Antibody drug conjugate targeting CLDN 18.2 and application thereof

Abstract

The invention relates to an antibody drug conjugate targeting CLDN 18.2 and application of the antibody drug conjugate. The antibody drug conjugate comprises an antibody targeting CLDN 18.2 or an antigen binding fragment of the antibody and load MMAE.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an antibody drug conjugate targeting CLDN 18.2 and application thereof. BACKGROUND

[0002] Claudin 18.2 (CLDN 18.2) is an important member of the Claudin family, which is only lowly expressed in differentiated epithelial cells in the gastric mucosa under normal physiological conditions, and its main function is to complete the connection between cells. Studies have found that CLDN 18.2 is highly expressed in various tumor tissues, such as gastric cancer (60-80%), pancreatic cancer (50%), esophageal cancer (30-50%) and lung cancer (40-60%) and the like, and is closely related to tumor progression and prognosis. CLDN18.2 has become one of the core popular targets in the field of tumor targeted therapy. At present, the monoclonal antibody VYLOY (zolbetuximab-clzb) targeting CLDN18.2 has been approved for the treatment of G / GEJ adenocarcinoma, and antibody drug conjugates (ADC) as a new treatment method also show great potential. SUMMARY

[0003] The present application provides an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof targeting CLDN 18.2 and a payload, which has one or more of the following advantages: (1) specifically recognizes and binds to CLDN18.2; (2) efficiently binds to CLDN18.2 without binding to CLDN18.1; (3) has good endocytosis activity; (4) has good cell killing activity; (5) has good bystander effect; (6) has high safety; and (7) has good inhibitory effect on the growth of tumor with high expression of CLDN18.2. In addition, the antibody drug conjugate described in the present application can maintain the expected cytotoxic effect under different ADCC effect intensities, and can selectively adapt to different ADCC intensities according to different tumor types or clinical needs while maintaining binding activity and efficacy, thereby having higher flexibility and application advantages in clinical development.

[0004] In one aspect, the present application provides an antibody drug conjugate (ADC) comprising an antibody or antigen binding fragment thereof targeting CLDN 18.2 and a payload, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1-3 and the VL comprises LCDR1-3, wherein the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 5, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 6, wherein the payload has the following structure:

[0005] (MMAE).

[0006] In certain embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 15.

[0007] In certain embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 16.

[0008] In certain embodiments, the VH and VL are selected from any one of the following groups of VH and VL: (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 8; or (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 15, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 16.

[0009] In certain embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region (CH) and a light chain constant region (CL).

[0010] In certain embodiments, the heavy chain constant region and the light chain constant region are both derived from human immunoglobulin.

[0011] In certain embodiments, the heavy chain constant region is selected from IgG1, IgG2, IgG3, or IgG4 subtypes, and the light chain variable region is of kappa type or lambda type.

[0012] In certain embodiments, the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 13, and the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 10.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NO: 11, 14, and 17.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 18.

[0015] In certain embodiments, the heavy chain and the light chain are selected from any one of the following groups of heavy chain and light chain: (1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 11, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; or (2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 14, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; or (3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 17, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 18.

[0016] In certain embodiments, the CLDN18.2 is human CLDN18.2.

[0017] In certain embodiments, the antibody is selected from one or more of the following group: a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0018] In certain embodiments, the antigen-binding fragment is selected from the following group: a Fab, a Fab', a F(ab)2, a Fv fragment, a F(ab')2, a scFv, a di-scFv, and a dAb fragment.

[0019] In certain embodiments, the antibody drug conjugate comprises a structure of Ab-[L1-L2- payload]m, wherein Ab represents the antibody or antigen-binding fragment thereof targeting CLDN 18.2, wherein L1 represents a linker connected to Ab, and L2 represents a linker connected to a payload.

[0020] In certain embodiments, the m is an integer from 1 to 10.

[0021] In certain embodiments, the m is 4.

[0022] In certain embodiments, the L1 and / or L2 is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid-based linker.

[0023] In certain embodiments, the L1 is attached to the Ab through a thiol, azido, or amido group on the Ab.

[0024] In certain embodiments, the L2 is selected from the group consisting of a polypeptide, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2- pyridyldithio)valerate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N- succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-4-(maleimidomethyl)cyclohexylcarboxamide succinimidyl ester (SMCC), N-sulfo(4-maleimidomethyl)cyclohexylcarboxamide sulfosuccinimidyl ester (sulfo-SMCC), and 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecan-1-oate (CX1-1).

[0025] In certain embodiments, the -L1-L2- is -Mc-VC-PAB.

[0026] In certain embodiments, the antibody drug conjugate has the structure Ab-[Mc-VC-PAB-MMAE]4.

[0027] In certain embodiments, the antibody drug conjugate has attenuated antibody-dependent cell-mediated cytotoxicity (ADCC).

[0028] In certain embodiments, the antibody drug conjugate has an amino acid modification at the N297 position.

[0029] In another aspect, the present application provides a pharmaceutical composition comprising the antibody drug conjugate described herein.

[0030] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0031] In another aspect, the present application provides use of the antibody drug conjugate described herein in the manufacture of a medicament for treating a CLDN 18.2 positive tumor.

[0032] In certain embodiments, the tumor comprises gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, cholangiocarcinoma, bladder cancer and / or leukemia.

[0033] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description in combination with the drawings. The illustrative embodiments described in the detailed description, given merely for the purposes of illustration, make this application sufficiently enabling. As will be realized, the application is capable of modifications in various obvious respects, all without departing from the application as recited in the claims. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0034] The specific features of the application involved are shown in the appended claims. The features and advantages of the application involved can be better understood by reference to the detailed description of the example embodiments and the accompanying drawings. The drawings are briefly described as follows:

[0035] Figure 1 Shown is the binding activity of ADCs formed by conjugating M19I with different payloads to CLDN 18.2.

[0036] Figure 2 Shown is the flow cytometry test result of specific binding of naked anti-M19I to CLDN18.2.

[0037] Figure 3 Shown is the fluorescence signal of specific binding of naked anti-M19I to CLDN18.2.

[0038] Figure 4 Shown is the endocytosis activity of M19I-MMAE detected using flow cytometry.

[0039] Figure 5 Shown is the cell-killing effect of three ADCs (M19I-DXD, M19I-MMAE and M19I-SN38) on NUGC4-CLDN18.2 overexpressing cell lines.

[0040] Figure 6 Shown is the cell-killing activity of three ADCs (M19I-DXD, M19I-MMAE and M19I-SN38) on NCI-N87-CLDN18.2 overexpressing cell lines.

[0041] Figure 7 Shown is the killing activity of M19I-MMAE (DAR 4) on BXPC3 cells with high expression of CLDN18.2.

[0042] Figure 8 Shown is the killing activity of M19I-MMAE (DAR 4) on NUGC4 cells with medium expression of CLDN18.2.

[0043] Figure 9 Shown is the killing activity of M19I-MMAE (DAR 4) on A375 cells without expression of CLDN18.2.

[0044] Figure 10 Shown is the bystander effect detection result of M19I-MMAE (DAR 4) at effector: target (E:T) = 5:1.

[0045] Figure 11 Shown is the bystander effect detection result of M19I-MMAE (DAR 4) at effector: target (E:T) = 10:1.

[0046] Figure 12 Shown is the cell killing activity of M19I-MMAE and M19I wt-MMAE on N87-CLDN18.2 overexpression cell line.

[0047] Figure 13 Shown is the cell killing activity of M19I-MMAE and M19I wt-MMAE on NUGC4-CLDN18.2 overexpression cell line.

[0048] Figure 14 Shown is the bystander effect of M19I-MMAE and M19I wt-MMAE at E:T = 5:1.

[0049] Figures 15-17 Shown is the endocytosis effect of different DAR value ADCs on different CLDN18.2 expression level cells. Figure 15 Shown is the endocytosis activity detection result of different drugs acting on different cells at the same concentration (40 nM). Figure 16 Shown is the endocytosis activity detection of the same drug acting on different cells. Figure 17 Shown is the endocytosis activity detection of the same drug at different concentrations on the same cells.

[0050] Figure 18 Shown is the killing activity of ADCs with different DAR values on BXPC3 cells.

[0051] Figure 19 Shown is the killing activity of ADCs with different DAR values on NUGC4 cells.

[0052] Figure 20 The tumor volume change of NUGC4-CLDN18.2 mice treated with different ADCs is shown.

[0053] Figure 21 The body weight change of NUGC4-CLDN18.2 mice treated with different ADCs is shown.

[0054] Figure 22 The detection results of ADCC effect of M19I, M19I wt and M19I mutant are shown. DETAILED DESCRIPTION

[0055] The following specific examples illustrate the embodiments of the present application, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure herein.

[0056] TERMS

[0057] In the present application, the term "Claudin 18.2", also known as Claudin 18.2, CLDN18.2, generally refers to subtype 2 of the Claudin 18 family. The CLDN18.2 described in the present application can include the complete CLDN18.2 protein and its functional fragments, functional variants, homologues and / or proteins, peptides or polypeptides having at least one common epitope with CLDN18.1. The CLDN18.2 described in the present application can include human CLDN18.2 and CLDN18.2 of other species (such as mouse, monkey, etc.). The amino acid sequence of CLDN18.2 (for example, human CLDN18.2) is known in the art. For example, the nucleotide sequence of human CLDN18.2 can be shown under GeneBank accession number NM_001002026.3.

[0058] In the present application, the term "antibody drug conjugate (ADC)", generally refers to a complex formed by connecting an antibody or its antigen binding fragment to a payload through a linker. The antibody or its antigen binding fragment in the antibody drug conjugate can specifically recognize and bind to the antigen on the surface of the cell, achieving precise delivery of the payload in vivo. For example, it can kill cancer cells while minimizing damage to normal cells.

[0059] In the present application, the term "payload" refers generally to an active molecule conjugated to an antibody or antigen-binding fragment thereof via a linker, which functions to exert the intended biological effect on a target cell, typically a cytotoxic activity. The payload can be a small molecule cytotoxic drug, for example, the payload can be a microtubulin inhibitor, a DNA damaging agent, or a topoisomerase inhibitor, among others, or other types of therapeutic or diagnostic active ingredients.

[0060] In the present application, the term "linker" refers to a structure that links an antibody or antigen-binding fragment thereof to a payload. The linker can be a chemically synthesized structure or a biosynthetic structure, and typically comprises a reactive group capable of forming a covalent bond with the antibody and / or the payload. Unless otherwise specified, the linkers described in the present application are not limited to a specific chemical structure or coupling method, and can include any structure known in the art that is capable of effectively linking an antibody to a payload.

[0061] In the present application, the term "Drug-to-Antibody Ratio" or "DAR" refers generally to the number of drugs linked to an antibody of an ADC. The DAR of an ADC can range from 1-8, or can be higher loading (e.g., 10), and the range of DAR can depend on the number of linking sites on the antibody. In the present application, the DAR can be the number of drugs loaded onto a single antibody, and the DAR can also be the average or mean DAR of a group of ADCs.

[0062] In the present application, the term "ADCC" or "Antibody-Dependent Cellular Cytotoxicity" is a function mediated through Fc receptor binding and refers to lysis of target cells by antibodies in the presence of effector cells as reported herein. The ability of antibodies to induce the initial steps that mediate ADCC is detected by measuring their binding to cells expressing Fc gamma receptors, such as cells recombinantly expressing Fc gamma RI and / or Fc gamma RIIA, or NK cells (which express Fc gamma RIIIA by nature). In the present application, "Fc" refers generally to the fragment crystallizable region of an antibody molecule, which is typically composed of the constant regions of the two heavy chains, typically including the CH2 and CH3 regions. In certain embodiments, the Fc also includes the hinge region.

[0063] In the present application, the term "CLDN18.2-positive tumor" refers to tumor cells that express CLDN18.2 protein on their surface. To determine whether a cell expresses CLDN18.2 protein on the surface, expression of CLDN18.2 mRNA can be determined by a method selected from the group consisting of in situ hybridization and RT-PCR (including quantitative RT-PCR), or CLDN18.2 protein expression on the cell surface can be determined using an antibody against CLDN18.2 protein in a method such as immunohistochemistry, FACS, and the like. In some embodiments, the CLDN18.2-positive tumor can be a mammal-implanted tumor, such as a mouse-implanted tumor.

[0064] In the present application, the term "tumor" generally refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. In the present application, a tumor can include a tumor associated with expression of CLDN18.2. For example, a tumor can include a CLDN18.2-positive tumor.

[0065] In the present application, the term "CDR" also known as "complementarity determining region" generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structurally defining loop. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camelid antibodies consisting of only heavy chains are also able to function normally and stably in the absence of light chains. Antibody CDRs can be determined by various encoding systems, such as CCG, Kabat, Chothia, IMGT, integrated consideration of Kabat / Chothia, and the like.

[0066] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 of the basic heterotetramer units along with an additional polypeptide called J chain, and contain 10 antigen binding sites. IgA antibodies, which are present as a dimmer when not complexed with J chain, contain 2-5 of the basic 4-chain units along with J chain, and can have 2-10 antigen binding sites. For IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while each of the H chains is disulfide ly linked to the other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VHand the CL is aligned with a first constant domain of a heavy chain (CHI). Particular amino acid residues are recognized as being at the interface between light and heavy chain variable domains. The VHand VLpair together to form a single antigen binding site. For structures and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of the constant domains of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively.

[0067] In the present application, the term "antigen binding fragment" generally refers to one or more fragments having the ability to specifically bind to an antigen. In the present application, the antigen binding fragment can include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0068] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions can also be allowed, as long as they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody can optionally comprise at least a portion of a human immunoglobulin constant region. The "humanized antibody" retains antigenic specificity similar to that of the original antibody. The "humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody that includes sequences derived from non-human immunoglobulin. In some cases, the CDR region residues of a human immunoglobulin (recipient antibody) can be replaced by those of a CDR region of a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate that has the desired properties, affinities and / or capabilities. In some cases, the FR region residues of a human immunoglobulin can be replaced by corresponding non-human residues. Furthermore, the humanized antibody can comprise amino acid modifications not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve the properties of the antibody, such as binding affinity.

[0069] In the present application, the term "treatment" generally includes preventing the onset of the disease, slowing or reversing the course of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated therewith, and / or preventing further increase in the severity of the disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects generally beneficial to the treated patient.

[0070] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or disorder. The pharmaceutical composition can comprise an antibody drug conjugate described herein. In certain embodiments, the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier. In addition, the pharmaceutical composition described herein includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0071] In the present application, the term "carrier" generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art. The pharmaceutical composition can further comprise one or more suitable formulations of (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the used dose and concentration.

[0072] In the present application, the term "subject" generally refers to a human or a non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0073] In the present application, the term "effective amount" generally refers to an amount of the antibody of the present application sufficient to prevent or slow down symptoms associated with a disease or a disorder (e.g., cancer). A therapeutically effective amount is related to the disease being treated, wherein a person skilled in the art can easily determine the actual effective amount.

[0074] In the present application, the protein, polypeptide and / or amino acid sequence referred to shall also be understood to at least include variants or homologues thereof having the same or similar function as the protein or polypeptide.

[0075] In the present application, the variant can be, for example, a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof capable of specifically binding to CLDN18.2). For example, the functional variant can comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions and / or insertions. The functional variant can substantially maintain the biological properties of the protein or the polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or the polypeptide before the change. For example, the substitution can be a conservative substitution.

[0076] In the present application, the homologue can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof capable of specifically binding to TMPRSS6).

[0077] In the present application, the homology generally refers to similarity, analogy or correlation between two or more sequences. The "percent sequence homology" can be calculated by comparing two sequences to be aligned in a comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence homology. The alignment for determining percent sequence homology can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve the maximum alignment over the full length sequence or a desired region of the sequence under comparison. The homology can also be determined by FASTA and BLAST.

[0078] In the present application, the term "about" as used herein generally refers to a variation within normal tolerances in the art, typically within 10% of the stated value, such as within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%. Unless otherwise indicated by context, all numerical values provided herein are modified by the term "about". DETAILED DESCRIPTION

[0080] In one aspect, the present application provides an antibody drug conjugate (ADC) comprising an antibody or antigen binding fragment thereof targeting CLDN 18.2 and a payload.

[0081] Antibodies or antigen-binding fragments thereof targeting CLDN 18.2

[0082] In the present application, the antibody or antigen-binding fragment thereof can comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1-3 and the VL comprises LCDR1-3, wherein the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 5, and the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 6 (wherein the CDRs are delineated in the manner of Kabat).

[0083] In certain embodiments, the VH can comprise an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 15.

[0084] In certain embodiments, the VL can comprise an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 16.

[0085] In certain embodiments, the VH and VL are selected from any one of the following sets of VH and VL: (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 8; or (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 15, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 16.

[0086] In certain embodiments, the VH can comprise an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to an amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 15. In certain embodiments, the VL can comprise an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to an amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 16.

[0087] In certain embodiments, the antibody or antigen-binding fragment thereof targeting CLDN 18.2 described herein is a murine antibody or antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof targeting CLDN 18.2 described herein is a humanized antibody or antigen-binding fragment thereof.

[0088] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a heavy chain constant region (CH) and a light chain constant region (CL). In certain embodiments, both the heavy chain constant region and the light chain constant region are derived from human immunoglobulin. In certain embodiments, the heavy chain constant region can be selected from the heavy chain constant region of IgG, IgA, IgM, IgD or IgE. In certain embodiments, the heavy chain constant region can be selected from the IgG1, IgG2, IgG3 or IgG4 subtypes. In certain embodiments, the light chain constant region can be of kappa or lambda type.

[0089] In certain embodiments, the heavy chain constant region can comprise the amino acid sequence as set forth in SEQ ID NO: 9.

[0090] In certain embodiments, the heavy chain constant region can be a wild-type sequence, or comprise one or more amino acid substitutions, deletions or insertions based on the wild-type sequence, to improve or alter the properties of the antibody, such as stability, binding affinity, effector function (such as ADCC, CDC), glycosylation pattern or half-life.

[0091] In certain embodiments, the heavy chain constant region can comprise amino acid mutations that enhance ADCC effect, such as amino acid substitutions that increase Fc receptor binding affinity.

[0092] In certain embodiments, the heavy chain constant region can comprise amino acid mutations that attenuate ADCC effect, such as amino acid substitutions that decrease Fc receptor binding or impair effector function. For example, in certain embodiments, the mutation site involves the glycosylation modification region of the Fc region, such as amino acid mutation of fucosylation site, to decrease Fc receptor binding capacity and attenuate ADCC effect.

[0093] In certain embodiments, the enhanced or attenuated ADCC effect of the antibody can be relative to an antibody with wild-type heavy chain constant region sequence. In certain embodiments, the comparison benchmark can also be the parent antibody without Fc engineering, or a homologous control of the same antibody (such as a human IgG1 homologous control antibody).

[0094] In some embodiments, the antibody-enhancing ADCC mutation may increase ADCC activity by approximately 1.5-fold, 2-fold, 3-fold, or higher relative to antibodies with a wild-type constant region. In some embodiments, the attenuating ADCC mutation may reduce ADCC activity by approximately 10%, 30%, 50%, or more relative to antibodies with a wild-type constant region. In some embodiments, the magnitude of the enhancement or attenuation can be characterized by changes in the percentage of target cell lysis induced by effector cells, the area under the dose-response curve (AUC), or the half-maximum effective concentration (EC50).

[0095] In some embodiments, the antibodies that enhance or weaken the ADCC effect can be prepared in different ways. In some embodiments, the enhancement or weakening of the ADCC effect can be achieved by introducing one or more amino acid mutations into the Fc region. For example, in some embodiments, introducing mutations that increase the affinity for Fcγ receptors (such as FcγRIIIa) (e.g., F243L, R292P, Y300L, L235V, and P396L, etc.) can enhance the ADCC effect; while introducing mutations that decrease the binding affinity of Fcγ receptors (e.g., L234F, L235E, P331S, etc.) can weaken the ADCC effect.

[0096] In some embodiments, the ADCC effect can also be modulated by controlling the antibody's glycosylation pattern. For example, reducing the fucosylation level in the Fc region or obtaining a fucosylated antibody can significantly enhance the ADCC effect; while altering the glycan structure by introducing mutations at glycosylation sites, adjusting cell culture conditions, or using specific host cell lines (such as engineered CHO cell lines) can be used to weaken the ADCC effect. In some embodiments, antibodies with different ADCC strengths can also be obtained through in vitro enzymatic modification, glycoengineering methods, or selective removal / addition of sugar residues.

[0097] In some embodiments, the antibody or its antigen-binding fragment has an enhanced ADCC effect. For example, the heavy chain constant region may contain one or more amino acid mutations from the following group: F243L, R292P, Y300L, L235V, and P396L. In some embodiments, the heavy chain constant region may simultaneously contain mutations of F243L, R292P, Y300L, L235V, and P396L.

[0098] In some embodiments, the antibody or its antigen-binding fragment has a weakened ADCC effect. For example, the heavy chain constant region may contain one or more amino acid mutations from the group consisting of L234F, L235E, and P331S. In some embodiments, the heavy chain constant region may contain L234F, L235E, and P331S simultaneously.

[0099] Unless otherwise stated, the amino acid numbers describing mutation sites in the constant region or Fc in this application are based on the sequence number of the human IgG1 heavy chain constant region, and the numbering method is in accordance with the EU numbering system (Kabat / IMGT).

[0100] In some embodiments, the heavy chain constant region may contain an amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 10.

[0101] In some embodiments, the antibody or its antigen-binding fragment may comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 14 and 17.

[0102] In some embodiments, the antibody or its antigen-binding fragment may comprise a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence as shown in SEQ ID NO:12 or SEQ ID NO:18.

[0103] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain and light chain are selected from any one of the following groups of heavy and light chains: (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 11 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 12; or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 14 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 12; or (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17 and the light chain comprises the amino acid sequence shown in SEQ ID NO: 18.

[0104] In some implementations, the CLDN18.2 may be a human CLDN18.2.

[0105] In some embodiments, the antibody may be selected from one or more of the following groups: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0106] In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, and dAb fragment.

[0107] In this application, the antibody targeting CLDN18.2 or its antigen-binding fragment may be greater than or equal to about 10. 5 M -1 (For example, greater than or equal to about 10) 5 M -1, greater than or equal to approximately 10 6 M -1 , greater than or equal to approximately 10 7 M -1 , greater than or equal to approximately 10 8 M -1 , greater than or equal to approximately 10 9 M -1 , greater than or equal to approximately 10 10 M -1 , greater than or equal to approximately 10 11 M -1 , greater than or equal to approximately 10 12 M -1 , greater than or equal to approximately 10 13 M -1 Ka (or higher) (i.e., the equilibrium association constant of the binding interaction, with a value of 1 / M); or, less than or equal to about 10 -5 M (for example, less than or equal to about 10) -5 M, less than or equal to approximately 10 -6 M, less than or equal to approximately 10 -7 M, less than or equal to approximately 10 -8 M, less than or equal to approximately 10 -9 M, less than or equal to approximately 10 -10 M, less than or equal to approximately 10 -11 M, less than or equal to approximately 10 -12 M, less than or equal to approximately 10 -13 The equilibrium dissociation constant Kd (M or smaller) binds to or associates with CLDN18.2. In this application, the affinity between an antibody or its antigen-binding fragment targeting CLDN18.2 and CLDN18.2 can be determined using conventional techniques in the art, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or by displacement assay of labeled ligands, or by surface plasmon resonance devices (such as the Biacore T100, which is available from Biacore, Inc., Piscataway, NJ) or optical biosensor technology.

[0108] Antibody drug conjugates (ADCs)

[0109] The antibody-drug conjugate described in this application comprises the structure Ab-[L1-L2-load]m, wherein Ab represents the antibody or its antigen-binding fragment targeting CLDN 18.2, and L1 represents the linker connected to Ab, and L2 represents the linker connected to the load.

[0110] In some implementations, m represents the DAR value and can be an integer from 1 to 10, for example, 2, 4, 6, or 8. In some implementations, m is 4.

[0111] In some embodiments, L1 and / or L2 are selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid-based linkers.

[0112] In some embodiments, L1 is linked to Ab via a thiol, azide, or amide group on Ab.

[0113] In some embodiments, the L2 is selected from the group consisting of: polypeptide sequences, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), and maleimide PEG. NHS, N-4-(maleimidemethyl)cyclohexylcarboxylic acid succinamide ester (SMCC), N-sulfono(4-maleimidemethyl)cyclohexylcarboxylic acid sulfosuccinate (sulfon-SMCC), and 2,5-dioxopyrrolidinyl-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolidinyl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecane-1-ester (CX1-1).

[0114] In some embodiments, the linker (-L1-L2-) may be selected from Mc-GGFG or Mc-VC-PAB, wherein Mc represents maleimide hexanoyl, GGFG represents glycine-glycine-phenylalanine-glycine tetrapeptide sequence, VC represents valine-citrulline, and PAB represents p-aminobenzyl alcohol.

[0115] In some embodiments, the antibody-drug conjugate has the following structure: Ab-[Mc-VC-PAB-MMAE]4.

[0116] In some embodiments, the payload may be a cytotoxic small molecule compound, which may include a tubulin inhibitor, a DNA damaging agent, and a topoisomerase inhibitor.

[0117] In some embodiments, the payload may be selected from one of the following groups: V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTOR inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, maytansine alkaloids, MetAP (methionine aminopeptidase), nuclear export inhibitors of protein CRM1, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphotransferase inhibitors, protein synthesis inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinin inhibitors, HDAC inhibitors, DNA disruptors, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, DHFR inhibitors, nucleoside analogs, HDAC inhibitors, anthracyclines, NAMPT inhibitors, SN-38 or derivatives thereof, etoposide phosphate, nitrogen mustard, protein body inhibitors, cytokines, Tubulysin B analogs, and Toll-like receptor agonists.

[0118] In some embodiments, the load may be selected from: DXD or its derivatives, SN-38 or its derivatives.

[0119] In some embodiments, the payload may be a tubulin inhibitor. In some embodiments, the tubulin inhibitor includes oliganthrines and maytansines. In some embodiments, the tubulin inhibitor is selected from the group consisting of: monomethyloliganthrine E (MMAE), monomethyloliganthrine F (MMAF), DM1 (Mestane), DM3 (maytansine DM3), and DM4 (Lavtansine).

[0120] In some embodiments, the load may have the following structure (MMAE).

[0121] In some implementations, the load may be R-SN38.

[0122] The antibody-drug conjugates described in this application are capable of killing tumors. These conjugates can target and recognize tumor-associated antigens through antibody or antigen-binding fragments, selectively delivering cytotoxic loads to tumor cells, thereby achieving targeted killing with both specificity and high efficiency. The killing effect can manifest in various forms, such as: in vitro, inhibiting tumor cell proliferation, reducing the number of tumor cells, and / or inducing apoptosis; in vivo, reducing tumor burden, slowing tumor growth, and / or reducing tumor tissue volume. In some embodiments, the tumor-killing effect can also be manifested as a decrease in the expression or serum levels of tumor-associated markers (such as CEA, CA19-9, AFP, etc.); at the clinical level, the tumor-killing effect can also be manifested as a prolongation of the subject's survival, such as an extension of progression-free survival (PFS), overall survival (OS), or disease-free survival (DFS). In this application, the terms "tumor killing" or "tumor cell killing" may also be collectively referred to as "antitumor activity" in some embodiments, and can be used to describe the ability of antibody-drug conjugates to inhibit, delay or reverse tumor progression in vivo and in vitro.

[0123] In some embodiments, the "killing effect" may refer to a decrease in any of the following indicators, such as the number of tumor cells, tumor volume, growth rate, weight, or number of metastases, observed under set experimental conditions, relative to a control group or an untreated group. For example, compared to the untreated group, the antibody-drug conjugate treated group may exhibit a reduction in the number of tumor cells of at least about 10%, 20%, 30%, 50%, or more; a reduction in tumor volume of at least about 15%, 30%, 50%, 70%, or more; and / or a decrease in tumor marker levels of about 10%, 30%, 50%, or more. In some embodiments, compared to subjects who did not receive the antibody-drug conjugate, subjects who received the antibody-drug conjugate had a survival time (e.g., progression-free survival, overall survival, disease-free survival) that was extended by about 10%, 30%, 50%, or more.

[0124] ADCC effect of antibody drug conjugates

[0125] In some embodiments, the antibody-drug conjugates described in this application, wherein the strength of the ADCC effect of the antibody does not affect its tumor-killing effect.

[0126] In some embodiments, the antibody-drug conjugates described in this application maintain good antitumor efficacy regardless of whether the ADCC effect of the antibody is enhanced or weakened. Fc-region-mediated effector functions (such as ADCC) play a crucial role in the efficacy of antibody drugs; enhancing the ADCC effect generally improves the antitumor activity of the drug, while weakening this function may lead to a decrease in overall antitumor activity. However, in the antibody-drug conjugates described in this application, although the ADCC effect is enhanced, the killing effect on tumor cells highly expressing CLDN18.2 is not increased; and even with a weakened ADCC effect, the antibody-drug conjugates still exhibit significant killing effects on tumor cells highly expressing CLDN18.2. The antibody-drug conjugates described in this application have stable antitumor effects, unaffected by significant changes in the strength of the ADCC effect, demonstrating the stability and broad clinical applicability of the antibody-drug conjugates described in this application.

[0127] In some embodiments, the ADCC effect of the antibody-drug conjugate is achieved through the Fc of the antibody therein.

[0128] The ADCC effect of the antibody or antibody-drug conjugate described in this application can be detected using methods commonly used in the art. For example, the antibody to be tested can be co-incubated with target cells expressing the target antigen, and the degree of target cell lysis can be measured in the presence of effector cells (such as natural killer (NK) cells derived from human peripheral blood or FcγRⅢa positive cells). Target cell lysis can be quantified by lactate dehydrogenase (LDH) release assay, chromium-51 release assay, flow cytometry, or a fluorescence / luminescent substrate-based detection method. This allows the determination of the ADCC effect intensity of the antibody relative to the control antibody.

[0129] In some embodiments, the enhanced or weakened ADCC effect is relative to antibody-drug conjugates containing wild-type heavy chain constant region sequences. In some embodiments, the comparison benchmark may further include: antibody-drug conjugates containing unengineered Fc-modified parent antibodies, or antibody-drug conjugates containing similar antibodies (e.g., human IgG1 isotype control antibodies).

[0130] In some embodiments, the antibody-drug conjugate exhibits enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). By enhancing the ADCC effect, the antibody-drug conjugate can exert cytotoxic effects not only through the drug-loaded portion but also through Fc-mediated effector function to achieve additional clearance of target cells, thereby improving antitumor or antipathogen activity in certain situations. For example, for cells with low levels of target antigen expression, the enhanced ADCC effect can compensate for insufficient drug delivery and improve overall therapeutic efficacy.

[0131] In some embodiments, the enhanced ADCC effect of the antibody-drug conjugate can be achieved by mutagenesis of the antibody's constant region. In some embodiments, the antibody in the antibody-drug conjugate may contain one or more amino acid mutations from the group consisting of F243L, R292P, Y300L, L235V, and P396L. In some embodiments, the antibody in the antibody-drug conjugate may contain mutations of F243L, R292P, Y300L, L235V, and P396L.

[0132] In some embodiments, the antibody-drug conjugate has attenuated antibody-dependent cell-mediated cytotoxicity (ADCC). By attenuating the ADCC effect, the antibody-drug conjugate can reduce non-target cytotoxicity due to immune effector function, decrease Fc receptor-mediated nonspecific binding or immune-related adverse reactions, thereby improving the selectivity and safety of drug delivery. For example, in some cases, the attenuated ADCC effect helps prolong the circulation time of the antibody in vivo and improve pharmacokinetic properties.

[0133] In some embodiments, the attenuated ADCC effect of the antibody-drug conjugate can be achieved by mutagenesis of the antibody constant region therein. In some embodiments, the antibody in the antibody-drug conjugate may contain one or more amino acid mutations from the group consisting of L234F, L235E, and P331S. In some embodiments, the antibody in the antibody-drug conjugate may contain mutations of L234F, L235E, and P331S.

[0134] In some embodiments, the reduced ADCC effect of the antibody-drug conjugate can be achieved by modifying the glycosylation site in the Fc region of the antibody. For example, fucosylation modification at the N297 site of the antibody can weaken its binding ability to the Fcγ receptor, thereby reducing ADCC. Furthermore, in some embodiments, the payload can be specifically bound to the N297 site via glycosylation coupling, thereby altering the glycosylation environment at this site while conjugating the drug, achieving the goal of reducing the ADCC effect.

[0135] Pharmaceutical compositions and uses

[0136] On the other hand, this application provides a pharmaceutical composition comprising the antibody-drug conjugate described in this application. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0137] In some embodiments, the pharmaceutical composition is used to treat a disease.

[0138] On the other hand, this application provides the use of the antibody-drug conjugate described herein in the preparation of a drug for treating a disease.

[0139] On the other hand, this application provides a method for treating a disease, which includes administering an effective amount of the antibody-drug conjugate described in this application to a subject in need.

[0140] On the other hand, this application provides the use of the antibody-drug conjugate described in this application in the treatment of diseases.

[0141] In some embodiments, the disease includes diseases associated with CLDN18.2. In some embodiments, the disease includes diseases associated with the upregulation of CLDN18.2 expression. For example, the disease can be a pathological condition in which CLDN18.2 is significantly elevated relative to normal levels in tissues or cells.

[0142] In some embodiments, the disease includes a tumor. In some embodiments, the disease includes a CLDN18.2 positive tumor. In some embodiments, the tumor includes gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer, and / or leukemia.

[0143] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0144] Example

[0145] Table 1 shows information on the commercial experimental materials (including but not limited to reagents, animals, cells, kits, and strains) used in the examples.

[0146] Table 1 shows the commercial experimental materials used in the examples.

[0147]

[0148] Example 1: Fabrication of an ADC targeting CLDN 18.2

[0149] In this embodiment, the ADC coupling was performed by Yantai Mabpharm International Biopharmaceutical Co., Ltd.

[0150] The amino acid sequences of the antibody HCDR1 are shown in SEQ ID NO: 1, HCDR2 in SEQ ID NO: 2, HCDR3 in SEQ ID NO: 3, LCDR1 in SEQ ID NO: 4, LCDR2 in SEQ ID NO: 5, and LCDR3 in SEQ ID NO: 6; the amino acid sequence of VH is shown in SEQ ID NO: 7, and the amino acid sequence of VL is shown in SEQ ID NO: 8. The amino acid sequence of the heavy chain of antibody M19I wt is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain is shown in SEQ ID NO: 12. Antibody M19I was obtained by mutating the Fc segment of M19I wt to F243L / R292P / Y300L / L235V / P396L. The amino acid sequence of the heavy chain of M19I is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is shown in SEQ ID NO: 12. The CDR is defined using the Kabat method.

[0151] ADCs were prepared by randomly conjugating cysteine ​​residues using Mc-GGFG-DXD, Mc-VC-PAB-MMAE, and Mc-VC-PAB-R-SN38 as linkers-loaders to antibody M19I or antibody M19I wt. The DAR values ​​of the ADCs obtained by conjugating antibody M19I or antibody M19I wt to MMAE, DXD, and SN38 were 4±0.5, 2±0.5, 4±0.5, and 8±0.5, respectively.

[0152] For M19I coupling, the three linker payloads were prepared and scaled up under different conditions. Mc-GGFG-DxD and Mc-VC-PAB-MMAE (DAR4) were prepared at 2.2 eq TCEP, 6 eq drug ratio, and 25℃; MC-VC-PAB-R-SN38 (DAR8) was prepared at 8.0 eq TCEP, 10 eq drug ratio, and 25℃. For M19I wt coupling, MMAE was prepared and scaled up under 2.4 eq TCEP, 6 eq drug ratio, and 25℃.

[0153] The purity of the prepared M19I-MMAE-DAR4, M19I-MMAE-DAR2, M19I wt-MMAE-DAR4, M19I wt-MMAE-DAR2, M19I-DxD-DAR4, and M19I-SN38-DAR8 was greater than 95% as determined by SEC-HPLC.

[0154] Example 2: Binding activity of ADCs with different loads to CLDN 18.2

[0155] The binding activity of three ADCs (M19I-DXD, M19I-MMAE, and M19I-SN38) to the antigen CLDN18.2 was detected by ELISA. 100 μL / well of human CLDN18.2-his (1 μg / mL) was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After reaching room temperature, the plates were washed six times with PBST (PBS containing 0.05% Tween-20) and blocked with 2% BSA at 37°C for 2 hours. The blocking solution was discarded, and the washing process was repeated. ADCs were serially diluted and added to the ELISA plates, incubated at 37°C for 2 hours, and then washed. 100 μL / well of Goat anti-human IgG Fc (HRP) was diluted 1:10000, incubated at 37°C for 2 hours, and then incubated with TMB for 5 minutes. The reaction was stopped by adding 50 μL / well of ELISA stop solution, and the OD450 was read by the ELISA reader. Four-parameter fitting was performed using GraphPad Prism, and the EC50 values ​​were compared.

[0156] Experimental results are as follows Figure 1 As shown, the results indicate that coupling with different loads does not affect the affinity of the antibody for the CLDN 18.2 antigen, and the ADC with MMAE load has better affinity than the ADCs with DXD and SN38 loads.

[0157] Example 3: Binding specificity of the naked antibody of the ADC to CLDN18.2

[0158] CHO-K1, CHO-K1-CLDN18.1, and CHO-K1-CLDN18.2 cell lines were collected, washed twice with RPMI 1640, and CellTrace was dissolved in DMSO to 10 mM. The CellTrace cells were then further diluted with RPMI 1640 to 25 μM, 2.5 μM, and 0.2 μM, respectively. 1e6 CHO-K1-CLDN18.2, CHO-K1-CLDN18.1, and CHO-K1 cells were incubated at 37°C for 1 h. After incubation, cells were washed twice with RPMI 1640 and reselected to 6E6 cells / ml. 50 μL of each of the three cell types were mixed together, centrifuged at 300 x g, and the supernatant was discarded. 100 μL of 200 nM M19I was added, and the cells were incubated at 4°C for 1 h. After washing twice with FACS buffer (PBS containing 2% FBS), AF488 anti-human IgG fluorescent secondary antibody was added at a ratio of 1:500, and the cells were incubated at 4°C for 30 min. After washing with FACS buffer, the cells were resuspended in 200 μL, and the PE and AF488 fluorescence signals were detected by flow cytometry.

[0159] Flow cytometry results as followsFigures 2-3 As shown, after staining cells with different concentrations of Celltrace, CHO-K1, CHO-K1-CLDN18.1, and CHO-K1-CLDN18.2 cells were distinguished by the intensity of PE fluorescence. Furthermore, the binding of M19I to CLDN18.1 or CLDN18.2 was evaluated using AF488 fluorescence signal in each cell population. The results showed that 25 μM, 2.5 μM, and 0.2 μM Celltrace effectively distinguished the three cell types. AF488 fluorescence signal was detected only in the CHO-K1-CLDN18.2 cell population, indicating that naked anti-M19I specifically recognizes CLDN18.2 but does not bind to CLDN18.1.

[0160] Example 4: Internalization activity of ADC (M19I-MMAE)

[0161] The endocytic activity of ADC (M19I-MMAE) was determined by flow cytometry. The NCI-N87-CLDN18.2 overexpressing cell line was collected, and the cell density was adjusted to 2e5 cells / ml. 100 μL / well was seeded into 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. The antibody and ADC were diluted to 160 nM with complete culture medium, and 4x pHrodo was prepared. The sample was mixed with 4x pHrodo at a 1:1 volume ratio and incubated at room temperature in the dark for 5 min. The labeled sample was then diluted 4-fold with complete culture medium, resulting in 6 concentration gradients. After discarding 50 μL / well of supernatant, 50 μL / well of the serially diluted sample was added to each well of the 96-well cell culture plate. The plates were incubated at 37°C with 5% CO2 for 48 h before detection. After culturing for 48 h, cells were collected into 96-well V plates, centrifuged at 300 x g for 5 min, the supernatant was removed, and the cells were washed twice with FACS buffer (PBS containing 2% FBS) and subjected to live / dead staining. The cells were then resuspended in 200 μL of FACS buffer and loaded onto the instrument. Endocytosis activity was detected by pHrodo signaling.

[0162] ADCs, after internalization, produce red fluorescence in the acidic intracellular environment. The intensity of the red fluorescence was measured by flow cytometry to compare the strength of endocytosis. Experimental results are as follows: Figure 4 As shown, compared with the negative control group (0 nM), the high concentration of ADC produced a significant red light shift, and the red light intensity was dose-dependent with the concentration of ADC. The results indicate that ADC (M19I-MMAE) exhibited good endocytic activity in NCI-N87-CLDN18.2.

[0163] Example 5: In vitro killing activity of ADCs with different loads

[0164] The killing effects of three ADCs (M19I-DXD, M19I-MMAE, and M19I-SN38) on CLDN18.2 overexpressing cell lines were compared. NUGC4-CLDN18.2 and NCI-N87-CLDN18.2 overexpressing cell lines were collected and cultured in complete medium (RPMI 1640 containing 10% FBS) to a cell density of 5e4 cells / ml. 100 μL / well (i.e., 5000 cells / well) was added to each well of a 96-well cell culture plate and incubated overnight at 37°C with 5% CO2. Different ADC drugs were serially diluted 5-fold using complete culture medium, resulting in 9 concentration points. 50 μL / well of each drug was added to a 96-well cell culture plate, resulting in final concentrations of 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.00256 nM. Positive control wells (cell-free, complete culture medium only) and negative control wells (cells only, no drug effect) were set up. Cell culture plates were incubated for 96 hours before cell viability was assessed. Before assay, CTG reagent was equilibrated to room temperature and 100 μL / well was added. After reacting at room temperature in the dark for 5 minutes, the liquid was transferred to a 96-well opaque white plate. Luminescence values ​​were read using a microplate reader. GraphPad Prism was used to perform a four-parameter fitting of drug concentration versus cell proliferation inhibition rate and compared with EC50. 50 value.

[0165] After treating CLDN18.2 overexpressing cells with different ADC drugs for 96 hours, cell viability was detected using CTG to compare the cell-killing activity of the drugs. The experimental results are as follows: Figures 5-6 As shown in the figure. The results indicate that, compared with the naked M19I antibody, all three ADC drugs exhibited strong cytotoxic effects in both cell lines, with M19I-MMAE showing significantly better cytotoxic effects than M19I-DXD and M19I-SN38.

[0166] Example 6: Relationship between the cytotoxic activity of the ADC drug M19I-MMAE (DAR4) and the expression level of CLDN18.2

[0167] The cytotoxic activity of M19I-MMAE (DAR4) against three cells with different CLDN18.2 expression levels was detected. BXPC3 was a cell line with high CLDN18.2 expression, NUGC4 was a cell line with moderate CLDN18.2 expression, and A375 was a cell line without CLDN18.2 expression. The three cell lines were collected, and the cell density was adjusted to 5e4 cells / ml in complete medium (RPMI 1640 containing 10% FBS). 100 μL / well (5000 cells / well) was added to each 96-well cell culture plate, and the cells were incubated overnight at 37°C with 5% CO2. M19I-MMAE was serially diluted 5-fold in complete culture medium to nine concentration points. 50 μL / well of each well was added to a 96-well cell culture plate, resulting in final concentrations of 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, and 0.00256 nM. Positive control wells (cell-free, complete culture medium only) and negative control wells (cells only, no drug effect) were set up. Cell culture plates were incubated for 96 hours before cell viability was assessed. Before assay, CTG reagent was equilibrated to room temperature and 100 μL / well was added. After reacting at room temperature in the dark for 5 minutes, the liquid was transferred to a 96-well opaque white plate. Luminescence values ​​were read using a microplate reader. GraphPad Prism was used to perform a four-parameter fitting of drug concentration-cell proliferation inhibition rate and compare the IC50 values. 50 value.

[0168] Table 2. In vitro killing activity of ADCs against cells with different CLDN18.2 expression levels.

[0169]

[0170] Experimental results are as follows Figures 7-9 As shown, compared with Isotype-MMAE, M19I-MMAE has a significantly stronger killing effect on CLDN18.2 positive cells BXPC3 and NUGC4 than on negative cells A375, and the higher the expression level of CLDN18.2, the more obvious the killing effect.

[0171] Example 7 Bystander effect of ADC drug M19I-MMAE (DAR4)

[0172] Antibody-adjuvant antibodies (ADCs) bind specifically to target antigens on the surface of tumor cells, enter tumor cells via endocytosis, are degraded in lysosomes, release their payload, and diffuse across the cell membrane to neighboring tumor cells, producing a bystander effect. Hek293 cells overexpressing Luciferase were co-incubated with NCI-N87-CLDN18.2 cells for 96 hours at controlled effector-to-target ratios (E:T) of 5:1 or 10:1. The killing effect of the ADC drug M19I-MMAE (DAR4) on Hek293 antigen-negative cells was evaluated by detecting Luciferase signaling. 20,000 NCI-N87-CLDN18.2 cells / well and 4,000 Hek293-Luciferase cells / well were added to 96-well cell culture plates. The ADC drug was serially diluted 5-fold, resulting in 9 concentration points with final system concentrations of 1500, 300, 60, 12, 2.4, 0.48, 0.096, 0.0192, and 0.00384 nM, respectively. After incubation for 96 hours, the luciferase signal was detected using a microplate reader.

[0173] The results are as follows Figures 10-11 As shown, the results indicate that M19I-MMAE (DAR4) exhibits a significant bystander effect at E:T ratios of 5:1 or 10:1, effectively inhibiting the proliferation of Hek293_Luciferase antigen-negative cells.

[0174] Example 8: Effects of Fc F243L / R292P / Y300L / L235V / P396L mutations in ADCs on tumor cell killing and bystander effect.

[0175] Using the CTG detection in Example 5 and the Luciferase detection in Example 7, the activity of Hek293_luciferase in tumor cells and antigen-negative cells after ADC treatment was detected, and the differences in tumor cell killing and bystander effect between M19I-MMAE (F243L / R292P / Y300L / L235V / P396L) and M19I wt-MMAE (wild-type Fc) were compared.

[0176] Experimental results are as follows Figures 12-14 As shown, the results indicate that while Fc mutation enhances ADCC activity, it does not increase the cytotoxicity or bystander effect of ADCs. With a DAR value of 4, the cytotoxic effects of the two ADCs on NCI-N87-CLDN18.2 and NUGC4-CLDN18.2 cells remained consistent. Under the same effector-to-target ratio, the two ADCs showed essentially no difference in their inhibitory effects on antigen-negative cells (Hek293_luciferase), and their bystander effects were also essentially the same.

[0177] Example 9: Comparison of different DAR values ​​in ADC

[0178] 9.1 Internalization effect of ADCs with different DAR values ​​on cells with different CLDN18.2 expression levels

[0179] Referring to Example 4, A375 (negative), NUGC4 (intermediate expression of CLDN18.2) and BXPC3 (high expression of CLDN18.2) cells were collected respectively. After 2e4 cells were co-incubated with pHrodo-labeled antibody or ADC for 48 h, pHrodo red fluorescence was detected by flow cytometry.

[0180] The results are as follows Figures 15-17 As shown, the results indicate that the endocytic activity of equal concentrations (40 nM) M19I wt-MMAE (DAR4) and M19I wt-MMAE (DAR2) in the same cell type was consistent with that of the naked antibody, meaning that endocytic activity was not affected by differences in DAR values. Endocytosis efficiency was only related to the expression level of CLDN18.2 on the cells and the drug concentration. When the same drug was applied to different cells, the higher the CLDN18.2 expression level, the better the endocytosis effect. In CLDN18.2-positive cells, the endocytosis effect was dose-dependent with the drug concentration.

[0181] 9.2 Cell-killing effects of ADCs with different DAR values ​​on cells with different CLDN18.2 expression levels

[0182] Referring to Example 6, NUGC4 (intermediate expression of CLDN18.2) and BXPC3 (high expression of CLDN18.2) cells were collected respectively. After 5e3 cells were treated with serially diluted drugs for 96 h, cell viability was detected by CTG.

[0183] Experimental results are as follows Figure 18 , 19 As shown, the results indicate that M19I wt-MMAE (DAR4) and M19I wt-MMAE (DAR2) did not show significant differences in killing effect on cells with different CLDN18.2 expression levels.

[0184] Example 10: In vivo tumor-suppressing effect of ADC

[0185] Balb / c nude mice were used to model NUGC4-CLDN18.2 cells for drug efficacy testing. 71 Balb / c nude mice (6-8 weeks old, female, catalog number: SM-014) were subcutaneously inoculated with NUGC4-CLDN18.2 cells (cell count: 5E6 / mouse + 30% Matrigel) and recorded as day 0. The survival status and subcutaneous tumor formation of the mice were observed every other day. Six days after tumor cell inoculation, mice were randomly divided into nine groups of six mice each, based on average tumor volume (~10⁹ mm³) and body weight. The average tumor volumes were as follows: Group 1, 109.22±3.66 mm³; Group 2, 109.46±2.57 mm³; Group 3, 109.35±2.54 mm³; Group 4, 109.43±2.44 mm³; Group 5, 109.47±2.38 mm³; Group 6, 109.44±2.41 mm³; Group 7, 109.69±2.53 mm³; Group 8, 109.98±2.43 mm³; and Group 9, 109.81±2.39 mm³. Groups were PG-D0 on the day of grouping. All mice were administered the drug via tail vein injection at a dose of 5 mL / kg. The drug was administered once.

[0186] Table 3. Dosage and mean tumor volume for each mouse group

[0187]

[0188] On day 31 after the start of drug administration, the mean tumor volume in the control group (Group 1) was 1531.38±163.54 mm3, and the mean tumor volumes in Groups 2, 3, 6, and 7 were 1014.15±197.16 mm3, 1361.62±191.76 mm3, 1044.73±205.29 mm3, and 1529.15±217.61 mm3, respectively. The tumor volume inhibition rates (TGI) were 36.39%, 11.95%, 34.23%, and 0.19%, respectively, with no statistically significant difference compared to Group 1 (P > 0.05). The mean tumor volumes in Groups 4, 5, 8, and 9 were 207.34±199.87 mm3, 7.30±1.98 mm3, 161.69±82.69 mm3, and 0.00±0.00 mm3, respectively. The tumor volume inhibition rate (TGI) of mm3 was 93.12%, 107.18%, 96.36% and 107.72%, respectively, which were statistically significant compared with Group 1 (P < 0.01).

[0189] On day 31 after the start of drug administration, the average weight gain rates of the mice in each group were 1.7%, 6.8%, 5.6%, 4.7%, 6.0%, 5.3%, 6.9%, 6.0%, and 6.2%, respectively.

[0190] At the end of the experiment (PG-D31), the animals were euthanized, and the tumors were removed and weighed. The average tumor weight of the control group (Group 1) was 1.2236 ± 0.1146 g; the average tumor weights of Groups 2, 3, 4, 5, 6, 7, 8, and 9 were 0.8461 ± 0.1613 g, 1.0165 ± 0.1429 g, 0.1612 ± 0.1508 g, 0.0137 ± 0.0028 g, 0.7914 ± 0.1538 g, 1.2352 ± 0.1795 g, 0.0979 ± 0.0517 g, and 0.0000 ± 0.0000 g, respectively. Significant differences in tumor weight were observed between the 3 mpk and 10 mpk administration groups and the Group 1 control group.

[0191] The effects of M19I wt-MMAE and M19I-MMAE on tumor growth were evaluated, and the experimental results are as follows: Figure 20 , 21 As shown, both M19I wt-MMAE and M19I-MMAE effectively inhibited tumor growth at single intravenous administration of 3 mg / kg and 10 mg / kg. No significant drug toxicity was observed during the experiment; the mice maintained good overall health and stable weight, and no mice died during the experiment, indicating that both M19I wt-MMAE and M19I-MMAE have good safety profiles.

[0192] Example 11: Killing effect of M19I mutant-MMAE (L234F / L235E / P331S) on CLDN 18.2 positive tumor cells

[0193] M19I mutant-MMAE (L234F / L235E / P331S) is an antibody against the Fc region of M19I wt, targeting the L234F, L235E, and P331S sites. The amino acid sequence of the heavy chain constant region of the naked anti-M19I mutant is shown in SEQ ID NO: 19, the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO: 20, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 21, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO: 12.

[0194] NCI-N87-CLDN18.2 cell lines were collected and the cell density was adjusted to 1E6 cells / ml with complete medium (RPMI 1640 containing 10% FBS). 40 μL / well was added to each well of a 96-well cell culture plate. The drug was serially diluted 10-fold with complete medium to eight concentration points, with 20 μL / well added to each well. The plates were centrifuged at 1000 rpm for 5 min and incubated at 38°C for 30 min. ADCC bioassay Effector cell V variant cells were collected and the cell density was adjusted to 2E6 cells / ml with complete medium. 40 μL / well (E:T = 2:1) was added to each well of a 96-well cell culture plate, resulting in final antibody concentrations of 200, 20, 2, 0.2, 0.02, 0.002, 0.0002, and 0.00002 nM. After centrifugation at 1000 rpm for 5 min, the plates were incubated at 37°C with 5% CO2 for 5 h. Before incubation, equilibrate the Luciferase assay reagent to room temperature. After incubation, add 100 μL / well of the assay reagent and react at room temperature in the dark for 5 min. Then, read the fluorescence using a microplate reader to detect the ADCC effect of M19I wt-MMAE and M19I mutant-MMAE (L234F / L235E / P331S).

[0195] Referring to Example 6, NUGC4 (intermediate expression of CLDN18.2) and BXPC3 (high expression of CLDN18.2) cells were collected respectively. After 5e3 cells were treated with serially diluted drugs for 96 h, cell viability was detected by CTG.

[0196] The detection results of ADCC effect of naked antibody are as follows Figure 22 As shown, compared with M19I, the ADCC effect of the M19I mutant (L234F / L235E / P331S) was significantly weakened, but its killing effect on tumor cells was not reduced.

[0197] Example 12: ELISA antigen-binding activity assay of purified humanized antibody proteins before and after humanization.

[0198] The amino acid sequence of antibody VH before humanization is shown in SEQ ID NO: 7, and the amino acid sequence of antibody VL is shown in SEQ ID NO: 8. The amino acid sequence of antibody VH after humanization is shown in SEQ ID NO: 15, and the amino acid sequence of antibody VL is shown in SEQ ID NO: 16.

[0199] The binding ability of purified humanized antibody proteins to human CLDN18.2-his protein was compared using ELISA. One day in advance, coat the plates. Dilute CLDN18.2-His to 1ug / ml using 1x ELISA coating buffer. Take an ELISA plate and add 100ul / well, incubate overnight at 4°C. Remove the plate and equilibrate to room temperature. Prepare PBST (0.05% Tween-20 in 1x PBS) and blocking buffer (2% BSA in PBST). Discard the supernatant, add 300ul / well of PBST to wash the plate, repeating 6 times. Add 300ul / well of blocking buffer and block at 37°C for 2 hours. Prepare dilution buffer (0.5% BSA in PBST) and use it to dilute the purified humanized antibody protein to 1ug / ml, performing a 5-fold serial dilution for a total of 8 concentration points. After blocking, wash the plate 6 times with PBST, add 100ul / well of the diluted antibody or humanized antibody supernatant, and incubate at 37°C for 2 hours. Dilute the detection antibody Goat anti-human IgG 1:10000 with the dilution buffer. Fc(HRP); After incubation, wash the plate 6 times with PBST, add 100 μL / well of detection antibody, and incubate at 37°C for 1 hour; After detection antibody incubation, wash the plate 6 times with PBST, add 100 μL / well of 1x TMB, and develop color at room temperature in the dark for about 3 minutes; Add 50 μL / well of ELISA stop solution to stop color development, and read the OD450 value using a multi-mode microplate reader.

[0200] The test results showed that the humanized antibody protein had a strong binding to the human CLDN18.2-his protein, and the humanized antibody did not affect the binding affinity.

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof targeting tight junction protein 18.2 (CLDN 18.2) and a payload, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1-3 and the VL comprises LCDR1-3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6, wherein the payload has the following structure: .

2. The antibody-drug conjugate according to claim 1, wherein the VH and VL are selected from any one of the following groups of VH and VL: (1) the VH contains the amino acid sequence shown in SEQ ID NO: 7 and the VL contains the amino acid sequence shown in SEQ ID NO: 8; or (2) the VH contains the amino acid sequence shown in SEQ ID NO: 15 and the VL contains the amino acid sequence shown in SEQ ID NO:

16.

3. The antibody-drug conjugate according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) and a light chain constant region (CL).

4. The antibody-drug conjugate according to claim 3, wherein both the heavy chain constant region and the light chain constant region are derived from human immunoglobulins.

5. The antibody-drug conjugate according to claim 4, wherein the heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 subtypes, and the light chain constant region is κ or λ type.

6. The antibody-drug conjugate according to claim 3, having attenuated antibody-dependent cell-mediated cytotoxicity (ADCC).

7. The antibody-drug conjugate according to claim 3, wherein the heavy chain constant region comprises one or more amino acid mutations from the group consisting of: L234F, L235E, and P331S.

8. The antibody-drug conjugate according to claim 3, wherein the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 13, and the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:

10.

9. The antibody-drug conjugate of claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain and light chain are selected from any one of the following groups of heavy and light chains: (1) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 11, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 12; or (2) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 12; or (3) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

18.

10. The antibody-drug conjugate according to claim 1, wherein the CLDN18.2 is human CLDN18.

2.

11. The antibody-drug conjugate according to any one of claims 1-10, comprising Ab-[L1-L2-loading] m The structure is defined as follows: Ab represents the antibody or antigen-binding fragment targeting CLDN 18.2, L1 represents the linker connected to Ab, and L2 represents the linker connected to the payload.

12. The antibody-drug conjugate according to claim 11, wherein m is an integer from 1 to 10.

13. The antibody-drug conjugate according to claim 12, wherein m is 4.

14. The antibody-drug conjugate of claim 11, wherein L1 and / or L2 are selected from the group consisting of: cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, and dicarboxylic acid-based linkers.

15. The antibody-drug conjugate of claim 11, wherein the L1 is linked to the Ab via a thiol, azide, or amide group on the Ab.

16. The antibody-drug conjugate according to claim 15, wherein the L2 is selected from the group consisting of: peptides, N-succinimide-3-(2-pyridyldithio)propionate (SPDP), N-succinimide-4-(2-pyridyldithio)valerate (SPP), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimide-iodoacetate (SIA), N-succinimide-4-iodoacetylaminobenzoate (SIAB), and maleimide PEG. NHS, N-4-(maleimidemethyl)cyclohexylcarboxylic acid succinamide ester (SMCC), N-sulfono(4-maleimidemethyl)cyclohexylcarboxylic acid sulfosuccinate (sulfon-SMCC), and 2,5-dioxopyrrolidinyl-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrolidinyl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaoctadecane-1-ester (CX1-1).

17. The antibody-drug conjugate according to claim 11, wherein -L1-L2- has a -Mc-VC-PAB structure.

18. The antibody-drug conjugate according to claim 11, having an Ab-[Mc-VC-PAB-MMAE]4 structure.

19. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1-18.

20. The pharmaceutical composition of claim 19, further comprising a pharmaceutically acceptable carrier.

21. Use of the antibody-drug conjugate of any one of claims 1-18 in the preparation of a medicament for treating CLDN 18.2 positive tumors.

22. The use according to claim 21, wherein the tumor includes gastric cancer, gastroesophageal junction cancer, gastroesophageal cancer, esophageal adenocarcinoma, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, head and neck cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, bile duct cancer, bladder cancer, and / or leukemia.

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