appreciate that, although in some embodiments, a reactive group (e.g., functional group A) is linked to the sugar chain of the antibody during antibody glycosylation modification, and thus an "antibody with modified glycosylation" is defined as an antibody comprising the reactive group, upon formation of the antibody-drug conjugate, the reactive group may react with the linker moiety to form a new group with the linker moiety, and thus, in the antibody-drug conjugate, the new group is considered to be part of the linker.

[0175] Preferably, the antibody of the present invention is a monoclonal antibody, more preferably an IgG antibody (eg, a four-chain IgG antibody), most preferably an IgG1 antibody. In one embodiment, the antibody is a complete antibody.

[0176] When the modified antibody is a complete antibody, the antibody preferably comprises two or more, more preferably two, GlcNAc-E(A)x substituents, wherein the GlcNAc-E(A)x substituents are optionally fucosylated. However, if the modified antibody is an antibody fragment, such as a Fab or Fc fragment, the antibody may have only one GlcNAc-E(A)x substituent, which is optionally fucosylated. The GlcNAc-E(A)x substituent may be located at any position in the antibody, provided that the substituent does not interfere with the binding of the antigen to the antigen-binding site of the antibody. In one embodiment, the GlcNAc-E(A)x substituent is located in the Fc domain of the antibody, more preferably, in the CH2 domain.

[0177] As described above, the antibody of the present invention having engineered glycosylation comprises 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 native conserved N-glycosylation site) of the antibody, such as a glycosylation site in the Fc region (more preferably, a CH2 domain). In 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 native conserved N-glycosylation site) of the IgG antibody. In another preferred embodiment, the native site is the Asn297-glycosylation site of the IgG antibody. The Asn297-glycosylation site is present 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 site 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 to the anti-human CLDN18.2 of the present invention. Preferably, the linker can achieve site-specific coupling of the ADC.

[0180] In some embodiments, the linker suitable for use in the present invention can be any linker that can achieve conjugation of an antibody to a drug. In some embodiments, the linker can be a linker used in a technology that can achieve site-specific conjugation.

[0181] In a preferred embodiment, the linker of the present invention is a linker connected to an antibody oligosaccharide. As defined herein, a "linker connected to an antibody oligosaccharide" refers to any linker that is connected to a reactive group on a sugar chain at an antibody glycosylation site to couple the antibody to a drug. The sugar chain on the antibody glycosylation site is generally an N-sugar chain, and is usually modified to transform the non-uniform structure N-sugar chain into a single structure N-sugar chain with a reactive group, and then the reactive group carried by the sugar chain is connected to the "linker" to achieve site-specific coupling of the drug to the antibody to obtain an antibody-drug conjugate. In a preferred embodiment, the N-glycosylation site of the antibody is the antibody Fc domain, preferably a conserved N-glycosylation site on the CH2 domain, such as Asn297. Thus, in one embodiment, the "linker attached to an antibody oligosaccharide" of the present invention is particularly any linker that can achieve site-specific coupling with a reactive group on an N-sugar chain at a conserved N-glycosylation site (e.g., Asn297) on an antibody Fc domain, such as the linker described in PCT / NL2013 / 050744 or the linker of PCT / EP2021 / 075401, which are incorporated herein by reference in their entirety. In one embodiment, the reactive group of the present invention is an azido group, a keto group, or an alkynyl group. In one embodiment, the linker of the present invention is a linker comprising an alkynyl group. In one embodiment, the reactive group of the present invention is an azido group, a keto group, or an alkynyl group, preferably an azido group, and the linker of the present invention is a linker comprising an alkynyl group. When the present invention refers to such linkers, since a new group is formed after the reactive group reacts with the group of the linker, the group formed after the reaction of the reactive group in the antibody-drug conjugate can also be defined as part of the "linker", for example, as shown in formula (II) of the present invention.

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

[0183] D in formula (I) of the present invention can be any antitumor compound, without particular limitation, as long as it has an antitumor effect and has a substituent or partial structure that can be attached to the linker structure. For example, the antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For antitumor compounds, a portion or all of the linker can preferably be cleaved within tumor cells, releasing the antitumor compound portion, thereby exhibiting an antitumor effect. When the linker is cleaved from the drug-attached portion, the antitumor compound is released in its unmodified structure, allowing it to exert its original antitumor effect.

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

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

[0186]

[0187] Ab represents an antibody as defined herein,

[0188] L1 is the connector;

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

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

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

[0192] b is 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, 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, most preferably GalNAc, such as 6-deoxy-2-acetamidogalactose, which is preferably linked to L1 through the 6-C atom.

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

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

[0199]

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

[0201] R1 is independently selected from hydrogen, halogen, -OR2, -NO2, -CN, -S(O)2R2, C1-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, and wherein the alkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, arylalkyl and heteroarylalkyl are optionally substituted, wherein two substituents R1 can be linked together to form a fused, bridged or spiro cycloalkyl or a fused or spiro 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, 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;

[0203] c is 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 in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by a heteroatom selected from O, N, and S, with the proviso that the O, N, and / or S are not directly attached to each other;

[0208] Each L4 is independently a cleavable linker.

[0209] It should be understood that when m is 0, it means that Q is absent and the relevant nitrogen atom of the triazole is directly linked to the E moiety via a covalent single bond.

[0210] In one embodiment, L4 are each independently

[0211] wherein 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 embodiment, L4 are each independently

[0214] In one embodiment, wherein -L1- has the structure

[0215]

[0216] In one embodiment, the antibody-drug conjugate has an average DAR of 1-15, eg, 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] Among them, 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 ADC molecules of the present invention

[0224] In one aspect, the present invention provides a method for preparing an antibody having modified glycosylation, the method comprising

[0225] (1) Preparation of glycosylated antibodies: culturing a host cell comprising a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium) to obtain an antibody comprising a core N-acetylglucosamine substituent (core-GlcNAc substituent), wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated;

[0226] (2) preparing a trimmed antibody: deglycosylation of the antibody prepared in step (1) in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated;

[0227] (3) contacting the trimmed antibody obtained in (2) with a compound of formula E(A)xP in the presence of a suitable catalyst to obtain an antibody comprising a GlcNAc-E(A)x substituent, wherein the GlcNAc-E(A)x substituent is bonded to the antibody via the C1 of the N-acetylglucosamine of the GlcNAc-E(A)x substituent; wherein the catalyst is a glycosyltransferase, wherein P is selected from uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP).

[0228] To perform step (1), nucleic acid encoding an antibody (e.g., an antibody described above, e.g., any one polypeptide chain and / or multiple polypeptide chains) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

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

[0230] The glycosyltransferase in step (3) is preferably a glycosyltransferase or a glycosyltransferase 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 from the phylum Nematoda, preferably from the class Chromadorea or the class Secernentea, or from the phylum Arthropoda, preferably from the class Insecta. More preferably, β-(1,4)-GalNAcT enzymes are from Caenorhabditis elegans, Ascaris suum, Trichoplusia ni, Drosophila melanogaster, saprophytic fruit nematodes, Caeno rhabditis briggsae, Wuchereria elegans, Loa filariae, Pseudomonas spp., Coptotermes spp., Camponotus floridianus, Crassostrea gigas, and Platyrrhineia rubripes. Preferably, the glycosyltransferase suitable for step (3) is the Trichoplusia ni β-(1,4)-GalNAcT enzyme (e.g., His-TnGalNacT) named TnGalNAcT disclosed in PCT / EP2016 / 059194.

[0231] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. "ADC" in the present invention is defined as an antibody coupled to a biologically and / or pharmaceutically active active substance (D) 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) as defined herein. Preferably, the linker-active substance is site-specifically coupled to the antibody.

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

[0233]

[0234] The meanings of the variables or symbols are as defined above;

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

[0236] Generating an antibody-drug conjugate of (Formula II)

[0237]

[0238] The meanings of the variables and symbols are as defined above.

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

[0240]

[0241] The variables are as defined above.

[0242] IV. Pharmaceutical Compositions

[0243] In some embodiments, the present invention provides a composition comprising any ADC molecule described herein or a pharmaceutically acceptable salt thereof, preferably a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., 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 (including pharmaceutical compositions) comprising the ADC molecules of the present invention or pharmaceutically acceptable salts thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0245] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0246] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0247] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0248] A medicament comprising the ADC described herein can be prepared by mixing the ADC molecule of the 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 composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication being treated, preferably having those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists). The active ingredient is suitably present in combination in an amount effective for the purpose.

[0250] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0251] VII. Pharmaceutical Combinations and Kits

[0252] In some embodiments, the present invention also provides a drug combination or drug combination product comprising an 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), etc.).

[0253] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0254] In one embodiment, the kit of parts of the present invention comprises in 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 an additional therapeutic agent.

[0257] VIII. Use and Methods

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

[0259] In some embodiments, the tumor (eg, cancer) patient has (eg, elevated levels, such as nucleic acid or protein levels) CLDN18.2. In some embodiments, the patient's tumor cells express CLDN18.2, such as moderate expression of CLDN18.2, preferably high expression of CLDN18.2.

[0260] In some embodiments, the tumor, such as cancer, includes solid tumors and blood tumors and metastatic lesions. In one embodiment, the example of a solid tumor includes a malignant tumor. The cancer can be in the early, middle or late stages or be a metastatic cancer.

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

[0262] In some embodiments, the tumor is an immune evasion tumor.

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

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

[0265] In other aspects, the present invention provides the use of an ADC molecule or a pharmaceutical composition or a pharmaceutical combination or a kit in the production or preparation of a medicament for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0266] In some embodiments, the ADC molecule or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of a disorder and / or symptoms associated with a disorder.

[0267] In some embodiments, the ADC molecules or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, such as for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0268] In some embodiments, the treatment modality includes surgery; radiation therapy, localized or focused irradiation, and the like.

[0269] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist).

[0270] Exemplary additional antibodies include antibodies that specifically bind to immune checkpoints.

[0271] Combination therapies of the invention encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the ADC molecules of the invention can occur prior to, concurrently with, and / or after administration of the other therapeutic and / or agents.

[0272] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscularly, intraocularly, intraarterially, intraportally or intralesionally; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

[0273] The composition can also be administered topically via an implant membrane, sponge, or another suitable material onto which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implant device is used, the device can be implanted in any suitable tissue or organ and the desired molecule can be delivered via diffusion, a timed-release bolus, or continuous administration.

[0274] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may 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 overexpressing cell lines

[0277] According to the manufacturer's instructions, use A cell line stably expressing human Claudin18.2 (abbreviated as CLDN18.2, hereinafter referred to as "CLDN18.2") was constructed using a kit (Invitrogen, A1369601). The full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was first constructed into the pCHO1.0 vector to construct a plasmid. The constructed plasmid was then transfected into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) using chemical transfection and electrofection, respectively. The transfected cells underwent two rounds of pressure screening to obtain cell pools expressing CLDN18.2. Then, cells highly expressing CLDN18.2 were sorted using a flow cytometer (MoFlo XDP, Beckman Coulter), and monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2 stably expressing CLDN18.2 were obtained by dilution method.

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

[0279] According to the manufacturer's instructions, use A cell line stably expressing human Claudin18.1 (abbreviated as CLDN18.1, hereinafter referred to as "CLDN18.1") was constructed using a kit (Invitrogen, A1369601). The full-length human CLDN18.1 gene (UniProt ID: P56856-1) was first constructed into the pCHO1.0 vector (Invitrogen, A1369601) to generate a plasmid. This plasmid was then chemically transfected into CHO-S cells (Invitrogen, A1369601). The transfected cells underwent two rounds of pressure selection to obtain pools of cells expressing CLDN18.1. Cells expressing high levels of CLDN18.1 were then isolated using a flow cytometer (MoFlo XDP, Beckman Coulter), and a monoclonal cell line, CHO-hCLDN18.1, stably expressing CLDN18.1, was obtained by dilution.

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

[0281] The full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the pWPT-GFP vector (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. The culture supernatants were collected after 48 and 72 hours of culture and concentrated using PEG8000. The concentrated virus was used to transfect pancreatic cancer DAN-G cells (CLS Cell Lines Service GmbH, 300162) and gastric cancer KATO III cells (ATCC, HTB-103). Then, cells expressing CLDN18.2 were sorted using a flow cytometer (MoFlo XDP, Beckman Coulter) to obtain the tumor cell lines DAN-G-hCLDN18.2 and KATO III-hCLDN18.2 stably transfected with CLDN18.2.

[0282] Example 1.2. Generation of CLDN18.2 Monoclonal Clones

[0283] The present invention uses hybridoma technology to immunize mice with H2L2 fully human antibody transgenic mice (purchased from Harbour BioMed) using the cells (CHO-hCLDN18.2) obtained in Example 1. Splenocytes from the mice are then electrofused with myeloma cells. The supernatant is then collected and screened using flow cytometry (FACS) to identify hybridoma cells that specifically express anti-CLDN18.2 antibodies and secrete antibodies that do not bind to CLDN18.1. The cells to be tested (HEK293-hCLDN18.2) obtained in Example 1 are counted and diluted to 1×10 6 Cells were plated at 100 μl / well in a U-bottom 96-well plate. The culture supernatant from the hybridoma 96-well plate was centrifuged at 500 g for 5 minutes, and the cell culture medium was removed. The supernatant from the hybridoma 96-well plate was added to the U-bottom plate and the cells were resuspended. 100 μl was added to each well and the cells were incubated on ice for 30 minutes. The supernatant was removed at 500 g for 5 minutes, and the cells were washed once with PBS. 100 μl of FITC-conjugated secondary antibody against mouse Fab (1:500 dilution in PBS) was added to each well. For the positive control antibody, 100 μl of FITC-conjugated secondary antibody against human Fab was added to each well. The cells were incubated on ice for 30 minutes in the dark. The supernatant was removed at 500 g for 5 minutes, and the cells were washed once with PBS. The cells were resuspended in 50 μl of 1× PBS and analyzed on a FACS machine. Positive clones were rescreened against CHO-hCLDN18.1 using the same method as above. 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 in HEK293 cells (Invitrogen, A14527) as full-length monoclonal antibodies.

[0286] First, expression vectors were constructed, with the heavy and light chain variable regions of HB37A6 and a control antibody (see sequence listing) placed at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 5) and light chain kappa constant region (SEQ ID NO: 10), respectively. These vectors were then incorporated into a pcDNA3.1 expression vector with an N-terminal signal peptide to generate the light and heavy chain expression vectors. The resulting 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 exchanged into PBS (Gibco, 70011-044). The concentration was determined 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 / ml. The cell density on the day of transfection was approximately 3×10 6 cells / ml. Take 1 / 10 of the final volume of Opti-MEM medium (Gibco, 31985-070) as the transfection buffer, add the appropriate plasmid to the transfected cells at 1.0 μg / ml, and mix well. Add the appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI in 293F cells is 1:3), mix well, and incubate at room temperature for 20 minutes to obtain a DNA / PEI mixture. Slowly add the DNA / PEI mixture to the cells, gently shaking the shaker while adding, and then place it in a 36.5°C, 8% CO2 incubator for culture. After seven days, obtain the cell feed solution and collect the cell supernatant for purification.

[0289] The Protein A column used for purification (Hitrap Mabselect Sure, GE, 11-0034-95) was treated with 0.1M NaOH for 2 hours. The glass bottles and other components were washed with distilled water and then dry-baked at 180°C for 4 hours. Prior to purification, the collected cell suspension 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 applied 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 eluate was collected. 80 μl of 2 M Tris-HCl was added to every 1 ml of eluate. The collected antibody was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the concentration was determined.

[0290] Example 1.4: Determination of CLDN18.2 Antibody Affinity by SPR

[0291] Surface plasmon resonance (SPR) was used to determine the equilibrium dissociation constant (K) of HB37A6 binding to human CLDN18.2. D ). According to the manufacturer's instructions, human Claudin18.2 (GenScrip, P50251802) was coupled to the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino coupling kit (GE Healthcare, BR-1006-33). After coupling, 1 M ethanolamine was injected to block the remaining active sites. According to the manufacturer's instructions, the affinity and kinetic constants were obtained by detecting the binding and dissociation between the antigen on the chip surface and the antibody in the mobile phase using Biacore (GE Healthcare, T200). The gradient diluted antibody (0-100 nM) was passed through the chip surface in an order from low concentration to high concentration, with an association time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). The data results were analyzed kinetically using Biacore T200 analysis software with a 1:1 binding model. As shown in Table 1, the affinity of HB37A6 is better than that of the control antibody Zmab.

[0292] Table 1. Affinity constants (equilibrium dissociation constants) of CLDN18.2 antibodies detected by SPR

[0293]

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

[0295] The binding of the anti-CLDN18.2 monoclonal antibody HB37A6 and the control antibody Zmab to the CHO-S cell lines stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1 (i.e., CHO-hCLDN18.2 and CHO-hCLDN18.1 prepared as described in Example 1) 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 Cells were plated at 100 μl / well of a U-bottom 96-well plate. The plate was centrifuged at 500 g for 5 minutes, and the cell culture medium was removed. Anti-CLDN18.2 monoclonal antibody HB37A6 and control antibody Zmab were added to the plate and the cells were resuspended. 100 μl was added to each well. The starting concentration of the antibodies was 900 nM, and then three-fold dilutions were made for a total of 10 concentrations. The cells were incubated on ice for 30 minutes. The supernatant was removed at 500 g for 5 minutes, and the cells were washed once with PBS. 100 μl of goat anti-human Fc PE-conjugated secondary antibody (Southern Biotech, J2815-5H87B) was added to each well. The cells were incubated on ice for 30 minutes in the dark. The supernatant was removed at 500 g for 5 minutes, and the cells were washed once with PBS. The cells were resuspended in 50 μl of 1× PBS and analyzed by FACS. The data were analyzed using GraphPad Prism software (Figures 1 and 2). As shown in Figures 1 and 2 , the antibodies specifically bind to human CLDN18.2 ( Figure 1 ) but do not bind to human CLDN18.1 ( Figure 2 ).

[0297] Example 1.6. Binding of CLDN18.2 Antibodies to Tumor Cell Lines

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

[0299] Example 1.7: In vivo anti-tumor effect of CLDN18.2 antibody

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

[0301] The HB37A6 antibody was used to test its anti-tumor efficacy in NOD-SCID mice bearing human pancreatic cancer (female NOD-SCID mice (15-18 g) purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). The human pancreatic cancer cell line DAN-G-hCLDN18.2 constructed in Example 1 was routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in 1× PBS to obtain a suspension with a cell density of 12 × 10^5 cells / ml. The cell suspension was mixed with matrigel gel in a 1:1 ratio 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 inoculated 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 The mice within the range were divided into groups according to the size of the tumor (8 mice per group).

[0303] Each mouse was administered hIgG (Equitech-Bio, lot number 160308-02), HB37A6, and the control antibody Zmab at a dose of 10 mg / kg on days 5, 9, 12, and 16 after inoculation. Tumor volume was monitored 2-3 times per week. Tumor volume was measured using a vernier caliper to measure the maximum longitudinal axis (L) and maximum width (W) of the tumor. Tumor volume was calculated using the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

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

[0305] The HB37A6 antibody was selected to test its anti-tumor efficacy in NOG mice bearing human gastric cancer (female NOG mice (15-18 g) purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.). PBMCs (Allcells) were revived, harvested by centrifugation, and dispersed in 1× PBS to a cell density of 2.5 × 10^6 cells / ml. On day 0, 0.2 ml of the cell suspension was injected into the ocular vein of NOG mice to establish the humanized NOG mouse model.

[0306] NUGC-4 cells were routinely revived and subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and dispersed in 1× PBS at a density of 12 × 10⁶ cells / mL. A 1:1 mixture of PBS and Matrigel gel was mixed to prepare a cell suspension with a concentration of 6 × 10⁶ cells / mL. On day 5, 0.2 mL of the cell suspension was subcutaneously inoculated into the right abdominal region of humanized NOG mice to establish a NUCG-4 tumor-bearing mouse model.

[0307] On the first day 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 each time on days 1, 5, 8, and 12 after inoculation. Tumor volume and body weight were monitored 2-3 times per week. Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper. 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 of inoculation using the following formula:

[0308] TGI%=100%*(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume of control group before administration).

[0309] 3. Results

[0310] The results, as shown in Figure 4, show that both HB37A6 and the control antibody Zmab inhibited tumor growth in the human pancreatic cancer DAN-G-CLDN18.2 mouse model, with a TGI of 28% for HB37A6 and 24% for Zmab. As shown in Figure 5, HB37A6 demonstrated superior anti-tumor efficacy compared to the control antibody Zmab in the human gastric cancer NUGC-4 mouse model, with a TGI of 31% for HB37A6 and 0% for Zmab.

[0311] Example 2.1 Synthesis of IEX019 ADC Molecule

[0312] IEX019

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

[0314] Example 2.1.1 Preparation of IEX019-02

[0315] 1) Preparation of Compound 6

[0316]

[0317] Under N2 atmosphere, to a solution of BCN-OH (5, 1.5 g, 10 mmol) in DCM (150 mL) were added chlorosulfonyl isocyanate (CSI) (0.87 mL, 1.4 g, 10 mmol), Et3N (2.8 mL, 2.0 g, 20 mmol) and 2-(2-aminoethoxy)ethanol (1.2 mL, 1.26 g, 12 mmol). The mixture was stirred for 10 minutes and quenched by adding aqueous NH4Cl (saturated, 150 mL). After separation, the aqueous layer was extracted with DCM (150 mL). The combined organic layers were dried (Na2SO4) and concentrated. The residue was purified by column chromatography. Product 6 (2.06 g, 5.72 mmol, 57%) was obtained as a light yellow thick 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] To a stirred solution of 6 (47 mg, 0.13 mmol) in DCM (10 mL) was added CSI (11 μL, 18 mg, 0.13 mmol). After 30 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 30 minutes, p-nitrophenyl chloroformate (52 mg, 0.26 mmol) and Et3N (54 μL, 39 mg, 0.39 mmol) were added. After an additional 4.5 hours, the reaction mixture was concentrated and the residue was purified by gradient column chromatography (33→66% EtOAc / heptane (1% AcOH)) to afford 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 exatecan 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 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) was added Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 7 (65 mg, 72 μmol) in DMF (1.4 mL). The reaction mixture was stirred for 24 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 light 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 to HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2

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

[0328] Next, the incubation mixture obtained above was purified using a 50 mL protA column (Hitrap Mabselect Sure, GE, 11-0034-95). The incubation mixture obtained above was 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 dialysis 3 times with PBS, the (modified) glycosylated antibody obtained was concentrated to 32.6 mg / mL using a Vivaspin Turbo 15 ultrafiltration device (Sartorius).

[0329] The obtained glycosylated antibodies were analyzed by mass spectrometry. The main steps are as follows: Before mass spectrometry analysis, the glycosylated antibodies were treated with IdeS to analyze the Fc / 2 fragment. 20 μg (modified) glycosylated antibody solution was mixed with IdeS (Fabricator TM ) (1.25 U / μL) in PBS pH 6.6 in a total volume of 10 μL at 37°C for 1 hour. The sample was diluted to 80 μL and analyzed on a JEOL AccuTOF (ESI-TOF) using Magtran software to obtain deconvoluted spectra. Mass spectrometric 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 demonstrated that HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 in which GlcNAc at Asn297 of both heavy chains was substituted with 6-azido-GalNAc (4-position substitution) was obtained.

[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 azide-modified HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 as a biomolecule. To this end, PBS pH 7.4 (808 μL), 1,2-propylene glycol (11.3 mL), and compound 1 (350 μL, 40 mM DMF solution) were added to a solution of HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 (10.8 mL, 350 mg, 32.6 mg / ml, PBS pH 7.4). The reaction was incubated at room temperature overnight, then filtered and dialyzed to PBS pH 7.4. Residual free payload was removed by adding charcoal (350 mg) and rotating overnight at room temperature. The charcoal was removed by centrifugation and filtration, and the ADC was purified on an AKTA Purifier-10 (GE Healthcare) with a Superdex200 26 / 600 SEC (GE Healthcare) column.

[0333] Mass spectrometry analysis of the IdeS digested sample revealed two major products, both corresponding to the resulting ADC, the HB37A6-SYNtecan E conjugate. The first peak, with an observed mass of 26,469 Da (calculated mass 26,465 Da), corresponds to the conjugated Fc / 2 fragment (2x closed lactone form of the payload). The second peak, with an observed mass of 26,499 Da (calculated mass 26,501 Da), corresponds to the bound Fc / 2 fragment (2x open carboxylate form of the payload).

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

[0335]

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

[0337] Among them, Ab is HB37A6.

[0338] Example 2.1.2 Preparation of IEX019-03

[0339]

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

[0341] Among them, Ab is HB37A6.

[0342] (a) A reducing agent solution (TCEP (Sigma, C4706), dissolved in water) was added to an HB37A6 solution (antibody HB37A6 was dissolved in PBS buffer (Thermo, 10010023)), and the reaction mixture was placed on an oscillator 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 4.5-6.5.

[0345] (iii) The optimal reaction temperature is 20-37°C.

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

[0347] (b) Excess linker-toxin MC-GGFG-DXD (purchased from Levena biopharma, SET0218, the structure of which can be shown below) was dissolved in DMSO and reacted with the thiol group of the antibody reduced in step (a). The reaction mixture was placed in an oscillator for 1-2 hours.

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

[0349] (ii) The optimal reaction temperature is 20-37°C,

[0350] The crude ADC product was obtained.

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

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

[0353]

[0354] Example 2.1.3 Preparation of IEX019-04

[0355]

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

[0357] Among them, Ab is HB37A6.

[0358] The molecule was prepared as follows.

[0359] (a) Add a 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 on an oscillator 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 was 1.9-2.7.

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

[0363] (iv) The optimal pH value 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) bearing 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 on a shaker for 1-2 h.

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

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

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

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

[0369] (e) The ADC product was tested by HIC and SEC-HPLC to determine the average DAR and SEC purity.

[0370]

[0371] Example 2.1.4 Preparation of IEX019-05

[0372]

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

[0374] Among them, Ab is HB37A6.

[0375] This molecule was prepared as follows.

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

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

[0378] (ii) the optimal molar ratio of linker-payload / mAb was 5.5–6.5;

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

[0380] (iv) The optimal reaction temperature is 20-37°C;

[0381] The crude ADC product was obtained.

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

[0383] (c) The DAR average value and SEC purity of the ADC product were determined by UV 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] The information of all mAbs 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 Experiment of IEX019 Molecule

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

[0394] Neither IEX019-01 nor IEX019-02 bound to non-target cell DAN-G, but had strong affinity for DANG-hCLDN18.2, demonstrating that antibody binding depends on target expression specificity and that exatecan conjugation does not affect antibody binding. Furthermore, the control molecule IEX019-06 (a negative control monoclonal antibody for IgG, conjugated to exatecan using the same technique) did not bind to target cells (Figure 6).

[0395] Example 2.3 IEX019 molecule endocytosis experiment

[0396] Strong endocytosis is a key characteristic of ADC drugs. After 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 a key indicator of its tumor suppressive efficacy.

[0397] In order to detect the internalization efficiency of antibodies after conjugation with small molecule compounds, we used flow cytometry to detect the internalization of different IEX019 molecules in DANG-hCLDNA18.2 cells. After digestion of DANG-hCLDNA18.2 cells, the cell density was adjusted to 1*10 5 Plate 100 cells / well in a 96-well plate. Centrifuge at 500g for 3 minutes and discard the supernatant. Take 100μl of the molecule to be tested and resuspend the cells (molecular concentration is 50nM). Set up 5 replicates for each sample (i.e., endocytosis time: 0h, 1h, 2h, 3h, 4h). Place on ice and incubate for 1h. After 1h, centrifuge at 500g for 3min and discard the supernatant. Add 200μl FACS buffer (1% FBS, 1X PBS) to each well and rinse twice. Take one set of samples, transfer it to a new 96-well plate, and incubate at 37℃ for 4h. Continue to incubate the remaining samples on ice. Repeat the previous operation and incubate the samples at 37℃ for 3h, 2h, 1h, and 0h. After the specific incubation time is completed, centrifuge at 500g for 3min and discard the supernatant. Add 100 μl of a 1:400 dilution of anti-hFC-PE antibody (Southern Biotech) to each well and incubate on ice for 30 minutes in the dark. After secondary antibody incubation, centrifuge at 500 g for 3 minutes and discard the supernatant. Rinse twice with 200 μl of FACS buffer per well, then resuspend the cells in 100 μl of PBS and analyze on an analyzer.

[0398] The experimental results are shown in Figure 7. Taking 0 hours of incubation at 37°C as the zero point of endocytosis, all molecules reached the maximum limit of endocytosis, approximately 60%, after 2 hours of incubation. This indicates that the ADC molecules designed and synthesized based on IEX019-01 maintain a strong ability to be internalized, consistent with the monoclonal antibody, after binding to tumor cells.

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

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

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

[0402]

[0403] After digesting the cells with EDTA / Trypsin, the cells were plated evenly in a 96-well plate at an adjusted density (Table 4). IEX019 samples (IEX019-02, IEX019-03, and IEX019-04) were diluted to specific concentrations (starting at 100 nM and diluted by a factor of 3). Wells without IEX019 served as controls. The plates were incubated at 37°C for 5 days. After 5 days, 100 μl of CellTiter-Glo detection reagent was added to each well, incubated at room temperature for 30 minutes, and assayed using a microplate reader. Relative cell viability was calculated (relative cell viability = sample / control * 100%), and curves were fitted using Graph Pad Prism 8.0.

[0404] As shown in Figure 8 , the ADC's ability to kill cell lines depends on the level of surface hCLDN18.2 expression. In hCLDN18.2-negative DANG cells, IEX019 had no significant effect on cell viability (Figure 8A). However, in cell lines with intermediate hCLDN18.2 expression (NUGC-4 and SNU620), IEX019 showed some cell killing (Figure 8B). Furthermore, in the high-expressing DAN-G hCLDN18.2 cell line, IEX019 exhibited significant killing effects (Figure 8C). This demonstrates the excellent selectivity and efficacy of IEX019.

[0405] Table 4. Cell seeding density

[0406]

[0407]

[0408] Example 2.5: Bystander killing effect

[0409] During the synthesis of ADC drugs, small molecule compounds can be attached to antibodies via cleavable linkers. After being internalized and entering the cell membrane, the linker is cleaved, releasing the small molecule and killing the target cell. After the target cell dies, the small molecule compound is released from the target cell into the intercellular space, further killing non-target cells within a certain range. This effect is called the bystander effect. Because cells within a tumor can have significant differences in target expression levels (tumor heterogeneity), the bystander effect is very important for effectively killing tumor cells and inhibiting tumor growth.

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

[0411] After the cells were digested with EDTA / Trypsin, the density was adjusted and a six-well cell culture plate was prepared. 7.5×10 DANG cells and 7.5×10 DANG-hCLDN18.2 cells were added. 4 , and co-culture the two cell types. Add 200 μl of the test sample (IgG (SEQ ID NO: 21, SEQ ID NO: 22), IEX019-02, IEX019-05, IEX019-06, Exatecan (Macklin, E881532)) to each well, with a final concentration of 50 nM, and set up 3 replicates for each sample. Place the cell culture plate in a 37°C incubator and culture for 5 days. After 5 days, discard the culture supernatant, rinse with PBS, and add Trypsin-EDTA to digest the cells. Collect all digested cells and transfer them to a 96-well plate. According to the antibody incubation process of flow cytometry detection technology, incubate with the primary antibody (IEX019-01, 100 nM) and the secondary antibody (anti-hFc-PE, SouthernBiotech) at 4°C for 1 hour and 0.5 hours respectively. After rinsing with PBS, the live / dead Violet dye (Thermo, L34964) was diluted 1:1000, and 100 μl was added to each well. Incubate at 4°C for 20 min. After rinsing with PBS, the cells were resuspended in 100 μl of PBS and analyzed. Live / dead dye was used to separate the populations within each sample. The IEX019-01-negative (i.e., hCLDN18.2-negative) population was designated as DAN-G cells, and the IEX019-01-positive (i.e., hCLDN18.2-positive) population was designated as DAN-G-hCLDN18.2. The number of both cell types in each sample was counted, and the relative cell viability of each cell type was calculated using the following equation. The curves were fitted using Graph Pad Prism 8.0.

[0412] Relative viability of DANG cells = number of DNAG cells in the sample group / number of DNAG cells in the IgG group * 100%

[0413] Relative viability of DANG-hCLDN18.2 cells = number of DNAG-18.2 cells in the sample group / number of DNAG-18.2 cells in the IgG group * 100%

[0414] The cell viability of the IgG group was set as 100%.

[0415] As shown in Figure 9, the negative control sample IEX019-06 had no killing effect on either cell type. The IEX019-05 molecule, which linked the DM1 toxin to the IEX019-01 mAb via a non-cleavable linker, lacked the ability to kill surrounding non-target cells, and therefore could only kill DANG-hCLDNA8.2 cells while having no effect on DANG cells. Only IEX019-02 exhibited a significant bystander effect, killing both target and non-target cells.

[0416] Example 2.6: Antitumor efficacy of IEX019 in the DAN-G-hCLDN18.2 mouse xenograft tumor model

[0417] To demonstrate the in vivo efficacy of IEX019, we used DANG-hCLDN18.2 cells to inoculate CB-17-SCID mice to determine the anti-tumor efficacy of the antibody. SPF-rated female CB-17-SCID mice (14-17 g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, with a certification number of 110011201108225246.

[0418] DANG-hCLDN18.2 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation and dispersed with PBS (1×) to prepare a cell concentration of 3×10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB-17 SCID mice to establish a DANG-hCLDN18.2 tumor-bearing mouse model.

[0419] On the 5th day after tumor cell inoculation, the tumor volume of all mice ranged from 50.16 to 136.68 mm 3 The mice were divided into groups (6 mice per group) and the dosage and method of administration were as shown in Table 5. The drugs were administered on the 5th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 10a and 10b. The monitoring lasted for 92 days.

[0420] The relative tumor inhibition rate (TGI%) was calculated on the 50th day after inoculation using the following formula:

[0421] TGI%=100%*(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume of control group before administration).

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

[0423] V=L*W 2 / 2. Body weight was measured using an electronic balance.

[0424] Table 5. Experimental design table (Note: single administration, 1 time in total)

[0425] Group Dosage Dosage times Dosage method IEX019-01 (control group) 10 mg / kg single dose intraperitoneal injection IEX019-02 (treatment group) 10 mg / kg single dose intraperitoneal injection IEX019-03 (treatment group) 10 mg / kg single dose intraperitoneal injection IEX019-04 (treatment group) 10 mg / kg single dose intraperitoneal injection

[0426] The tumor inhibition results are shown in Table 6 and Figure 10A. At day 50 post-inoculation, a single dose of IEX019-02 achieved a tumor inhibition rate of 103.60%, significantly superior to IEX019-03 and IEX019-04, which had tumor inhibition rates of 93.70% and 35.20%, respectively. 82 days after inoculation, 100% of the mice in the IEX019-02 group had complete tumor regression. Mouse body weights were also measured, and as shown in Figure 10B, no significant differences in weight were observed.

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

[0428]

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

[0430] In order to demonstrate the in vivo efficacy of the IEX019 molecule, we used NUGC-4 cells to inoculate CB-17-SCID mice to determine the anti-tumor efficacy of the antibody molecule of the present invention.

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

[0432] NUGC-4 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS (1×) to prepare a cell concentration of 3×10 7 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB-17 SCID mice to establish a NUGC-4 tumor-bearing mouse model.

[0433] On the 5th day after tumor cell inoculation, the tumor volume of all mice was 72.25-140.50 mm 3The mice were divided into groups (6 mice per group) and the dosage and method of administration were shown in Table 7. The drug was administered on the fifth day after inoculation. The tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 11A and 11B. The monitoring was terminated after 40 days.

[0434] The relative tumor inhibition rate (TGI%) was calculated on the 33rd day after inoculation using the following formula:

[0435] TGI%=100%*(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume of control group before administration).

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

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

[0438] Table 7. Experimental design table (Note: single administration, 1 time in total)

[0439]

[0440] The tumor inhibition rates are shown in Tables 8 and 11A. On day 33 after inoculation, the tumor inhibition rates of IEX019-02 and IEX019-03, compared to the negative control IEX019-06, were 80.04% and 54.31%, respectively. Mouse body weights were also measured, and as shown in Figure 11B, no significant differences were observed in the mice's weights.

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

[0442]

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

[0444] To demonstrate the in vivo efficacy of IEX019, we measured the antitumor efficacy of the molecule of the present invention (IEX019-02) in CB-17-SCID mice inoculated with SNU620 cells. SPF-rated female CB-17-SCID mice (18-20 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were used, with a certification number of 110011211102179364.

[0445] SNU620 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation and dispersed with PBS (1×) to prepare a cell concentration of 3×10 7On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of CB-17 SCID mice to establish the SNU620 tumor-bearing mouse model.

[0446] On the 7th day after tumor cell inoculation, the tumor volume of all mice was 58.1-117.3 mm 3 The mice were divided into groups (6 mice per group) and the dosage and administration method were shown in Table 9. The mice were given the drug on the 7th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 12a and 12b. The monitoring was terminated after 39 days.

[0447] The relative tumor inhibition rate (TGI%) was calculated on the 39th day after inoculation using the following formula:

[0448] TGI%=100%*(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume of control group before administration).

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

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

[0451] Table 9. Experimental design table (Note: single administration, 1 time in total)

[0452]

[0453] The tumor inhibition results are shown in Table 10 and Figure 12A. On day 39 after inoculation, a single dose of IEX019-02 (10 mg / kg) resulted in a 143.77% tumor inhibition rate, compared to the hIgG control group. Tumors completely regressed in 100% of the mice. Mouse body weights did not differ significantly between the treatment groups and the hIgG control group (Figure 12B).

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

[0455]

[0456] Sequence information:

[0457]

[0458]

[0459]

Claims

1. An antibody-drug conjugate having the formula I: Ab-(L-(D) r ) p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is an antibody or a fragment thereof that binds to CLDN18.2 (such as human CLDN18.2); L is a linker; D is a drug, such as an anti-tumor compound; and p is from 1 to 10, such as 1-9, 2-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r is 1-5, preferably 1 or 2; wherein the Ab in formula (I) comprises HCDR1, HCDR2, HCDR3 shown by the following amino acid sequences respectively: SEQ ID NO:1, 2 and 3, and LCDR1, LCDR2 and LCDR3 shown by the following amino acid sequences respectively: SEQ ID NO:6, 7 and 8.

2. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein Ab in formula (I) comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:4 or consists of said amino acid sequence; or (ii) comprises the amino acid sequence of SEQ ID NO:4 or consists of said amino acid sequence; or (iii) comprises an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4 and consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions; and / or the light chain variable region (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:9 or consists of said amino acid sequence; or (ii) comprises the amino acid sequence of SEQ ID NO:9 or consists of said amino acid sequence; or (iii) comprises an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO:9 and consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions.

3. The antibody-drug conjugate according to any one of claims 1-2, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab in formula (I) further comprises a heavy chain constant region and / or a light chain constant region.

4. The antibody-drug conjugate according to any one of claims 1-3, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab in formula (I) is an IgG antibody.

5. The antibody-drug conjugate according to any one of claims 1-4, or a pharmaceutically acceptable salt or solvate thereof, wherein the heavy chain constant region of the Ab in formula (I) is from IgG1 or IgG2 or IgG3 or IgG4, preferably IgG1.

6. The antibody-drug conjugate according to any one of claims 1-5, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab in formula (I) comprises a heavy chain and a light chain, wherein the heavy chain (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 11 or consists of said amino acid sequence; (ii) comprises the amino acid sequence of SEQ ID NO: 11 or consists of said amino acid sequence; or (iii) comprises an amino acid sequence having 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11 or consists of said amino acid sequence; and the light chain (i) comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 12 or consists of said amino acid sequence; (ii) comprises the amino acid sequence of SEQ ID NO: 12 or consists of said amino acid sequence; or (iii) comprises an amino acid sequence having 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12 or consists of said amino acid sequence.

7. The antibody-drug conjugate according to any one of claims 1-6, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab in formula (I) is a human antibody.

8. The antibody-drug conjugate according to any one of claims 1-7, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab in formula (I) is an antigen-binding fragment, such as Fv, Fab, Fab’, Fab’-SH, F(ab’)2; dAb (domain antibody); linear antibody; single-chain antibody (such as scFv); single-domain antibody such as VHH; bivalent antibody or a fragment thereof; or camelid antibody.

9. The antibody-drug conjugate according to any one of claims 1-8, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker is MC-VC-PAB, vc-PAB, SMCC or MC-GGFG.

10. The antibody-drug conjugate according to any one of claims 1-8, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody is an antibody with modified glycosylation, such as an antibody obtained by enzymatically modifying the sugar chain in vitro (such as modifying the sugar chain by glycosidase (such as endoglycosidase or glycosyltransferase)), preferably, the antibody with modified glycosylation refers to an antibody in which the sugar chain at the antibody glycosylation site is modified from a heterogeneous structure N-glycan to a single-structure N-glycan with a reactive group (such as any reactive group capable of reacting with the linker moiety, such as azide, keto group and alkyne group), more preferably, the N-glycosylation site is a conserved N-glycosylation site on the antibody Fc domain, such as Asn297.

11. The antibody-drug conjugate according to any one of claims 1-8, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker is a linker capable of achieving site-specific conjugation, preferably a linker conjugated to an antibody oligosaccharide, preferably, the oligosaccharide linker is a linker containing an alkyne group, most preferably, the oligosaccharide linker is a linker capable of achieving specific site conjugation with a reactive group (such as azide) on the N-glycan at a conserved N-glycosylation site (such as Asn297) on the antibody Fc domain.

12. The antibody-drug conjugate according to any one of claims 1-11, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-tumor compound is a cytotoxic agent or a chemotherapeutic agent, such as Exatecan, Dxd, MMAE or DM1.

13. An antibody-drug conjugate having formula II or a pharmaceutically acceptable salt or solvate thereof wherein Ab is as defined in any one of claims 1-8, L1 is a linker; E is a sugar or sugar derivative, preferably 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); and GlcNAc is N-acetylglucosamine and Fuc is fucose; D, r are as defined in formula I of any one of claims 1-8; b is 0 or 1; x is 1, 2, 3 or 4; y is from 1 to 20.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein x is 1 or 2; and y is from 1 to 10; preferably x is 1 and y is 2.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein E is GalNAc.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 15, wherein E is 6-deoxy-2-acetamidogalactose and is linked to L1 through the C atom at position 6.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-15, wherein the GlcNAc linked to Ab is present at the Asn297-glycosylation site of the two heavy chains of the antibody.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 11-17, wherein L1 has the following structure Q is -N(H)C(O)CH2- or -CH2-; R1 is independently selected from hydrogen, halogen, -OR2, -NO2, -CN, -S(O)2R2, C1-C 12 alkyl, aryl, heteroaryl, C1-C 12 alkylaryl, C1-C 12 alkylheteroaryl, arylC1-C 12 alkyl and heteroarylC1-C 12 alkyl, and 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 spiro cycloalkyl or a fused or spiro arene or heteroarene substituent, and wherein R2 is independently selected from hydrogen, halogen, C1-C 24Alkyl, aryl, heteroaryl, C1-C 12 Alkylaryl, C1-C 12 Alkylheteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 Alkyl; R3 and R4 are each independently selected from hydrogen, halogen, C1-C 24 Alkyl, aryl, heteroaryl, C1-C 12 Alkylaryl, C1-C 12 Alkylheteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 Alkyl; c is 0, 1, 2, 3 or 4; m is 0 or 1; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Y is O, S or NR1; L2 and L3 are each independently C1-C 12 Alkyl, one or more carbon atoms in which 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; L4 are each independently a cleavable linker.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 18, wherein L4 are each independently wherein R5 and R6 are each independently selected from hydrogen and C1-C 12 Alkyl; and Ar is selected from aryl.

20. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 19, wherein L4 are each independently 21. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13-17, wherein -L1- has the following structure 22. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 21, wherein the antibody-drug conjugate has an average DAR of 1-15, such as 2-10, 2-8 or 3-5.

23. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 13, the antibody-drug conjugate being selected from: wherein Ab is HB37A6 wherein q represents the average DAR, in IEX019-02, IEX019-04, IEX019-05, q is 2-5, such as 3-5 or 3.5-4.5, in IEX019-03, q is 5-11, such as 7-9 or 7.5-8.

5.

24. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 23 or a pharmaceutically acceptable salt or solvate thereof, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

25. A pharmaceutical combination comprising the antibody-drug conjugate of any one of claims 1 to 23 or a pharmaceutically acceptable salt or solvate thereof, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

26. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody-drug conjugate of any one of claims 1 to 23 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of claim 24, or the pharmaceutical combination of claim 25.

27. The method of claim 26, wherein the tumor is cancer, preferably, the cancer has an elevated level of (e.g., at the nucleic acid or protein level) CLDN18.2, preferably, the tumor cells of the subject moderately or highly express CLDN18.2, the cancer such as epithelial cancer or gastrointestinal cancer, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.

28. The method of any one of claims 26-27, wherein the method further comprises administering to the patient one or more therapies, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modality includes radiotherapy or surgery, or the therapeutic agent includes chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

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