An antibody, pharmaceutical conjugate thereof and use thereof

Abstract

Antibodies and antibody-drug conjugates, relate to antibodies targeting Claudin 18.2 (CLDN18.2) and antibody-drug conjugates (ADCs) and compositions containing the antibodies and ADC molecules and therapeutic applications thereof.

Description

Technical Field

[0001] This invention relates to antibodies and antibody-drug conjugates, and more particularly to antibodies and antibody-drug conjugates (ADCs) targeting Claudin18.2 (CLDN18.2), as well as compositions containing said antibody or ADC molecules and their therapeutic applications. Background Technology

[0002] Gastric cancer is the third leading cause of cancer death worldwide, with an overall 5-year survival rate of less than 20% for patients with metastatic gastric cancer and gastroesophageal junction cancer (EGJA). Despite numerous studies, the need for gastric cancer treatment remains unmet due to the large number of patients. This is particularly true for HER2-negative patients who lack effective targeted therapies, resulting in limited treatment options and a precarious survival outlook.

[0003] Claudin18.2 is a member of the Claudin family of tight junction proteins, mediating tight junctions between cells. Claudin18.2 is highly expressed in gastric and pancreatic cancers, but is only expressed in limited quantities in gastric mucosal cells in normal tissues and is not expressed in other normal tissues. Therefore, it can serve as an ideal target for the development of ADC (antibody-drug conjugate) drugs.

[0004] Claudin18.2 is a four-transmembrane protein containing two extracellular loops and one intracellular loop. Its sequence is very similar to that of Claudin18.1, a protein in the same family, differing only in the first extracellular loop by eight amino acids. Claudin18.1 is specifically expressed in the lungs, therefore developing ADC drugs that recognize only Claudin18.2 and not Claudin18.1 presents significant challenges.

[0005] Data presented at the 2016 ASCO meeting showed that Astellas's Claudin18.2-targeting chimeric antibody IMAB362, combined with chemotherapy, achieved remarkable results in treating patients with locally advanced or metastatic gastric cancer, extending progression-free survival and overall survival by nearly half. However, no monoclonal antibody or ADC targeting Claudin18.2 has yet been approved for marketing. Therefore, developing differentiated, superiorly targeted anti-Claudin18.2 antibodies and ADCs with excellent in vivo tumor-killing effects could provide gastric cancer patients with a wider range of better treatment options. Invention Overview

[0007] In a first aspect, the present invention provides antibody-drug conjugates (ADCs) having the following formula (I) or pharmaceutically acceptable salts or solvates thereof:

[0008] Ab-[LD] q (I)

[0009] in,

[0010] Ab indicates anti-Claudin18.2 antibody.

[0011] L represents the connector.

[0012] D indicates a cytotoxic or cell-inhibiting drug, such as a topoisomerase I inhibitor, and

[0013] q = 1 to 20, for example, q = 1-8, 2-8, 4-8, or 6-8.

[0014] The Ab contains three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 1 or 3 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 2, preferably wherein the CDRs are defined according to Kabat or IMGT or a combination thereof.

[0015] In a second aspect, the present invention provides compositions comprising the ADC of the present invention or pharmaceutically acceptable salts or solvates thereof.

[0016] In a third aspect, the present invention provides the use of the ADC of the present invention or its pharmaceutically acceptable salts or solvates and compositions thereof in the treatment of Claudin18.2 positive tumors.

[0017] In a fourth aspect, the present invention provides an anti-Claudin18.2 antibody, pharmaceutical compositions thereof, and uses thereof. Attached Figure Description

[0018] Figure 1A-1I The results showed that the expression positivity rates of Claudin18.2 or Claudin18.1 in different cell lines of Example I-1 were detected using FACS technology and fluorescently labeled antibodies. An isotype control antibody was used as the control. Among them: Figure 1A The HEK293T-Claudin18.2 (human) stable cell line was detected using PE fluorescently labeled anti-human Claudin18.2 antibody. Figure 1B The HEK293T-Claudin18.1 (human) stable cell line was detected using APC fluorescently labeled anti-human Claudin18.1 antibody. Figure 1C The L929-Claudin18.2 (human) stable cell line was detected using APC fluorescently labeled anti-human Claudin18.2 antibody. Figure 1D The stable CHO-Claudin18.2 (human) cell line was detected using APC fluorescently labeled anti-human Claudin18.2 antibody. Figure 1EThe HEK293T-Claudin18.2 (cynomolgus monkey) stable cell line was detected using PE fluorescently labeled anti-cynomolgus monkey Claudin18.2 antibody. Figure 1F The HEK293T-Claudin18.2 (rat) stable cell line was detected using APC fluorescently labeled anti-rat Claudin18.2 antibody. Figure 1G The HEK293T-Claudin18.2 (mouse) stable cell line was detected using APC fluorescently labeled anti-mouse Claudin18.2 antibody. Figure 1H The NUGC4-Claudin18.2 (human) stable cell line was detected using APC fluorescently labeled anti-human Claudin18.2 antibody. Figure 1I The NUGC4 tumor cell line was detected using APC fluorescently labeled anti-human Claudin18.2 antibody.

[0019] Figure 2A-2B The results showed that in the cell ELISA assay, the anti-human Claudin18.2 humanized antibody and the reference antibody IMAB362 were detected in relation to NUGC4-Claudin18.2 (human) (…). Figure 2A ) and NUC4 Figure 2B Cell affinity.

[0020] Figures 3A-3C The results showed that flow cytometry was used to detect anti-human Claudin18.2 humanized antibody and reference antibody IMAB362 versus HEK293T-Claudin18.2 (human) Figure 3A ), NUC4-Claudin18.2 (people) Figure 3B ) and NUC4 Figure 3C Cell affinity.

[0021] Figures 4A-4C The results showed that flow cytometry was used to detect the anti-human Claudin18.2 humanized antibody and the reference antibody IMAB362 against HEK293T-Claudin18.2 (cynomolgus monkey). Figure 4A HEK293T-Claudin18.2 (rat) Figure 4B ) and HEK293T-Claudin18.2 (mice) ( Figure 4C Cell affinity.

[0022] Figure 5 The results show the ADCC killing activity of the anti-human Claudin18.2 humanized antibody against HEK293T-Claudin 18.2 (human) cells.

[0023] Figure 6 The results show the assay results of the CDC killing activity of the anti-human Claudin18.2 humanized antibody against HEK293T-Claudin 18.2 (human) cells.

[0024] Figure 7 The pharmacokinetic assay of the anti-Claudin18.2 humanized antibody in rats was shown.

[0025] Figures 8A-8B The results showed that the anti-Claudin18.2 humanized antibody ADC interacted with L929-Claudin18.2 cells before and after toxin conjugation. Figure 8A ) and NUGC4 cells ( Figure 8B The combination of ).

[0026] Figures 9A-9E The results showed that, in in vitro experiments, the anti-Claudin18.2 humanized antibody ADC drug was effective against HEK293T-Claudin 18.2 cells (…). Figure 9A and 9B HEK293T-Claudin 18.1 cells Figure 9C and 9D ) and NUGC4-Claudin18.2 cells ( Figure 9E (The killing effect of)

[0027] Figure 10 The study demonstrated the tumor-suppressive efficacy of the anti-Claudin18.2 humanized antibody ADC in the NUGC-4CDX model.

[0028] Figure 11 The study showed the effect of the anti-Claudin18.2 humanized antibody ADC drug on the body weight of mice. Invention Details

[0030] definition

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0032] When a trade name is used in this document, unless the context otherwise indicates, the trade name includes the product formulation of the trade name product, the generic name of the drug, and the active pharmaceutical ingredient.

[0033] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0034] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0035] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0036] In this paper, the terms "cludin 18," "Claudin 18," or "CLDN18" are used interchangeably to refer to the CLDN18 member of the Claudin family of tight junction proteins. CLDN18 is a four-terminal transmembrane protein with both its N- and C-termini located in the cytoplasm. It has two extracellular loops, CLDN1 and CLDN2. The first extracellular loop (i.e., extracellular loop 1), located near the N-terminus, consists of approximately 53 amino acids on average, while the second extracellular loop (i.e., extracellular loop 2) consists of approximately 30 amino acids. CLDN18 includes two splice variants, CLDN18.1 and CLDN18.2, which differ by 8 amino acids in extracellular loop 1. In normal tissues, CLDN1 expression is confined to the lungs, while CLDN2 expression is confined to the gastric mucosa.

[0037] In this document, the term "CLDN18.1" preferably refers to human CLDN18.1, such as human CLDN18.1 having the amino acid sequence under NCBI_057453.1. The term also covers Claudin18.1 from other species such as cynomolgus monkeys, mice and rats, but unless otherwise specified, the term refers to human CLDN18.1 in this document.

[0038] In this document, the term "CLDN18.2" preferably refers to human CLDN18.2, such as human CLDN18.2 having the amino acid sequence under NCBI_001002026.1. The term also covers Claudin18.2 from other species, such as cynomolgus monkey Claudin18.2 (e.g., amino acid sequence under XP_015300615.1), mouse Claudin18.2 (e.g., amino acid sequence under NP_001181850.1), and rat Claudin18.2 (e.g., amino acid sequence under NP_001014118.1). Unless otherwise specified herein, the term refers to human CLDN18.2.

[0039] In this paper, the terms “CLDN18”, “CLDN18.1”, and “CLDN18.2” also cover post-translational modification variants and conformational variants, as well as mutants, especially naturally occurring variants, allele variants, and species homologs.

[0040] In this document, the term "CLDN18.2 positive" cell refers to a cell that is positive for CLDN18.2 expression on its cell surface, such as cancer cells, modified cancer cells, or modified non-tumor cells. The CLDN18.2 expression level on the cell surface can be determined by any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection methods or immunofluorescence staining methods; and optionally, cells positive for CLDN18.2 expression on their cell surface can be determined by comparison with a predetermined reference value. Preferably, the CLDN18.2 expression level measured on positive cells is at least two times higher than the predetermined reference value. The predetermined reference value can be determined by those skilled in the art using conventional methods. In one embodiment, the predetermined reference value can be the CLDN18.2 expression level value measured on normal cells outside the gastric mucosa using the same assay method. In another embodiment, the predetermined reference value can be determined using the FACS method as described in Example I-1 of this application by comparing the fluorescence staining intensity produced on cells by the specific CLDN18.2 antibody relative to the isotype control antibody. For example, the predetermined reference value can be set to be at least 10 times greater than the average fluorescence staining intensity of the isotype control antibody.

[0041] In some embodiments, preferably, CLDN18.2 positive cells express sufficiently high levels of CLDN18.2 on their surface, thereby being able to bind to CLDN18.2 specific antibodies in detection methods such as immunofluorescence staining or FACS, and generate detection signals higher than predetermined reference values, and / or generate signals significantly different from non-cancerous normal cells other than the gastric mucosa (e.g., at least 2 times higher, or preferably at least 10 times or 100 times higher, or more).

[0042] In this document, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), chimeric or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0043] In this article, "whole antibody" (which may be used interchangeably with "full-length antibody," "complete antibody," and "intact antibody") refers to an immunoglobulin molecule containing at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The variable region is a domain in the antibody's heavy or light chain that participates in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen but exhibits various effector functions. The antibody's light chain can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant region. The antibody's heavy chain can be divided into five main types based on the amino acid sequence of its constant region: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further subdivided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2.

[0044] In this document, the terms "antibody fragment" and "antigen-binding fragment" are used interchangeably to refer to a molecule that is not a complete antibody but contains the portion of the complete antibody used to bind the antigen bound by that complete antibody. As those skilled in the art will understand, for the purpose of antigen binding, antibody fragments typically contain amino acid residues from the "complementarity-determining region" or "CDR". Antibody fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Examples of antibody fragments include, but are not limited to, Fab, scFab, disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv, disulfide-linked scFv, diabody, triabody, tetrabody, and minibody. In some embodiments of the invention, antibody fragments contain cysteine ​​residue portions for forming interchain disulfide bonds between the light and heavy chains, such as cysteine ​​residues in the Fab region and / or hinge region of an IgG1 antibody, to provide amino acid residue sites that can be used for thiol coupling chemistry. In other embodiments of the invention, the antibody fragment includes cysteine ​​residues introduced into the Fc region to provide amino acid residue sites that can be used for thiol coupling chemistry.

[0045] In this paper, the term "immunoglobulin" refers to a protein with a structure that naturally forms antibodies. For example, IgG immunoglobulins are heterotetrameric proteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). From the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). Accordingly, in this paper, when an antibody is referred to as an IgG antibody, it means that the antibody is a heterotetrameric protein with an IgG-like immunoglobulin structure. In IgG antibodies, typically the VH-CH1 of the heavy chain pairs with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG antibody essentially consists of two Fab molecules linked by immunoglobulin hinge regions and two dimerized Fc regions. IgG immunoglobulins can be subdivided based on the sequence of their heavy chain constant regions, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), and γ4 (IgG4). The light chains of IgG immunoglobulins can also be subdivided into one of two types, referred to as κ and λ, based on the amino acid sequence of their constant domains. In some embodiments, the antibody according to the invention is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In other embodiments, the antibody according to the invention is an IgGκ or IgGλ antibody, for example, an IgG1κ or IgG1λ antibody.

[0046] In this document, the terms "complementarity-determining region" or "CDR region" or "hypervariant region" are used interchangeably to refer to regions within the antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. In this document, the CDRs of the antibody heavy and light chains are sequentially numbered starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. CDRs located within the antibody heavy chain variable domain are also referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. Within a given amino acid sequence of a light chain variable region or heavy chain variable region, the CDR sequence can be determined using various methods known in the art. For example, annotations of CDRs in a given light chain variable region or heavy chain variable region can be obtained at http: / / www.abysis.org / abysis / , including CDR sequences defined based on Kabat, AbM, Chothia, Contact, and IMGT. Furthermore, CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence. Unless otherwise stated, in this invention, when referring to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it means the numbering 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)).

[0047] In this paper, "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that participates in the binding of the antibody to its antigen. The heavy chain variable region (VH) and light chain variable region (VL) can be further subdivided into hypervariable regions (HVR, also known as complementarity-determining regions (CDRs)) interspersed with more conserved regions (i.e., framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some aspects, one or more residues in one or both of the two variable regions (i.e., VH and / or VL) can be modified, for example, by modifying one or more CDR regions and / or by modifying one or more framework regions, especially by substituting conserved residues, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen-binding ability) of the antibody molecule before the modification. In still other aspects, antibody variable regions can be modified by CDR transplantation. Since the CDR sequence is responsible for most antibody-antigen interactions, recombinant antibody variants that mimic the properties of known antibodies can be constructed. In these antibody variants, the CDR sequence from a known antibody is grafted onto the scaffold region of a different antibody with different properties. The properties of the mutated and / or modified antibody or ADC conjugate containing it can be evaluated in in vitro or in vivo assays, such as target antigen binding properties or other desired functional properties, such as ADC endocytosis, pharmacokinetics, and in vivo tumor-killing activity.

[0048] The term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0049] In this document, the term "humanized antibody" refers to an antibody to which a CDR sequence derived from another mammalian species, such as a mouse lineage, is appended to a human framework sequence. Additional framework region modifications can be made within the human framework sequence, and / or additional amino acid modifications can be made to the CDR sequence, for example, to perform antibody affinity maturation. In some embodiments herein, the antibody of the present invention is a humanized antibody having a framework region sequence "derived" from a specific human lineage sequence. Here, "derived" means that the amino acid sequence of the antibody framework region has at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identity with the corresponding framework region amino acid sequence encoded by the human lineage immunoglobulin gene, and that the antibody retains antigen-binding activity, for example, having an equivalent (e.g., ±10%) CLDN18 binding affinity with 25C7A5-HZ1 or -HZ2, or with 2D3A11-HZ1 or -HZ2.

[0050] In this document, the term "isolated" antibody refers to an antibody that has been separated from its components in its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which can be determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0051] The term "epitope" refers to the antigenic region to which an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein. In this invention, preferably, the antibody according to the invention binds to the natural epitope of human Claudin18.2; more preferably, it binds to the natural epitope of human Claudin18.2 expressed on the cell surface. Therefore, in a preferred embodiment, a cell-based assay is used to detect the binding of the antibody of the invention to the natural epitope of human Claudin18.2.

[0052] In this document, the term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the cell ELISA assay described in the embodiments herein, and another specific method is the FACS assay described in the embodiments herein. In one embodiment, in a cell-based assay, such as when performed as described in Example I, an antibody is considered to have high affinity for Claudin18.2 if it has an EC50 value that is equivalent to (i.e., ±10%) or smaller and / or a maximum binding signal value that is equivalent to (i.e., ±10%) or higher than that of the reference antibody IMAB362.

[0053] The term "specific binding" indicates that an antibody selectively or preferentially binds to a target antigen relative to a non-target antigen. In this document, if an antibody binds to human Claudin18.2 expressed on the cell surface (particularly with high affinity) but does not bind to or substantially does not bind to human Claudin18.1 expressed on the cell surface when measured in an antigen binding assay, such as in a FACS assay or cell ELISA as described in the examples, then the antibody should be considered to "specifically bind to human Claudin18.2". The binding of an antibody to human Claudin18.2 or human Claudin18.1 expressed on the cell surface can be measured in a cell ELISA or FACS to determine the antibody's binding specificity to Claudin18.2.

[0054] In this document, an antibody is referred to as not binding or substantially not binding to human Claudin 18.1 when it does not have significant affinity for human Claudin 18.1 in cell-based assays and does not bind significantly to human Claudin 18.1, particularly not producing a detectable signal significantly distinct from the background (this is also referred to herein as "no nonspecific binding" to human Claudin 18.1). Preferably, in this invention, the nonspecific binding of the antibody according to the invention to Claudin 18.1-expressing cells is determined by FACS assay, for example, as described in Example I, using recombinant cell lines (such as the recombinant HEK293T stable cell line) with a Claudin 18.1 expression positivity rate greater than 95% or greater than 98%. Depending on the specific assay method chosen, those skilled in the art can select suitable negative and / or positive controls. For example, isotype controls can be used to eliminate background staining caused by nonspecific binding of the antibody to the cells.

[0055] However, as those skilled in the art will understand, antibodies that specifically bind to human Claudin18.2 and have no nonspecific binding to human Claudin18.1 may be cross-reactive with Claudin18.2 proteins from other species. Hereinafter, the term "cross-reactivity" refers to the ability of an antibody to bind to Claudin18.2 from different species. For example, in some embodiments, antibodies specific to human Claudin18.2 according to the invention may also bind to Claudin18.2 from other species (e.g., cynomolgus monkey, mouse, and / or rat Claudin18.2). Methods for determining cross-reactivity include those described in the examples and standard assays known in the art, such as those using flow cytometry or cell ELISA. Species cross-reactivity of antibodies is advantageous in some cases. For example, when a target antibody exhibits species cross-reactivity with preclinical experimental animals, such as mice, rats, or primates, it will facilitate preclinical safety and efficacy evaluation of the target antibody and its ADC conjugates prior to their therapeutic or diagnostic applications in humans.

[0056] In this document, the term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. For example, the antibody according to the invention may be an IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibody, having a heavy chain constant region of the aforementioned immunoglobulin type. For example, the antibody of the invention may be an IgG1 antibody having a human IgG1 constant region. Furthermore, the invention contemplates not only antibodies employing natural sequence constant regions but also antibodies containing variant sequence constant regions.

[0057] The term "Fc region" in this article is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions.

[0058] In this document, the term "native sequence Fc region" encompasses the Fc region sequences of various naturally occurring immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to immunoglobulin functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In still other embodiments, the human IgG heavy chain Fc region carries at the N-terminus a hinge sequence or a partial hinge sequence of a native immunoglobulin, such as the sequence E216 to T225 or the sequence D221 to T225 according to EU designations.

[0059] In this document, the term "variant sequence Fc region" refers to a polypeptide containing a modified Fc region relative to the native Fc region sequence. The modification can be the addition, deletion, or substitution of amino acid residues. Substitution can include both naturally occurring and non-natural amino acids. The purpose of the modification may be to alter the binding of the Fc region to its receptor and the effector function thereby induced.

[0060] In this paper, the term "effective function" refers to those biological activities attributable to the Fc region of immunoglobulins that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Depending on the intended use of the antibody molecule, the effector function of a target antibody can be altered relative to an antibody molecule possessing a wild-type Fc region, for example, by reducing or eliminating ADCC or CDC activity.

[0061] In this document, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. ADCC in humans is primarily mediated by natural killer cells (NK cells). In ADCC, an antibody binds to an antigen displayed on the surface of a target cell. The FcγRIIIA region on the surface of NK cells recognizes the Fc region of the antibody, thereby activating the NK cells and releasing perforin and granolithase, leading to lysis and apoptosis of the target cells. The ADCC activity of an antibody can be evaluated using a luciferin reporter system such as that described in Example I. In some cases, when the ADCC effector activity of an antibody is not desired, the ADCC activity of the antibody can be removed by modifying the Fc region of the antibody.

[0062] In this paper, the term "CDC" refers to complement-dependent cytotoxicity. In CDC, the Fc region of an antibody binds to the C1q complement molecule, thereby forming a membrane attack complex that leads to the clearance of target cells. IgM is the most potent isotype for complement activation. Both IgG1 and IgG3 are also highly effective in guiding CDC via the classical complement activation pathway. The CDC activity of an antibody can be detected using a guinea pig serum killing assay, as described in Example I. In some cases, when the CDC effector activity of an antibody is not desired, the CDC activity of the antibody can be removed by modifying the Fc region.

[0063] In this document, the term "receptor-mediated endocytosis" refers to the process triggered by the binding of a ligand to a corresponding receptor on the cell surface, in which the ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment. The endocytosis rate can be determined, for example, by the method described in Example I, to characterize the receptor-mediated endocytic activity of the antibody. As shown in the examples, the antibody of the present invention can initiate Claudin18.2 receptor-mediated endocytosis upon binding to Claudin18.2 expressed on the cell surface, and the antibody of the present invention possessing this endocytic property can be effectively used as a tool for delivering antitumor drugs into cancer cells after conjugation with a drug (e.g., a small molecule toxic drug molecule) to form an ADC.

[0064] In this paper, “sequence identity” refers to the degree of sequence similarity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. The “sequence identity percentage” can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence identity percentage. Optimal alignments for determining the sequence identity percentage can be performed 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 suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequence being compared or within the target sequence region.

[0065] In this invention, the percentage of amino acid sequence identity, with respect to the antibody sequence, is determined by optimally aligning the candidate antibody sequence with the given antibody sequence, preferably according to the Kabat numbering rules. In this document, without specifying a comparison window (i.e., the target antibody region to be compared), alignment along the entire length of the given antibody sequence will be applicable. In some embodiments, sequence identity may be distributed across the entire heavy chain variable region and / or the entire light chain variable region, or the percentage sequence identity may be limited only to the framework region, while the sequence corresponding to the CDR region remains 100% identical.

[0066] In this document, "reference antibody IMAB362" refers to the anti-Claudin18.2 antibody constructed using the amino acid sequences of the heavy and light chain variable regions of the 175D10 monoclonal antibody disclosed in patent CN 101312989 B. In the context of comparison with reference antibody IMAB362, the reference antibody IMAB362 will have the same antibody structure as the antibody to be compared in the portions outside the variable regions, for example, when both have heavy and light chain constant region structures, they will have the same heavy and light chain constant region sequences.

[0067] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, for example, fluorine or chlorine.

[0068] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms. Specifically, alkyl groups have 1 to 10 carbon atoms, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, as used herein, the term "C1-C6 alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms, examples of which include methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, or tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), hexyl (including n-hexyl, 2-methylpentyl, 3-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl), etc.

[0069] As used herein, the term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, containing at least one double bond. Specifically, alkenyl groups have 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0070] As used herein, the term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one triple bond. Specifically, an alkynyl group has 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkynyl" refers to a straight-chain or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc.

[0071] As used herein, the term "alkylene" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight-chain or branched saturated alkane. Specifically, alkylenes have 1 to 10 carbon atoms, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, as used herein, the term "C1-C6 alkylene" refers to a straight-chain or branched alkylene having 1 to 6 carbon atoms, including, but not limited to, methylene, ethylene, propylene, butylene, etc.

[0072] As used herein, the term "alkenyl" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight-chain or branched unsaturated alkene containing at least one double bond. Specifically, alkenyl groups have 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinylidene, propenide, allylidene, butenide, pentenide, and hexenide.

[0073] As used herein, the term "ynynyl" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight-chain or branched unsaturated alkyne containing at least one triple bond. Specifically, ynynyl groups have 2 to 8, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 ynynyl" refers to a straight-chain or branched ynynyl group having 2 to 6 carbon atoms, such as ethynylene, propynylene, propynylene, butynylene, penynylene, and hexynylene.

[0074] As used herein, the term "cycloalkyl" refers to a monocyclic, fused, bridged, or spirocyclic non-aromatic monovalent hydrocarbon ring structure having a specified number of ring atoms. It can be saturated or unsaturated, for example, containing one or more double bonds. The cycloalkyl group may contain three or more carbon atoms in the ring, for example, 3-18, 3-10, or 3-8 carbon atoms, such as C1. 3-10 cycloalkyl, C 3-8 cycloalkyl, C 3-6 cycloalkyl, C 5-6 Cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0075] As used herein, the term "heterocyclic" or "heterocyclic group" refers to a 5-20 membered (e.g., 5-14, 5-8, 5-6) aromatic or non-aromatic monocyclic, bicyclic, or polycyclic ring system having 1-4 independent heteroatom ring members selected from N, O, or S. One or more N, C, or S atoms in the heterocycle may be oxidized. Preferably, the heterocycle is a 5-10 membered ring system, and is a monocyclic or fused bicyclic ring. Representative examples include, but are not limited to, pyrrolidine, azacyclic butane, piperidine, morpholine, tetrahydrofuran, tetrahydropyran, benzofuran, benzothiophene, indole, benzopyrazole, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazine, and isoxazole. It should be understood that this term includes heteroaryl groups as defined herein.

[0076] The term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example, 6-12 carbon atoms in the ring moiety. Preferably, the aryl group is (C6-C6) 10Aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which may optionally be substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamide, heterocyclic, etc.

[0077] The term "heteroaryl" refers to a 5-20 membered (e.g., 5-14, 5-8, 5-6) aromatic monocyclic or polycyclic ring system containing 1-4 heteroatoms selected from N, O, or S, which may be substituted or unsubstituted. Preferably, the heteroaryl is a 5-10 membered ring system, which is a monocyclic or fused bicyclic ring. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furanyl, 2- or 3-pyrroleyl, 2-, 4- or 5-imidazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isooxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridinyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl.

[0078] The term "heteroalkyl" refers to a stable straight-chain or branched hydrocarbon that is fully saturated or contains 1 to 3 degrees of unsaturation, consisting of the indicated number of carbon atoms and one to ten, preferably one to three, heteroatoms selected from O, N, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, Si, and S may be located at any internal position of the heteroalkyl group or at the position where the heteroalkyl group is attached to the rest of the molecule. Representative examples of heteroalkyl groups include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. At most two heteroatoms can be consecutive, such as -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Typically, C1 to C4 heteroalkyl or heteroalkylene groups have 1 to 4 carbon atoms and 1 or 2 heteroatoms, while C1 to C3 heteroalkyl or heteroalkylene groups have 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some respects, heteroalkyl and heteroalkylene compounds are saturated.

[0079] Unless otherwise specified, the term “substituted” as used in defining various groups herein means that the corresponding group can be substituted by, for example but not limited to, the following groups: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, halogen, cyano, nitro, azide, carboxyl, hydroxyl, mercapto, amino, mono- or dialkylamino, mono- or dicycloalkylamino, mono- or diarylamino, mono- or diheterocyclicamino, mono- or diheteroarylamino, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-oxy, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-thio, alkyl- or cycloalkyl The groups are alkyl- or heterocyclic- or heteroaryl- or aryl-acyl, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-acylamino, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-acyloxy, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-sulfonyl, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-sulfonyloxy, alkyl- or cycloalkyl- or heterocyclic- or heteroaryl- or aryl-sulfonylamino, or the optional substituted amino-formyl group described above, and each of these groups being further substituted by the remaining optional substituents, wherein the various groups are as defined herein. Examples of substituents include, but are not limited to, one or more groups independently selected from the following: halogen, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, C(O)-amino, OCOCH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO2, phenyl, piperidinyl, piperazine, and pyrimidinyl.

[0080] As used herein, the term “substitution” or “substituted” means that one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a specified atom are replaced by a specified group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and forms a stable compound. Combinations of substituents and variables are only permitted if such combinations form a stable compound.

[0081] In this document, the term "linker" refers to the bifunctional portion in a drug-antibody conjugate that links a drug to an antibody. The linkers of the present invention may have multiple components (e.g., in some embodiments, a linker responsible for conjugating the antibody; a degradable peptide unit; and optionally a spacer).

[0082] In this document, the term "PEG unit" refers to an organic moiety comprising repeating ethylene-oxygen subunits (PEG or PEG subunits), which can be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxygen subunits). Polydisperse PEG is a non-homogeneous mixture of size and molecular weight, while monodisperse PEG is typically purified from a non-homogeneous mixture and therefore has a single chain length and molecular weight. Preferred PEG units comprise discrete PEG, which is a compound synthesized stepwise rather than via a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length.

[0083] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention, and that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugates of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0084] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0085] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0086] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) coupled to the Ab portion (described herein) to the Ab portion in an ADC conjugate. In some embodiments described herein, the DAR may be determined by q in Formula I, for example, the DAR may be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the small molecule drug portion (D) coupled to the Ab portion (described herein) in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 20, for example 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example 1.0-8.0, 2.0-6.0, for example 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, a range with two of these values ​​as endpoints.

[0087] The term "drug" as used herein encompasses any substance effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants).

[0088] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or destruction. Examples of cytotoxic agents include, but are not limited to, camptothecin derivatives, oliguria, chlortetracycline, maytansine alkaloids, ricin, ricin A chain, carbetastatin, pyruvic acid, sea haretoxin, doxorubicin, doxorubicin, daunomycin, tacrolimus, cisplatin, CC1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraquinone, actinomycin, diphtheria toxin, pseudomonadoxime exotoxin (PE)A, PE40, absinthecin, absinthecin A chain, arbuscular mycotoxin A chain, and α-arbuscular mycotoxin. Sarcin, white tree toxin, mitogellin, retstrictocin, phenolmycin, inomycin, curicin, croton toxin, kazimycin, Sapaonaria officinalis inhibitors, glucocorticoids and other chemotherapeutic agents, and radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioactive isotopes of Lu, including Lu177.

[0089] The term "small molecule drug" refers to low-molecular-weight organic compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0090] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

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

[0092] The terms "drug combination" or "combination product" refer to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the ADC molecule or antibody of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. In some embodiments, the ADC molecule or antibody of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0093] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0094] In this document, the terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, 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 particular, a subject is a human.

[0095] The terms “tumor” and “cancer” are used interchangeably herein to refer to a physiological disorder in mammals characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the present invention include gastric cancer, pancreatic cancer, or gastroesophageal junction cancer, including metastatic forms of those cancers. The term also encompasses all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0096] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0097] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0098] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0099] When used herein, the term "effective amount" refers to such an amount or dose of the antibody-drug conjugate or a combination thereof of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention.

[0100] As used herein, the term "therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody-drug conjugate or its composition or combination thereof are less than the therapeutically beneficial effects. Relative to untreated individuals, the "therapeutic effective amount" preferably achieves at least about 30%, and more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% inhibition of measurable parameters (e.g., tumor volume).

[0101] When used in this context, the term "preventive effective dose" refers to the amount at which the desired preventive outcome is effectively achieved, at the required dose and for the required duration. Typically, because the prophylactic dose is administered in subjects before or at an early stage of the disease, the preventive effective dose will be less than the therapeutic effective dose.

[0102] In this invention, the term "CLDN18.2 positive" tumor refers to a tumor organ or tissue in which at least a portion of cancer cells are positive for CLDN18.2 cell surface expression. In some embodiments, the proportion of CLDN18.2 positive cancer cells in the cancer tissue or cancer cell population is at least 10% or 20%, preferably at least 30%, 40%, 50%, 60%, 70%, 75%, or 80%. In still other embodiments, at least 40% or at least 50%, preferably 60%, 70%, 80%, or 90% of cancer cells in the tumor tissue or organ are positive for CLDN18.2 surface expression. In some embodiments, compared to the corresponding tissues and organs of healthy subjects, cancer subjects treated according to the method of this invention have an increased level of CLDN18.2 cell surface expression in diseased tissues or organs, for example, an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, or more. In one embodiment, the CLDN18.2-positive tumor is selected from: gastric cancer, esophageal cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, and their metastases, particularly gastric cancer metastases. In one embodiment, the cancer is adenocarcinoma, particularly advanced adenocarcinoma. Particularly preferred cancers are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung, and ovary. In some embodiments, the cancer treated according to the invention is HER2-negative. In yet another embodiment, the CLDN18.2-positive tumor is a digestive system cancer or its metastases. In one embodiment, the cancer is selected from gastric cancer, esophageal cancer (particularly lower esophageal cancer), cancer of the esophagogastric junction, and gastroesophageal cancer. In a preferred embodiment, the cancer is, for example, CLAUDIN 18.2-positive and HER2-negative gastric cancer. In another preferred embodiment, the cancer is gastroesophageal cancer, such as metastatic, refractory, or recurrent advanced gastroesophageal cancer; in yet another embodiment, the cancer is metastatic gastric cancer or gastroesophageal junction cancer (EGJA).

[0103] Various aspects of the present invention will be further described in the following sections.

[0104] ADC section of this invention

[0105] I. Antibody-drug conjugates

[0106] In one aspect, the present invention provides an antibody-drug conjugate (ADC) having the following formula (I) or a pharmaceutically acceptable salt or solvate thereof:

[0107] Ab-[LD] q (I)

[0108] in,

[0109] Ab indicates anti-Claudin18.2 antibody.

[0110] L represents the connector.

[0111] D indicates a cytotoxic or cell-inhibiting drug, such as a topoisomerase I inhibitor, and

[0112] q represents an integer or decimal from 1 to 20, for example, q = 1-10, 1-8, 2-8, 4-8, or 6-8.

[0113] The components of the ADC coupling of the present invention and the ADC couplings of the present invention composed therefrom are described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the ADC coupling of the present invention may include any such combination of features.

[0114] Antibody Ab Unit

[0115] Through in-depth research, the inventors have developed and provided a humanized anti-Claudin18.2 monoclonal antibody with excellent properties. As shown in the examples, the antibody of the present invention not only exhibits high binding affinity and high specificity against cells expressing human Claudin18.2 (especially cells with low expression levels), but also possesses favorable properties such as Claudin18.2 antigen-mediated endocytic activity, stability, and / or pharmacokinetic properties. Therefore, it is suitable as a molecular component for conjugation with cytotoxic drugs to form antibody-drug conjugates (ADCs). As shown in the examples, the ADC conjugates formed from the antibodies of the present invention exhibit potent in vivo tumor-killing effects while also demonstrating good drug tolerability.

[0116] Therefore, in some aspects, the present invention provides antibody-drug conjugates (ADCs) comprising the antibody of the present invention that specifically binds to Claudin18.2 as the Ab unit of the conjugate of formula I of the present invention.

[0117] In some embodiments, Ab in formula (I) of the present invention comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO: 1 or 3 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO: 2, and preferably, wherein the CDRs are defined according to Kabat or IMGT or a combination thereof.

[0118] In some embodiments, Ab in formula (I) of the present invention comprises three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs), wherein:

[0119] (i) As defined by IMGT, HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 6, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 7, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 8, LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 9, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 11; or

[0120] (ii) According to Kabat's definition, HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 12, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 13, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 14, LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 15, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 17.

[0121] In one embodiment, the Ab in formula (I) of the present invention comprises the heavy chain and light chain variable region sequences or variants thereof of either the exemplary antibodies 25C7A5-HZ1 and -HZ2 of the present invention, for example, an antibody having the same CDR sequence as one of the exemplary antibodies and having the same or different framework region sequences.

[0122] In one embodiment, Ab in formula (I) of the present invention comprises a heavy chain variable region, wherein: the heavy chain variable region comprises:

[0123] -The amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it, or

[0124] -The amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0125] In one embodiment, Ab in formula (I) of the present invention comprises a light chain variable region, wherein: the light chain variable region comprises:

[0126] -The amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0127] In some preferred embodiments, the Ab in formula (I) of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 1 or 3, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. Preferably, the antibody sequence is humanized. More preferably, the heavy chain variable region of the antibody has a human-derived framework region sequence, and preferably has amino acid Q or E at position H6 according to the Kabat number in the heavy chain variable region, preferably having amino acid Q.

[0128] In some further preferred embodiments, Ab in formula (I) of the present invention comprises the heavy chain variable region of SEQ ID No: 1 and the light chain variable region of SEQ ID No: 2.

[0129] In some further preferred embodiments, Ab in formula (I) of the present invention comprises the heavy chain variable region of SEQ ID No: 3 and the light chain variable region of SEQ ID No: 2.

[0130] In some embodiments, the Ab in formula (I) of the present invention preferably further comprises a heavy chain constant region and / or a light chain constant region of the antibody. Preferably, the heavy chain constant region is a heavy chain constant region derived from human immunoglobulins. Preferably, the light chain constant region is a light chain constant region derived from human immunoglobulins. In some aspects, the heavy chain constant region contained in the Ab can be any isotype or subtype, such as the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4 isotypes, and preferably the IgG1, IgG2 or IgG4 heavy chain constant region, especially the human IgG1 heavy chain constant region. In still other aspects, the light chain constant region contained in the Ab can be a κ light chain constant region or a λ light chain constant region, especially the human κ light chain constant region.

[0131] In some embodiments, Ab in formula (I) of the present invention comprises a human IgG1 heavy chain constant region. Preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence comprising at least one, two, or three, but not more than 20, 10, or 5 amino acid changes relative to the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 4.

[0132] In some embodiments, Ab in formula (I) of the present invention comprises a human κ light chain constant region. Preferably, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence comprising at least one, two, or three, but not more than 20, 10, or 5 amino acid changes relative to the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 5.

[0133] In some embodiments, the Ab in formula (I) of the present invention is a full-length antibody comprising a heavy chain constant region and a light chain constant region. In some embodiments, the Ab in formula (I) of the present invention has a tetrameric structure formed by two light chains and two heavy chains. In still other embodiments, the Ab in formula (I) of the present invention is an IgG antibody, particularly an IgG1 antibody. In a further embodiment, the Ab in formula (I) of the present invention is a humanized antibody.

[0134] In some preferred embodiments, the Ab in formula (I) of the present invention comprises a heavy chain and a light chain, wherein: the heavy chain comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 18 or 19, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In other preferred embodiments, the Ab in formula (I) of the present invention comprises a heavy chain and a light chain, wherein: the light chain comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0135] In some preferred embodiments, Ab in formula (I) of the invention comprises heavy chains and light chains selected from:

[0136] (a) A heavy chain containing the amino acid sequence of SEQ ID NO: 18, and a light chain containing the amino acid sequence of SEQ ID NO: 20; and

[0137] (b) A heavy chain containing the amino acid sequence of SEQ ID NO: 19, and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0138] This invention also considers variants of any of the antibodies described above. The antibody variants of this invention preferably retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the original antibody. More preferably, the modification does not result in the antibody variant losing its binding to the antigen, but optionally imparts properties such as increased antigen affinity and different effector functions. It is understood that the heavy chain variable region or light chain variable region of the antibody, or each CDR region, can be modified individually or in combination. Furthermore, the Fc region of the antibody can also be modified. Modification of the Fc region can be performed alone or in combination with the modifications to the framework and / or CDR regions described above. The Fc region can be modified, for example, to alter one or more functions of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the antibodies of this invention can be chemically modified (e.g., linked to PEG) or their glycosylation patterns can be altered.

[0139] To form an ADC conjugate, the antibody according to the invention can be conjugated to a toxin-linker using some natural linker sites thereon. Such natural linker sites include the thiol group of cysteine ​​and the amino group of lysine. Typically, a relatively defined drug-antibody ratio (DAR) can be achieved by applying a disulfide bridge between the heavy and light chains of the antibody. Therefore, in one embodiment, after reducing the interchain disulfide bonds of the antibody, the toxin is conjugated to the antibody of the invention via thiol chemistry to form an ADC conjugate of formula (I). Alternatively, introducing artificial linker sites into the antibody can also be considered to achieve more site-specific conjugation.

[0140] Drug D Unit

[0141] The drug D unit of the antibody-drug conjugate is also referred to herein as the payload of the ADC drug. There are no particular limitations on the drug D that can be used in the ADC of this invention; it can be any drug or prodrug that is toxic or inhibitory to cells. Those skilled in the art can select appropriate drug molecules as the payload of the ADC based on the desired mechanism of action and cell-killing effect.

[0142] In some embodiments, drug D is a cytotoxic agent. Various cytotoxic agents with different mechanisms suitable as payloads have been reported in the art, including, but not limited to,

[0143] (1) Microtubule inhibitors / disruptors: for example, but not limited to, auristatin class (e.g., MMAE or MMAF), maytansin derivatives (e.g., DM2, DM4), tubulosynins, cryptomycins, antimitotic EG5 inhibitors (e.g., spindle kinesin KSP inhibitors).

[0144] (2) DNA damaging agents: for example, but not limited to, pyrrolobenzodiazepines (e.g., pyrrolo[2,1-c][1,4]benzodiazepine (PBD)), ducamycin, indolinobenzodiazepine; Duocannycins; Calicheamicins;

[0145] (3) Topoisomerase inhibitors: for example, but not limited to, camptothecins (e.g., ixotecan and its derivative Dxd);

[0146] (4) Others: apoptosis inducers (Bcl-xL inhibitors), thailanstatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, carbamycin.

[0147] Drug D in formula (I) of the present invention can be any compound selected from the above. In some embodiments, drug D is an antitumor growth inhibitor selected from maytansine derivatives, calichiomycin derivatives, and olritamine derivatives. In one embodiment, drug D is a microtubule inhibitor / stabilizer, such as vinca alkaloids, vincristine, paclitaxel, and docetaxel. In one embodiment, drug D is a DNA synthesis inhibitor, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, and cladribine. In one embodiment, drug D is a DNA topoisomerase inhibitor, such as topoisomerase I inhibitors (e.g., camptothecin derivatives) and topoisomerase II inhibitors (e.g., actinomycin D, doxorubicin, mitoxantrone).

[0148] In some preferred embodiments, the drug D of the ADC according to the invention is a topoisomerase I inhibitor. A typical example of a topoisomerase I inhibitor is a camptothecin class of drugs, such as, but not limited to, camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, exatecan, topotecan, belotetcan, irinotecan, SN-38, and FL118, and derivatives thereof. See, for example, Vesela Kostova et al., The Chemistry Behind ADCs, Pharmaceuticals 2021, 14, 442. https: / / doi.org / 10.3390 / ph14050442 References to WO2019 / 195665 are incorporated herein by reference.

[0149] In some embodiments of the present invention, the drug D of the ADC of the present invention is selected from the following camptothecin class of drugs:

[0150] - Camptothecin and its derivatives, for example

[0151] Where R A Selected from, for example, hydrogen, optionally substituted alkyl groups, wherein the substituents include, but are not limited to, hydroxyl and amino groups, wherein the amino or hydroxyl moiety may be substituted or unsubstituted, for example, substituted by alkyl, alkyl acyl, or alkyl sulfonyl groups; in one embodiment, the drug D is

[0152]

[0153] -10,11-methylenedioxy CPT (also known as MDCPT or FL118) and its derivatives, for example,

[0154] Wherein RB is selected from, for example, hydrogen, cycloalkyl, phenyl, optionally substituted alkyl, wherein the substituents include, but are not limited to, halogen, hydroxyl, optionally substituted alkoxy, cycloalkyl, heterocycloalkyl, phenyl, amino, wherein the amino moiety may optionally be substituted; in one embodiment, the drug D is

[0155]

[0156] -10-hydroxy CPT (also known as HCPT) and its derivatives, for example,

[0157]

[0158] Among them, R C Selected from, for example, substituted alkyl and cycloalkyl groups; R' C Selected from, for example, H, alkyl acyl, or optionally substituted heterocyclic alkyl acyl groups; in one embodiment, the drug D is 7-ethyl-10-hydroxy CPT (SN-38) or its prodrug irinotecan (CPT-11), or the drug D is topotecan.

[0159]

[0160] -Exatecan and its derivatives, for example

[0161] Among them, R D Selected from, for example, H, optionally substituted alkyl groups, optionally substituted alkyl-C(=O)-, wherein the substituent is, for example, -OH or a substituted or unsubstituted amino group; in one embodiment, the drug D is:

[0162] In one implementation scheme, the drug D is:

[0163]

[0164] F118 ((20S)-10,11-methylenedioxycamptothecin) was obtained by high-throughput screening of a compound library. Compared with other camptothecin derivatives, FL118 has been shown to have much higher in vivo and in vitro anticancer activity in many different cancer types. In addition to inhibiting topoisomerase I, FL118 can also selectively inhibit the gene promoter activity and endogenous expression of anti-apoptotic proteins such as survivin, XIAP, cIAP2 and Mcl-1. See Xiang Ling et al., A Novel Small Molecule FL118 That Selectively Inhibits Survivin, Mcl-1, XIAP and cIAP2 in a p53-Independent Manner, Shows Superior Antitumor Activity, PLoS ONE7(9):e45571.doi:10.1371 / journal.pone.0045571. Although the use of FL118 alone is severely hampered by its extremely poor water solubility and toxic side effects, as shown in the examples, the conjugate formed by combining this compound with the antibody and linker of the present invention not only effectively exerts the tumor-killing effect of the compound, but also overcomes the defects of the compound. The resulting conjugate has low aggregation and good animal tolerability.

[0165] Therefore, in some preferred embodiments of the present invention, the drug D of the ADC of the present invention is an FL118 derivative, especially FL118 substituted at the 7-position.

[0166] In some embodiments, drug D in formula (I) of the present invention is a camptothecin drug comprising the structure of formula D:

[0167]

[0168] in,

[0169] R x R y Each is independently selected from H, halogen, -OH, C1-C6 alkyl, or R. x and R y Together with their respective attached carbon atoms, they form 5-6 membered heterocycles with one or two selected from N, S, and O;

[0170] R aSelected from H, halogen, -OH, optionally substituted C1-C8 alkyl or C3-C8 alkynyl or C3-C8 alkenyl, optionally substituted C1-C8 alkoxy, optionally substituted C3-C8 cycloalkyl, optionally substituted phenyl, optionally substituted heterocyclic alkyl, optionally substituted heteroaryl.

[0171] Preferably, R a Selected from:

[0172] hydrogen;

[0173] C3-C8 cycloalkyl;

[0174] Phenyl;

[0175] C1-C8 alkyl or C3-C8 alkynyl or C3-C8 alkenyl groups optionally substituted with the following substituents: halogen, hydroxyl, C1-C4 alkoxy groups optionally substituted with NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2, C3-C8 cycloalkyl, heterocyclic alkyl, phenyl, and NR. 1 R 2 ,

[0176] Where R 1 and R 2 Selected independently from each other

[0177] hydrogen;

[0178] C1-C8 alkyl groups optionally substituted with substituents selected from the following: hydroxyl, amino, amino group substituted with one or two C1-C4 alkyl groups, amino group substituted with one or two C1-C4 hydroxyalkyl groups, amino group substituted with (C1-C4 hydroxyalkyl) and (C1-C4 alkyl) groups.

[0179] By 1 or 2 C3-C 10 cycloalkyl, C3-C 10 Heterocyclic alkyl, phenyl, or heteroaryl-substituted C1-C4 alkyl groups;

[0180] C3-C 10 cycloalkyl;

[0181] C3-C 10 Heterocyclic alkyl

[0182] C2-C6 heteroalkyl groups;

[0183] Mixed aromatics;

[0184] Optional halogenated phenyl groups;

[0185] C1-C8 alkyl-C(=O)-, optionally substituted with hydroxyl or amino groups;

[0186] Or, R 1 and R2 These atoms combine with their respective nitrogen atoms to form 5-, 6-, or 7-membered heterocycles having 0 to 3 substituents selected from halogens, C1-C4 alkyl, OH, C1-C4 alkoxy, NH2, NH(C1-C4 alkyl), and N(C1-C4 alkyl)2.

[0187] Each of the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl groups is independently and optionally substituted by 0 to 3 substituents selected from the following: OH, C1-C4 alkyl, C1-C4 alkoxy, NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2.

[0188] In some preferred embodiments, R x and R y Each is independently represented by H. In some other preferred embodiments, R... x For F and R y It is methyl. In some further preferred embodiments, R is... x and R y Together with their respective attached carbon atoms, they form a 5-membered heterocycle containing two oxygen atoms.

[0189] In some embodiments, the drug D in formula (I) of the present invention comprises the following formula D a or formula D b Camptothecin-based drugs with structural characteristics:

[0190] Especially formula (D) a Camptothecin-type drugs,

[0191] Among them, R a Selected from:

[0192] hydrogen;

[0193] C3-C8 cycloalkyl;

[0194] Phenyl;

[0195] C1-C8 alkyl, C3-C8 alkynyl, or C3-C8 alkenyl groups optionally substituted with the following substituents: halogen, hydroxyl, C1-C4 alkoxy, C3-C8 cycloalkyl, heterocyclic alkyl, phenyl, and NR, optionally substituted with NH2, NH(C1-C4 alkyl), and N(C1-C4 alkyl)2. 1 R 2 ,

[0196] Where R 1 and R 2 Selected independently from each other

[0197] hydrogen;

[0198] C1-C8 alkyl groups optionally substituted with substituents selected from the following: hydroxyl, amino, amino group substituted with one or two C1-C4 alkyl groups, amino group substituted with one or two C1-C4 hydroxyalkyl groups, amino group substituted with (C1-C4 hydroxyalkyl) and (C1-C4 alkyl) groups.

[0199] By 1 or 2 C3-C 10 cycloalkyl, C3-C 10 Heterocyclic alkyl, phenyl, or heteroaryl-substituted C1-C4 alkyl groups;

[0200] C3-C 10 cycloalkyl;

[0201] C3-C 10 Heterocyclic alkyl

[0202] C2-C6 heteroalkyl groups;

[0203] Mixed aromatics;

[0204] Optional halogenated phenyl groups;

[0205] C1-C8 alkyl-C(=O)-, optionally substituted with hydroxyl or amino groups;

[0206] Or, R 1 and R 2 These atoms combine with their respective nitrogen atoms to form 5-, 6-, or 7-membered heterocycles having 0 to 3 substituents selected from halogens, C1-C4 alkyl, OH, C1-C4 alkoxy, NH2, NH(C1-C4 alkyl), and N(C1-C4 alkyl)2.

[0207] Each of the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl groups is independently and optionally substituted by 0 to 3 substituents selected from the following: OH, C1-C4 alkyl, C1-C4 alkoxy, NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2.

[0208] In some implementation schemes, R a It is hydrogen.

[0209] In some implementation schemes, R a It is a C3-C8 cycloalkyl group (e.g., cyclopropyl, cycloheptyl, cyclopentyl).

[0210] In some implementation schemes, R a It is phenyl. In some other embodiments, R a It is a phenyl group substituted with NH2 or NH (C1-C4 alkyl).

[0211] In some implementation schemes, R aIt is a C1-C6 alkyl group, such as methyl, ethyl, propyl, 2-methylpropyl, butyl, isobutyl, 2,2-dimethyl-propyl, 2,2-dimethyl-butyl, n-hexyl, n-pentyl, 3-ethyl-pentyl.

[0212] In some implementation schemes, R a It is a C1-C4 alkyl group, which is optionally substituted with a hydroxyl group or a C1-C4 alkoxy group, wherein the alkoxy group is optionally further substituted with -NH2, -NH(C1-C4 alkyl) and -N(C1-C4 alkyl)2.

[0213] In some implementation schemes, R a It is a C2-C4 alkenyl or C2-C4 alkynyl group, which may optionally be substituted with a hydroxyl or amino group.

[0214] In some implementation schemes, R a It is a C1-C4 alkyl group substituted with one or two 5-6 membered heterocyclic alkyl groups selected from N, S and O, wherein the heterocyclic alkyl portion is optionally further substituted with C1-C4 alkyl groups, preferably, the heterocyclic alkyl group is piperazine or morpholino.

[0215] In some implementation schemes, R a It is -NR 1 R 2 Substituted C1-C4 alkyl groups, especially -methylene-NR 1 R 2 , where R 1 and R 2 Selected independently from:

[0216] hydrogen;

[0217] -C1-C4 alkyl;

[0218] -C1-C4 alkyl groups substituted with the following substituents:

[0219] -OH; -NH2; -NH(C1-C4 alkyl); -N(C1-C4 alkyl)2; -phenyl; -phenylene-NH2; -phenylene-C1-C4 alkoxy; -diphenyl; -C3-C 10 cycloalkyl; -C3-C 10 Cycloalkylene -C1-C4 alkyl; -C3-C 10 Heterocyclic alkyl groups, wherein the heterocyclic alkyl group has one or two heteroatoms selected from N, S, and O, preferably the heterocyclic alkyl group is piperidinyl:

[0220] Phenyl: Halogenated phenyl;

[0221] C3-C 10 cycloalkyl;

[0222] -C(=O)-C1-C4 alkyl-OH;-C(=O)-C1-C4 alkyl-NH2;

[0223] In some implementation schemes, R a It is -NR 1 R 2 Substituted C1-C4 alkyl groups, especially -methylene-NR 1 R 2 , where R 1 and R 2 It combines with the nitrogen atoms it is attached to to form a 5-, 6-, or 7-membered heterocyclic alkyl ring. In some respects, R 1 and R 2 The nitrogen atoms attached to each atom combine to form a 6-membered ring. In some aspects, the 6-membered ring is morpholino or piperazine.

[0224] In some preferred embodiments, R a Selected from: -C1-C4 alkylene-OH, -C1-C4 alkylene-O-C1-C4 alkylene-NH2, -C1-C4 alkylene-NH2, -C1-C4 alkylene-NH(C1-C4 hydroxyalkyl), -C1-C4 alkylene-N(C1-C4 hydroxyalkyl)2, -C1-C4 alkylene-N(C1-C4 aminoalkyl)(C1-C4 hydroxyalkyl), -C1-C4 alkylene-NH(C1-C 4-aminoalkyl), -C1-C4 alkylene-NH-C(=O)-C1-C4 alkylene-OH; -C1-C4 alkylene-NH-C(=O)-C1-C4 alkylene-NH2, -C1-C4 alkylene-N(C1-C4 alkyl)(C(=O)-C1-C4 alkylene-OH); -C1-C4 alkylene-N(C1-C4 alkyl)(C(=O)-C1-C4 alkylene-NH2).

[0225] In some further preferred embodiments, R a It is -C1-C4 alkylene-NH-C(=O)-C1-C4 alkylene-OH; -C1-C4 alkylene-NH-C(=O)-C1-C4 alkylene-NH2.

[0226] In some further preferred embodiments, R a It is -C2-C4 imene-NH2 or -C2-C4 imene-OH, for example, -CH=CH-CH2-NH2 or -CH=CH-CH2-OH.

[0227] In some further preferred embodiments, R a It can be -C1-C4 alkylene-OH, -C1-C4 alkylene-O-C1-C4 alkylene-NH2, or -C1-C4 alkylene-NH2.

[0228] In some preferred embodiments, R a It is a -C1-C4 alkylene-OH. In some respects, R a It is hydroxymethyl. In some respects, R a It is hydroxyethyl. In some respects, R a It is hydroxypropyl.

[0229] In some other, more preferred embodiments, R a It is a -C1-C4 alkylene-NH2. In some respects, R a It is an aminomethyl group. In some respects, R a It is aminoethyl. In some respects, R a It is aminopropyl.

[0230] Preferably, drug D is linked to linker L via its free hydroxyl or amino groups to couple to the antibody. More preferably, formula D or formula D a Or D b Drug D via R a The hydroxyl or amino group on the antibody is linked to the linker L to couple it to the antibody. Preferably, after linking to the linker, R... a It has a structure selected from the following:

[0231] Wherein, n is an integer from 1 to 4, preferably n = 2 or 3, where R' is H or C1-C4 alkyl, preferably H, where the wavy line on the left indicates the position connected to the linker L, and the asterisk on the right indicates the position connected to the camptothecin core;

[0232] More preferably, R after being connected to the connector a It has a structure selected from the following:

[0233]

[0234] Most preferably, R after being connected to the connector a It has a structure selected from the following:

[0235]

[0236] The wavy line on the left indicates the position where it connects to the connector L, and the asterisk on the right indicates the position where it connects to the camptothecin nucleus.

[0237] In some preferred embodiments, the LD unit in formula (I) of the present invention comprises the following structure:

[0238]

[0239] More preferably, the LD unit in formula (I) comprises the following structure:

[0240]

[0241] Connector L unit

[0242] The linker L suitable for use in this invention can be any linker capable of conjugating the antibody of this invention to a drug. Suitably, the addition of the linker should ensure adequate stability of the ADC of this invention in the circulatory system, while providing rapid and effective release of the active form of the toxic drug at the target site (e.g., tumor cells or tumor environment).

[0243] In some embodiments, the linker in formula (I) of the present invention is a non-degradable linker. Examples of non-degradable linkers include, but are not limited to, N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC). Typically, ADCs containing such linkers must be internalized into cells, where the antibody portion of the ADC is degraded by intracellular lysosomal proteases to release the active pharmaceutical molecule.

[0244] In some further embodiments, the linker in Formula (I) of the present invention is a degradable linker. Drug release from an ADC containing such a linker is triggered by the nature of the cleavage site in the linker. Therefore, the cleavage site of such a linker can be designed according to the characteristics of the target therapeutic site (e.g., tumor cell lysosomes and / or the tumor environment). In most cases, the degradable linker can consist of a coupling portion, a degradable portion, and optionally a spacer portion. The coupling portion is responsible for the connection between the antibody and the linker-drug and can be selected according to the desired antibody-drug conjugation chemistry. The degradable portion will contain a peptide or peptide analog that can be recognized by an enzyme in an enzyme-based release mechanism, such as oligopeptides or oligopeptide analogs such as Val-Ala, Val-Cit, Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly, cyclobutyl-Ala, and cyclobutyl-Cit that can be degraded by proteolytic enzymes. In some cases, the properties of an ADC can be improved by introducing modifications at peptide residue positions adjacent to enzyme cleavage sites in the linker, such as the stability of the ADC in blood circulation and / or the efficacy of the ADC at the target site. In some cases, spacer groups can be introduced between the degradable portion of the linker and the drug D, as needed, to promote the release (especially, traceless release) of the remaining portion of the drug active molecule self-coupling, for example, a p-aminobenzylcarbamate (PABA) or aminomethyl (-NHCH2-) spacer group that is spontaneously eliminated in acidic media. Furthermore, in cases where the drug is highly hydrophobic, the addition of units such as PEG can be considered (but is not necessary) to improve the properties of the ADC, such as reducing precipitation and aggregation. Linkers applicable to this invention include, but are not limited to, the linkers disclosed in WO2022 / 170971 (which is hereby incorporated by reference).

[0245] In some embodiments, the connector L in formula (I) of the present invention has the structure of formula (II):

[0246] -ZYM- (II),

[0247] in

[0248] Z is the linker base connected to Ab.

[0249] Y is a peptide of 2-5 amino acids, preferably a dipeptide, tripeptide, or tetrapeptide.

[0250] M is absent, or it is a spacer group used to link with drug D.

[0251] Z-connector base

[0252] Typically, the thio group of antibody cysteine ​​exists as a disulfide bond. Opening the disulfide bond of the antibody provides a free thiol group as a coupling site. One way to form an antibody-drug conjugate (ADC) by coupling with the antibody thiol group is to allow the free thiol group on the antibody to undergo a Michael addition reaction with a heterocyclic (e.g., maleimide) linker. Another way is to allow a linker containing a leaving group-substituted heterocyclic ring to undergo a nucleophilic substitution reaction with a free thiol group in the antibody molecule to obtain an antibody-drug conjugate. Both methods are applicable to the ADC conjugates of the present invention. Therefore, the linker according to the present invention may, in some aspects, contain a Z-linker of a heterocyclic (e.g., maleimide) or heterocyclic (e.g., pyrimidine) class; and, in some cases, it is preferred to contain a heterocyclic (e.g., pyrimidine) linker to provide a conjugate with higher stability in blood circulation.

[0253] In some implementations, Z in equation (II) has the following structure:

[0254] -Z1-Z2-Z3-Z4-,

[0255] Z1 represents the sulfur atom in Ab.

[0256] Z2 is a 5-10 membered heterocyclic group, preferably containing 1 or 2 heteroatoms selected from N, S and O;

[0257] Z3 is selected from key, -C(=O)-, -C1-C 10 Alkylene -C(=O)-, -C3-C 10 Imyynyl-C(=O)-, -C3-C10 imynyl-C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 cycloalkyl-C(=O)-, -O-C1-C8 alkylene-C(=O)-, -arylene-C(=O)-, -C1-C 10 alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-C3-C8 cycloalkylene-C(=O)-, -C3-C8 cycloalkylene-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-C3-C8 heterocyclic-C(=O)-, -C3-C8 heterocyclic-C1-C 10 Alkylene-C(=O)-, -heteroaryl-C(=O)-, -C1-C 10 alkylene-heteroaryl-C(=O)-, -heteroaryl-C1-C 10 Alkylene-C(=O)-,

[0258] Z4 is a bond or a PEG unit represented by the following formula.

[0259] Among them, R5 is selected from C 1-4 Alkylene, -NH-, -NH-C 1-4 alkylene-heteroaryl-, wherein the heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group, preferably a triazolyl group; R6 is -C(=O)-, -C 1-4 Alkylene, -C 1-4 Alkylene -C(=O)-, -NH-C(=O)-(CH2OCH2)-C(=O)-, -C 1-4 Alkylene -NH-C(=O)-(CH2OCH2)-C(=O)-, where m is an integer from 2 to 12, for example, m = 2, 4, 6, or 8.

[0260] In one embodiment, Z2 is a 5-10 membered heteroaryl group. In one embodiment, Z2 is selected from one or more groups independently selected from hydrogen, halogen, nitro, C. 1-6 Alkyl and Halogenated C 1-6 Alkyl-substituted pyrimidines, thiazoles, phenylprophiazoles, oxazoles, quinazolines, and pyrrolo[2,3d]pyrimidines. In some embodiments, Z2 is a heteroaryl group selected from the following:

[0261] Z2 is connected to Z1 via a carbon atom adjacent to the heteroatom. In a preferred embodiment, Z2 is a pyrimidine group, preferably... More The wavy line on the left indicates the position connected to Z1; the wavy line on the right indicates the position connected to Z3.

[0262] In some implementations, Z2 is a maleimide group. The wavy line on the left indicates the position connected to Z1; the wavy line on the right indicates the position connected to Z3.

[0263] In some implementations, Z3 is a -C3-C8 heterocyclic group -C(=O)- or -C1-C 10 Alkylene-C3-C8 heterocyclic-C(=O)-, -C3-C8 heterocyclic-C1-C 10 The alkylene group is -C(=O)-, preferably wherein the heterocycle in the heterocyclic group is a heteroaromatic ring, such as a triazole, pyrazole, thiazole, oxazole, isoxazole, or pyridazine heteroaromatic ring. In some embodiments, Z3 is -heteroaryl-C(=O)-, -C1-C 10 alkylene-heteroaryl-C(=O)-, -heteroaryl-C1-C 10 Alkylene-C(=O)-.

[0264] In some implementations, Z3 is -C3-C8 heteroaryl-C1-C 10 Alkylene-C(=O)-, especially,

[0265]

[0266] Where n' is 1-6, preferably, Z3 is... More

[0267] The wavy line on the left indicates the position connected to Z2, and the wavy line on the right indicates the position connected to Z4. Preferably, Z2 is a pyrimidine group.

[0268] In some embodiments, Z3 is arylene- or heteroarylene-C(=O)-, especially,

[0269] Z3 is connected to Z4 via -C-(=O)-, and preferably, Z2 is a pyrimidinyl group.

[0270] In other implementations, Z3 is -(C≡C)-C 1-5 Alkylene -C(=O)-, -(CH=CH)-C 1-5 Alkylene -C(=O)-, -C 1-6 Alkylene -C(=O)-, or -C 3-8 Cycloalkylene-C(=O)-, wherein Z3 is connected to Z4 via -C-(=O)-.

[0271] In some preferred embodiments, Z2 is pyrimidinyl and Z3 is -C(=O)-.

[0272] In some preferred embodiments, Z2 is a pyrimidinyl group and Z3 is -(C≡C)-C. 1-5 Alkylene -C(=O)- or -(CH=CH)-C 1-5 Alkylene -C(=O)-, especially -(C≡C)-C 1-5 Alkylene-C(=O)-.

[0273] In some preferred embodiments, Z2 is a maleimide group and Z3 is a -C group. 1-6 Alkylene -C(=O)-, or -C 3-8 Cycloalkylene-C(=O)-.

[0274] In some implementations, Z4 is the key, and Z3 is directly connected to Y in equation (II).

[0275] In some embodiments, Z4 is a unit containing 2-12 PEGs. In some embodiments, Z4 is...

[0276] Where m = 1-8, for example, 2, 3, 4, 5, 6, 7 or 8.

[0277] In some implementations, Z4 is selected from:

[0278] The wavy line on the left indicates the position connected to Z3; the wavy line on the right indicates the position connected to Y in Equation II.

[0279] In some embodiments, Z in formula (I) of the present invention has the following structure:

[0280] Where x1 = 1 - 8, for example, x1 = 3.

[0281] In some embodiments, Z in formula (I) of the present invention has the following structure:

[0282] Where x2 = 1 - 6.

[0283] In some preferred embodiments, Z in formula (I) of the present invention has the following structure:

[0284]

[0285] Where S is the sulfur atom in Ab, and R b For unsubstituted or substituted -C 3-10 alkynyl group -C(=O)- or -C 3-10 alkenyl-C(=O)- or -heteroaryl-C1-C 10 Alkylene-C(=O)-,

[0286] Preferably, R b yes

[0287]

[0288] The wavy line on the left indicates the position where it is connected to the pyrimidin group; the wavy line on the right indicates the position where it is connected to the Y group.

[0289] More preferably, Z has the following structure:

[0290]

[0291] In some embodiments, Z in formula (I) of the present invention has the following structure:

[0292] Where R E It is hydrogen, C 1-6 Alkyl, C 1-6 aminoalkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, wherein y = 0-4, for example, 0, 2, or 5, wherein the alkyl, amino, and hydroxyl moieties are optionally substituted; in some embodiments, R E It is an optional substituted aminoalkyl group, for example, substituted with -C 1-4 Alkylene -NH2, -C 1-4 Alkylene NHRF and -C 1-4 Alkylene N(R) F Replaced by )2, where each RF is independently selected from C 1-6 Alkyl and C 1-6 Halogenated alkyl, or two R F The groups combine with the nitrogen to which they are attached to form nitrogen heterocyclic butyl, pyrrolidinyl, or piperidinyl groups.

[0293] In some embodiments, Z in formula (I) of the present invention has the following structure:

[0294]

[0295] Y-peptide unit

[0296] To limit or minimize the off-target toxicity of ADCs while ensuring the release of toxin molecules at the target tumor site, it is advantageous in some respects to design antibody-drug conjugates (ADCs) that, while highly selectively targeting tumor cells, can be degraded by proteolytic enzymes in the tumor cells or environment (especially enzymes with significantly higher activity in tumor cells and / or the tumor environment compared to blood). For this purpose, oligopeptides or oligopeptide analogs that can be recognized and cleaved by such proteolytic enzymes can be included in the linker of such ADC conjugates.

[0297] In one embodiment, the Y unit, thus part of the linker of the present invention, is a biodegradable peptide-containing portion, for example, a biodegradable peptide linker containing two or more (e.g., 2-12, such as 2, 3, 4, 5, or 6) consecutive or discontinuous amino acids. Each amino acid of the peptide-containing unit may be independently selected from natural or non-natural amino acids, for example selected from: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, substituted lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, β-alanine, citrulline, and derivatives thereof. In some embodiments, each amino acid is independently selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, and derivatives thereof. In some embodiments, each amino acid is independently selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan and valine, N-methylglycine, β-alanine, and derivatives thereof. In some aspects, after the ADC is internalized into tumor cells, the amide bond in the Y unit is recognized and degraded by enzymes in the lysosomes of the tumor cells, releasing drug fraction D. In other aspects, the amide bond in the Y unit can be recognized and degraded by enzymes in the tumor environment, releasing drug fraction D. By designing antibody-drug conjugates that are biased towards tumor distribution, linkers with such Y-peptide units will promote the release of toxin molecules in tumor cells and the environment.

[0298] In some embodiments, Y comprises a unit selected from the following: Val-Cit, Phe-Lys, Val-Ala, Val-Lys-Gly, Ala-Ala-Ala, Val-Ala, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly, cyclobutyl-Ala, cyclobutyl-Cit. In some embodiments, Y comprises a unit selected from the following: Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Cit-Val, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn.

[0299] In some preferred embodiments, Y is a dipeptide, tripeptide, tetrapeptide, or pentapeptide containing a substituted lysine. In one embodiment, the substituted lysine is:

[0300]

[0301] Among them, R3 and R4 are independently selected from: H, C 1-6 Alkyl group, -CO-NH2, -CONH(C 1-6 Alkyl), and -CONH(C 1-6 Alkyl)2, wherein the alkyl group is optionally substituted with a group selected from the group consisting of halogen, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, 6-10 aryl and 5-14 heteroaryl.

[0302] In some preferred embodiments, Y is a peptide having the following amino acid sequence from the N-terminus to the C-terminus;

[0303] Xaa1-Xaa2-Xaa3-Xaa4-Xaa5,

[0304] Xaa1 is absent, or it is an amino acid selected from valine, glycine, alanine, and glutamic acid.

[0305] Xaa2 is an amino acid selected from phenylalanine, leucine, alanine and valine, with valine being preferred;

[0306] Xaa3 is either unsubstituted or substituted lysine;

[0307] Xaa4 is an amino acid selected from leucine, glycine, and alanine.

[0308] Xaa5 is absent, or it may be an amino acid selected from glycine and alanine.

[0309] In this embodiment, the N-terminus of the amino acid sequence is connected to the Z unit of the linker, and the C-terminus is connected to the M unit or directly to the drug D.

[0310] Preferably, Xaa3 is a lysine with an ε-amino group mono- or di-substituted by a C1-C3 alkyl group.

[0311] More preferably, Y is a peptide selected from the following: Phe-Lys-Gly, Leu-lys-Gly, Gly-Val-Lys-Gly, Val-Lys-Gly-Gly, Val-Lys-Gly, Val-Lys-Ala, Val-Lys-Leu, wherein the Lys residue is an unsubstituted or C1-C3 alkyl monosubstituted or disubstituted lysine.

[0312] M spacer base

[0313] In some embodiments, the ADC conjugate of the present invention has a spacer group (M) between the releasable peptide unit (Y) and the drug (D). The spacer group may be a functional group that facilitates the connection between the peptide unit (Y) and the drug D, or may provide additional structural components to further facilitate the release of the drug D from the remainder of the conjugate (e.g., a self-dissolving group, such as a p-aminobenzyl (PAB) component).

[0314] In some implementations, the M spacer base connecting the Y and drug D units can be selected from:

[0315] And preferably The wavy line on the left indicates a connection to Y, and the wavy line on the right indicates a connection to drug unit D.

[0316] In other embodiments, M is a covalent bond, and Y in Formula I is directly connected to drug D, such as through an amide bond.

[0317] In a preferred embodiment, the YM unit in the connector L includes the following structure:

[0318]

[0319] Preferably, the YM unit is connected to the following Z unit:

[0320]

[0321] Exemplary connector L

[0322] In some preferred embodiments, L in formula (I) of the present invention is a connector having the following structure:

[0323]

[0324] R3 and R4 are each independently selected from methyl, ethyl, and propyl. In some more preferred embodiments, R3 and R4 are both methyl; or R3 and R4 are both ethyl; or R3 and R4 are both propyl.

[0325] In some embodiments, the LD unit of Formula I of the present invention is linked to the antibody by forming a thioether bond with the thiol group of the free cysteine ​​residue of the light chain and / or heavy chain of the antibody.

[0326] Exemplary ADC Coupler

[0327] In some embodiments, the present invention provides ADC conjugates having the following structures or pharmaceutically acceptable salts or solvates thereof:

[0328]

[0329] Where q represents the average DAR value from 1 to 20, for example, the average DAR values ​​of approximately 2, 4, 6, and 8.

[0330] In some embodiments, the ADC according to the present invention has at least one or more of the following advantages:

[0331] - Compared to the distribution of ADCs in the bloodstream, it significantly promotes the distribution and accumulation of ADCs in the tumor environment;

[0332] - Effectively limits premature release of the payload into the plasma, exhibiting high circulatory stability.

[0333] - It can provide effective release of the active form of toxic drugs in the tumor environment;

[0334] - It imparts a highly effective tumor-killing effect in animals; and

[0335] - Good animal tolerance.

[0336] In some further embodiments, the ADC according to the present invention may also have at least one or more of the following advantages:

[0337] The addition of the toxin-linker does not induce aggregation and allows for high drug loading, with DAR up to 8; and

[0338] - Acceptable PK characteristics.

[0339] II. Preparation of Antibody-Drug Conjugates

[0340] The generation of antibody-drug conjugates can be accomplished by any technique known to those skilled in the art. In some aspects, the conjugation of the drug-linker to the antibody is accomplished by reacting with the amino acid residues of the antibody. In some embodiments, a heteroaryl linker L with a leaving group is used to conjugate drug D to a cysteine ​​residue of the antibody to prepare the conjugate of formula I of the present invention. In some embodiments, the interchain disulfide bonds of the antibody can be disrupted and free thiol groups exposed for conjugation with the heteroaryl linker-drug by controlling the conditions of treating the antibody with a reducing agent such as tris(2-hydroxyethyl)phosphine (TCEP). For IgG1 type antibodies, up to four linking disulfide bonds can be reduced, thereby generating up to eight reactive thiol groups for conjugation. Conjugates prepared by this method can contain zero, one, two, three, four, five, six, seven, or eight drugs in each antibody molecule.

[0341] When the prepared conjugate is a composition of conjugates with different drug conjugation sites and / or numbers, the drug loading of the conjugate is expressed as the average DAR, which is the average number of drug molecules per antibody. The average number of drug molecules per antibody in the prepared antibody-drug conjugate composition can be characterized by conventional methods, such as mass spectrometry, ELISA assay, and HPLC. In other embodiments, the quantitative distribution of the antibody-drug conjugate, expressed as q, can also be determined. The separation, purification, and characterization of homogeneous antibody-drug conjugates where q is a certain value from antibody-drug conjugates with other drug loadings can be achieved by methods such as reversed-phase HPLC or electrophoresis.

[0342] Therefore, in one aspect of the invention, q represents the number of drug-linker (LD) portions conjugated to a single antibody (Ab), and is preferably an integer from 1 to 16, 1 to 12, 1 to 10, or 1 to 8. In this case, the individual ADC conjugate may also be referred to as an ADC compound. In any embodiment herein, on the ADC compound according to the invention, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 drug-linker portions may be conjugated to a single antibody.

[0343] In another aspect of the invention, q represents the average DAR of the prepared coupling composition. In this case, q can be an integer or a decimal in the range of 1 to about 16, 1 to about 12, 1 to about 10, or 1 to about 8, 2 to about 16, 2 to about 12, 2 to about 10, or 2 to about 8. In some aspects, q represents an average DAR of about 6. In some aspects, q represents an average DAR of about 8.

[0344] III. Pharmaceutical Composition

[0345] In some embodiments, the present invention provides compositions comprising any ADC described herein or a pharmaceutically acceptable salt or solvate thereof, preferably pharmaceutical compositions or pharmaceutical formulations. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example a pharmaceutical composition, comprises an ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof, and a combination of one or more other therapeutic agents.

[0346] The present invention also includes compositions comprising the ADC of the present invention or pharmaceutically acceptable salts or solvates thereof (including pharmaceutical compositions). These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0347] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0348] For information on the use and applications of pharmaceutical excipients, see also “Hahdbook of Pharmaceutical Excipients”, 8th edition, R.C. Rowe, P.J. Seskey and S.C. Wen, Pharmaceutical Press, London, Chicago.

[0349] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0350] A drug comprising the ADC described herein can be prepared by mixing the ADC of the present invention, or a pharmaceutically acceptable salt or solvate thereof, having the desired purity, with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0351] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). The active ingredients are suitably combined in amounts effective for the intended use.

[0352] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0353] IV. Drug combinations and pillboxes

[0354] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof, 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)).

[0355] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0356] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0357] - A first container containing a pharmaceutical composition comprising the ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof;

[0358] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0359] V. Uses and Methods

[0360] One aspect of the present invention provides a method for preventing or treating tumors (e.g., cancer) in a subject, comprising administering to the subject an effective amount of the ADC of the present invention or a pharmaceutically acceptable salt or solvate thereof, a pharmaceutical composition, a pharmaceutical combination, or a cassette.

[0361] CLDN18.2 is selectively expressed on the surface of cancer cells from various sources, making it a suitable target for developing cancer immunotherapies. CLDN18.2 has been found to be highly selectively expressed in digestive system cancers such as pancreatic cancer, esophageal cancer, and gastric cancer. Therefore, in one aspect, the present invention provides the use of the antibody of the present invention in the prevention and / or treatment of CLDN18.2-positive tumors in subjects, including administration of the antibody-drug conjugate of the present invention at a preventive and / or therapeutically effective amount. In some embodiments, the antibody-drug conjugate of the present invention may be used as the sole active agent or may be administered in combination with other therapies or therapeutic agents. These other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells and eliminate tumors by binding to and / or blocking these molecules; and drugs that activate the subject's immune system, prompting it to spontaneously eliminate tumors.

[0362] In some embodiments, the tumor treated according to the present invention can be early, intermediate, or late-stage cancer, or metastatic cancer. In some embodiments, the tumor treated according to the present invention is a tumor that has previously received treatment and has experienced immune escape.

[0363] In the method of this invention, the administration of the antibody-drug conjugate of this invention may include 1) a therapeutic measure that cures, slows, alleviates, reduces or stops the progression of a diagnosed pathological condition or disease; or 2) a preventative or preventive measure that prevents and / or slows the development of a pathological condition or disease. In some embodiments, in the method of this invention, an individual will benefit from the said therapeutic or preventive measure and, compared to an individual who has not received said treatment, exhibit a reduction or improvement in the occurrence, recurrence, or development of disease, condition, symptom, and / or symptoms. In some embodiments, prior to receiving treatment using the method of this invention, the subject is receiving or has received other treatments, such as chemotherapy and / or radiotherapy, or other immunotherapies.

[0364] In one specific embodiment, the antibody-drug conjugate of the present invention can kill CLDN18.2 positive tumor cells and / or inhibit the proliferation of CLDN18.2 positive tumor cells.

[0365] The antibody-drug conjugates of this invention can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it can be administered via any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0366] For the prevention or treatment of disease, the appropriate dosage of the antibody-drug conjugate of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the specific type of antibody-drug conjugate, the severity and course of the disease, the purpose of treatment, previous treatments, the patient's clinical history and response to the antibody-drug conjugate, and the judgment of the attending physician. The antibody-drug conjugate of the present invention can be appropriately administered to the patient as a single treatment or as part of a series of treatments.

[0367] In some embodiments, the present invention also provides the use of the antibody-drug conjugate of the present invention in the preparation of medicaments for the aforementioned treatment and prevention methods.

[0368] The antibody portion of this invention

[0369] This invention also provides a humanized anti-Claudin18.2 monoclonal antibody with a variety of excellent properties, encoding a nucleic acid, a vector and host cell containing said nucleic acid, and an immunoconjugate containing said antibody, a multispecific antibody, a pharmaceutical composition, and uses. The antibody of this invention not only exhibits high binding affinity and high specificity against cells expressing human Claudin18.2 (especially cells with low expression levels), but also possesses favorable properties such as ADCC and / or CDC activity, Claudin18.2 antigen-mediated endocytic activity, stability, and / or pharmacokinetic properties. Therefore, it is suitable for use alone or in combination with other anticancer drugs for cancer treatment, and is also suitable as a molecular component for forming new anticancer molecules targeting cancer tissues, for example, by conjugating with cytotoxic drugs to form antibody-drug conjugates.

[0370] I. Antibody of the present invention

[0371] This invention provides antibodies or antigen-binding fragments thereof that specifically bind to Claudin18.2, preferably human Claudin18.2 protein (e.g., the human Claudin18.2 sequence NM_001002026), particularly humanized antibodies or antigen-binding fragments thereof. In one embodiment, the anti-Claudin18.2 antibody or antigen-binding fragment thereof according to the invention comprises:

[0372] (i) the HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 1 or 3, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 2, or

[0373] (ii) the HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 21, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 22, or

[0374] (iii) The HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 23, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 24, or

[0375] (iv) The HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 38 or 40, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 39, or

[0376] (v) The HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 44, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 45, or

[0377] (vi) The HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 46, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 47, or

[0378] (vii) The HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 48, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 49, or

[0379] (viii) The HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 50, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 51, or

[0380] (ix) The HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 52, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 53. Preferably, the CDRs are defined according to Kabat or IMGT or a combination thereof.

[0381] In some embodiments, preferably, the present invention provides an antibody or antigen-binding fragment of Claudin18.2 that binds to the Claudin18.2, comprising three complementarity-determining regions (HCDRs) of the heavy chain variable region and three complementarity-determining regions (LCDRs) of the light chain variable region, wherein:

[0382] (i) As defined by Kabat, HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 12, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 13 or 25, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 14, LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 15, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 16, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 17; or

[0383] (ii) As defined by IMGT, HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 6, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 7, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 8, LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 9, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 10, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 11; or

[0384] (iii) According to Kabat's definition, HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 26, HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 27 or 37, HCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 28, LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 32, LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 33, and LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 34.

[0385] (iv) According to Kabat's definition, HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 29, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 30, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 31, LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 35, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 36, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 34.

[0386] The present invention also considers antibodies comprising one of the CDR sequence combinations described in (i)-(iv) above. Preferably, the variant comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conserved substitutions) in the six CDR regions, and preferably the heavy chain CDR3 remains unchanged.

[0387] In some further embodiments, the present invention provides an anti-Claudin18.2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region selected from the following:

[0388] (i) the heavy chain variable region as shown in SEQ ID NO: 1 or 3, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0389] (ii) the heavy chain variable region as shown in SEQ ID NO: 21 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 22 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0390] (iii) the heavy chain variable region as shown in SEQ ID NO: 23 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 24 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0391] (iv) The heavy chain variable region as shown in SEQ ID NO: 38 or 40, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 39, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0392] (v) the heavy chain variable region as shown in SEQ ID NO: 44 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 45 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0393] (vi) The heavy chain variable region as shown in SEQ ID NO: 46 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 47 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0394] (vii) The heavy chain variable region as shown in SEQ ID NO: 48 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 49 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0395] (viii) The heavy chain variable region as shown in SEQ ID NO: 50 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 51 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with it, or

[0396] (ix) The heavy chain variable region as shown in SEQ ID NO: 52 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with it, and the light chain variable region as shown in SEQ ID NO: 53 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with it.

[0397] In one embodiment, preferably, the anti-Claudin18.2 antibody of the present invention or its antigen-binding fragment comprises the heavy chain and light chain variable region sequences or variants thereof of any of the exemplary antibodies 25C7A5 and its humanized antibodies (HZ1 and HZ2) and 2D3A11 and its humanized antibodies (HZ1 and HZ2), for example, antibodies having the same CDR sequence as one of the exemplary antibodies and having the same or different framework region sequences.

[0398] In some preferred embodiments, the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 21 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID No: 21 and the light chain variable region of SEQ ID No: 22.

[0399] In some further preferred embodiments, the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 1 or 3, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. Preferably, the antibody is a humanized antibody. More preferably, the heavy chain variable region of the antibody has a human-derived framework region sequence, and preferably has amino acid Q or E at position H6 according to the Kabat number in the heavy chain variable region, preferably having amino acid Q.

[0400] In some further preferred embodiments, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID No: 1 and the light chain variable region of SEQ ID No: 2.

[0401] In some further preferred embodiments, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID No: 3 and the light chain variable region of SEQ ID No: 2.

[0402] In some preferred embodiments, the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 23 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. Preferably, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID No: 23 and the light chain variable region of SEQ ID No: 24.

[0403] In some further preferred embodiments, the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence described in SEQ ID NO: 38 or 40, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity therewith. Preferably, the antibody is a humanized antibody. More preferably, the heavy chain variable region of the antibody has a human-derived framework region sequence, and preferably has amino acid Q or E at position H6 according to the Kabat number in the heavy chain variable region, preferably having amino acid Q.

[0404] In some further preferred embodiments, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID No: 38 and the light chain variable region of SEQ ID No: 39.

[0405] In some further preferred embodiments, the antibody or antigen-binding fragment comprises the heavy chain variable region of SEQ ID No: 40 and the light chain variable region of SEQ ID No: 39.

[0406] In some embodiments, the antibody of the present invention may comprise a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region is a heavy chain constant region derived from human immunoglobulin. Preferably, the light chain constant region is a light chain constant region derived from human immunoglobulin.

[0407] The heavy chain constant region contained in the antibody of the present invention can be any isotype or subtype, such as the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4 isotypes. In some embodiments, therefore, the present invention provides an anti-Claudin18.2 antibody comprising an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, especially a human IgG1 heavy chain constant region.

[0408] The light chain constant region contained in the antibody of the present invention can be a κ light chain constant region or a λ light chain constant region. Therefore, in some embodiments, the present invention provides an anti-Claudin18.2 antibody containing a κ light chain constant region or a λ light chain constant region, preferably the antibody of the present invention contains a human κ light chain constant region.

[0409] In some embodiments, the antibody according to the invention is an IgG1 antibody, more particularly an IgG1κ or IgG1λ isotype. More preferably, the antibody according to the invention is a human IgG1κ antibody.

[0410] In some embodiments, the anti-Claudin18.2 antibody of the present invention comprises a human IgG1 heavy chain constant region. Preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence comprising at least one, two, or three, but not more than 20, 10, or 5 amino acid changes relative to the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 4.

[0411] In some embodiments, the anti-Claudin18.2 antibody of the present invention comprises a human κ light chain constant region. Preferably, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence comprising at least one, two, or three, but not more than 20, 10, or 5 amino acid changes relative to the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 5.

[0412] In some preferred embodiments, the antibody of the present invention comprises a heavy chain sequence and a light chain sequence selected from the following:

[0413] (a) A heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 18 and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 20;

[0414] (b) A heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 19 and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 20;

[0415] (c) A heavy chain sequence containing the amino acid sequence of SEQ ID NO: 41 and a light chain sequence containing the amino acid sequence of SEQ ID NO: 43;

[0416] (d) A heavy chain sequence comprising an amino acid sequence selected from SEQ ID NO: 42, and a light chain sequence comprising an amino acid sequence comprising SEQ ID NO: 43. The present invention also provides variants of antibodies comprising any one of (a)-(d) above, wherein said variants have amino acid alterations in the heavy chain sequence and / or the light chain sequence. Preferably, the variant comprises, compared to the corresponding antibody, at least one, two, or three, but not more than 20, 10, or 5 amino acid alterations in the heavy chain sequence or the light chain sequence, or both, an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or higher identity. Preferably, the amino acid alterations do not occur in the CDR region, more preferably not in the variable region.

[0417] This invention also provides variants of any antibodies described herein, particularly variants of either the exemplary antibody 25C7A5 and its humanized antibodies (HZ1 and HZ2) or 2D3A11 and its humanized antibodies (HZ1 and HZ2). The antibody variants of this invention preferably retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the original antibody. More preferably, the modification does not result in the antibody variant losing its binding to the antigen, but optionally imparts properties such as increased antigen affinity and different effector functions. It is understood that the heavy chain variable region or light chain variable region of the antibody, or each CDR region, can be modified individually or in combination. Furthermore, the Fc region of the antibody can also be modified. Modification of the Fc region can be performed alone or in combination with the modifications to the framework and / or CDR regions described above. The Fc region can be modified, for example, to alter one or more functions of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. In addition, the antibodies of the present invention can be chemically modified (e.g., linked to PEG) or their glycosylation patterns can be altered.

[0418] In some embodiments, the antibodies of the present invention are preferably monospecific antibodies. However, in some embodiments, the antibodies of the present invention may also be multispecific antibodies, comprising a first binding specificity against Claudin18.2 and a binding specificity against another antigen, for example, a second and / or a third binding specificity against another antigen expressed on the surface of tumor cells.

[0419] In some embodiments, the anti-Claudin18.2 antibody or its antigen-binding fragment of the present invention has one or more of the following characteristics:

[0420] (i) It binds with high affinity to human Claudin18.2 expressed on the cell surface, but does not bind or barely binds to human Claudin18.1;

[0421] (ii) It exhibits cross-reactivity with Claudin 18.2 in cynomolgus monkeys, mice, or rats;

[0422] (iii) Killing cells that express Claudin18.2 on their surface, especially tumor cells such as gastric cancer cells;

[0423] (iv) It has ADCC activity;

[0424] (v) It has CDC activity;

[0425] (vi) It has Claudin18.2 receptor-mediated endocytosis activity.

[0426] In some embodiments, the antibodies of the present invention exhibit high binding affinity and high binding specificity for Claudin 18.2. Preferably, the binding affinity and binding specificity of the antibodies of the present invention for Claudin 18.2 and / or for Claudin 18.1 are determined by FACS or cellular ELSA assays (e.g., the assays described in Examples I-7). In one embodiment, in a cell-based assay, the binding affinity of the antibody of the present invention for Claudin 18.1 is at least 10-fold, 100-fold, or 10-fold lower than the binding affinity of the antibody for Claudin 18.2. 3 times, 10 4 times, 10 5 times or 10 6 In one embodiment, preferably, the antibody of the present invention does not bind or substantially does not bind to Claudin18.1 expressing cells.

[0427] In one embodiment, in cell-based assays, such as cellular ELISA assays using recombinant cell lines expressing human Claudin 18.2 with a positivity rate greater than 95% or 98%, the antibody of the present invention exhibits a high binding affinity for human Claudin 18.2, having an EC50 value of less than 300 ng / ml, particularly less than about 100 ng / ml, for example about 1 ng / ml to 50 ng / ml, and preferably less than about 20 ng / ml. Preferably, the assay is performed according to the cellular ELISA assay method described in Examples I-7. Preferably, the assay is performed using a recombinant HEK-293T cell line stably expressing human Claudin 18.2.

[0428] In another embodiment, in a cell-based assay, the antibody of the present invention exhibits comparable or stronger binding affinity to human Claudin 18.2 compared to the reference antibody IMAB362. In some embodiments, the antibody of the present invention exhibits comparable cell-binding affinity to cells expressing a high positivity rate (e.g., positivity rate greater than 95%) on the surface of human Claudin 18.2 compared to the reference antibody IMAB362; in other embodiments, the antibody of the present invention exhibits stronger cell-binding affinity to cells expressing a low positivity rate (e.g., positivity rate less than 50%, such as approximately 2-30%) on the surface of human Claudin 18.2. In the assay, mammalian cell lines recombinantly expressing human Claudin 18.2, such as HEK29, L929, or NUGC4 cells, can be used; tumor cell lines endogenously expressing human Claudin 18.2, such as NUGC4 cells, can also be used. Preferably, a cell ELISA assay is performed as described in Examples I-7; or a FACS assay is performed as described in Examples I-8. Preferably, compared with the reference antibody IMAB362, the antibody of the present invention exhibits stronger cell-binding activity for cells with an average low level of Claudin18.2 expression on the cell surface, such as NUGC4 cells that endogenously express human Claudin18.2.

[0429] In one embodiment, in a cell-based assay, the antibody of the present invention exhibits non-specific binding to Claudin 18.1. This binding property of the antibody to Claudin 18.1-expressing cells can be measured in an FACS assay, optionally relative to negative and / or positive controls. Preferably, in the assay method, the percentage of positive cells stained by the antibody and / or the fluorescence staining intensity value of the cells are determined. In one embodiment, a recombinant cell line with a positive expression rate of human Claudin 18.1 greater than 95% or greater than 98% is used, the positive cell rate of the antibody of the present invention is less than 3% (more preferably less than 2%, more preferably less than 1%, and even more preferably less than 0.7%), and preferably, the median and / or mean fluorescence signal intensity generated by the antibody of the present invention is comparable to the median / mean signal intensity generated by the isotype control antibody (e.g., ±20%, or preferably ±10%). Preferably, the assay is performed according to the FACS assay method described in Examples I-10. Preferably, a recombinant HEK-293T cell line stably expressing human Claudin 18.1 is used in the assay.

[0430] In one embodiment, the antibody of the present invention has ADCC activity. The ADCC activity of the antibody of the present invention can be determined according to Examples I-11. Preferably, in the determination, the antibody of the present invention exhibits an EC50 value of less than about 100 ng / ml, for example 80-50 ng / ml, or more preferably less than about 50 ng / ml.

[0431] In some further embodiments, the antibodies of the present invention possess CDC activity. The CDC activity of the antibodies of the present invention can be determined according to Examples I-12. Preferably, in the determination, the antibodies of the present invention exhibit CDC activity of less than about 5 μg / ml, for example, about 1-4 μg / ml.

[0432] In some further embodiments, the antibody of the present invention exhibits endocytic activity upon binding to the receptor Claudin18.2 on the cell membrane surface. The endocytic activity of the antibody of the present invention can be determined according to Examples I-13. Preferably, in the endocytic activity evaluation assay, the endocytosis rate of the antibody of the present invention is, for example, at least 10% after 4 hours at 37°C, for example, 10-25%, preferably at least 20%.

[0433] In yet another embodiment, the anti-Claudin18.2 antibody or its antigen-binding fragment of the present invention also has one or more of the following characteristics: (vii) good stability; and (viii) favorable pharmacokinetic properties.

[0434] In one embodiment, the stability of the antibody of the present invention can be determined according to the method described in Examples I-14. Preferably, in the accelerated stability assay, after being placed at 37°C for 3-14 days, the SEC-HPLC purity of the antibody is not less than 95%, preferably not less than 97%; and / or the non-reduced CE-SDS purity is not less than 90%, preferably not less than 93%; and / or the reduced CE-SDS purity is not less than 90%, preferably not less than 95%. More preferably, in the repeated freeze-thaw assay, after repeated freeze-thaw cycles at -80°C 4 or 8 times, the SEC-HPLC purity of the antibody is not less than 95%, preferably not less than 97%.

[0435] In another embodiment, the pharmacokinetic properties of the antibody of the present invention can be determined according to the method described in Examples I-15.

[0436] II. Polynucleotides, Vectors, and Hosts

[0437] In another aspect, the present invention provides a nucleic acid encoding any of the above-mentioned anti-Claudin18.2 antibodies or fragments thereof. A vector comprising said nucleic acid is also provided; and a host cell comprising said nucleic acid or said vector.

[0438] In one embodiment, the present invention provides one or more vectors comprising the nucleic acids of the present invention, including cloning vectors and expression vectors. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors that can be used in the present invention include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC).

[0439] In one embodiment, the present invention provides a host cell comprising the vector of the present invention. Suitable host cells for cloning or expressing the vector encoding an antibody include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression in bacteria such as *Escherichia coli*, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In yet another embodiment, the host cell is a eukaryotic cell. In yet another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. Examples of useful mammalian host cell lines include monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293HEK or 293 cells); and Chinese hamster ovary (CHO) cells, including DHFR. - CHO cells; and myeloma cell lines such as Y0, NS0 and Sp2 / 0.

[0440] III. Antibody Preparation

[0441] In another aspect, the present invention provides a method for preparing the anti-Claudin18.2 antibody of the present invention. In one embodiment, the method of the present invention includes culturing a host cell containing a nucleic acid encoding the antibody of the present invention under conditions suitable for antibody expression, and optionally recovering the antibody from said host cell (or host cell culture medium). For recombinant production of the anti-Claudin18.2 antibody, isolated or artificially synthesized or recombinantly synthesized nucleic acid encoding an antibody (e.g., the antibody described above) can be inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody, from hybridoma cells.

[0442] IV. Immunoconjugates

[0443] In another aspect, the present invention provides immunoconjugates generated by conjugating the antibody of the present invention to a heterologous molecule. In one embodiment, in the immunoconjugate, the antibody of the present invention (or an antigen-binding fragment thereof) is conjugated to a therapeutic agent, diagnostic agent, or detectable agent. In some embodiments, the antibody of the present invention may be conjugated to a heterologous molecule in the form of a full-length antibody or an antibody fragment. In the conjugate, linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a “cleavable linker” that facilitates the release of the polypeptide upon delivery to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0444] In embodiments conjugated with therapeutic agents, suitable therapeutic agents for the conjugate include, but are not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), drugs, or radioisotopes. In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies to detectable agents, including but not limited to, various enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0445] V. Pharmaceutical compositions and pharmaceutical preparations

[0446] The present invention also includes compositions comprising anti-Claudin18.2 antibodies or immunoconjugates thereof (including pharmaceutical compositions or pharmaceutical formulations) and compositions comprising polynucleotides encoding anti-Claudin18.2 antibodies or immunoconjugates thereof. These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art. For information on the use and applications of excipients, see “Handbook of Pharmaceutical Excipients,” 5th edition, RCRowe, PJSeskey, and SCOwen, Pharmaceutical Press, London, Chicago. In some embodiments, pharmaceutical formulations comprising the present invention can be prepared by mixing an anti-Claudin18.2 antibody or immunoconjugate of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)).

[0447] In the pharmaceutical compositions and formulations of the present invention, the antibodies of the present invention may be the sole active agent or may be combined with other therapeutic agents. Therapeutic agents that may be combined with the antibodies of the present invention include, but are not limited to, therapeutic agents that have beneficial therapeutic effects on the disease and / or condition to be treated. For example, the active ingredient may be required for the specific indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, other pharmaceutical ingredients that can provide anticancer activity may be present in the pharmaceutical compositions and formulations in a suitable combination with the active ingredients in an amount effective for the intended use.

[0448] VI. Combination Products

[0449] In another aspect, the present invention also provides a combination product comprising the antibody or antigen-binding fragment thereof of the present invention, a multispecific antibody or immunoconjugate, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, antitumor drugs, etc.). The components constituting the combination product, such as the antibody of the present invention and other therapeutic agents, may be formulated separately in different formulations and preferably contained in different containers. Depending on the disease to be treated and individual conditions, those skilled in the art can determine the administration method and order of administration of the components of the combination product. The combination product of the present invention can be used in the treatment methods of the present invention. In some embodiments, the present invention provides a combination product wherein the other therapeutic agent is, for example, an antibody that effectively stimulates an immune response to further enhance, stimulate, or upregulate the immune response of the subject. In some embodiments, the combination product is used for the prevention or treatment of Claudin18.2 positive tumors.

[0450] VII. Methods and Applications

[0451] In one aspect, the present invention provides methods and uses for applying the Claudin18.2 antibody of the present invention or its antigen-binding fragment, for example, in vivo and in vitro for:

[0452] (1) Targeted binding to tumor cells expressing CLAUDIN 18.2; and / or

[0453] (2) Induces ADCC and / or CDC activity against tumor cells expressing CLAUDIN 18.2, and

[0454] (3) Inhibit and / or kill tumor cells that express CLAUDIN 18.2 on their surface.

[0455] In some embodiments, the methods and uses of the present invention relate to the treatment of a disease in an individual subject. In other embodiments, the methods and uses of the present invention relate to the detection of the presence of Claudin18.2 in a sample from, for example, a subject. In still other embodiments, the present invention also provides the use of the anti-Claudin18.2 antibody of the present invention or an antigen-binding fragment thereof in the preparation of products (e.g., pharmaceutical compositions or pharmaceutical products or combination products or detection products) for the above-described uses.

[0456] Treatment methods and uses

[0457] In one aspect, the present invention provides the use of the antibody of the present invention in the prevention and / or treatment of CLAUDIN 18.2 positive tumors in subjects, including administration of the antibody of the present invention in a preventive and / or therapeutically effective amount.

[0458] In the method of this invention, the administration of the antibody may include 1) a therapeutic measure that cures, slows, alleviates, or reduces the symptoms of a diagnosed pathological condition or disease and / or stops the progression of the diagnosed pathological condition or disease; or 2) a preventive or preventative measure that prevents and / or slows the development of the pathological condition or disease. Therefore, in the method of this invention, the subject may be an individual who already has a disease, an individual susceptible to a disease, or an individual who wishes to prevent a disease. The individual will benefit from the therapeutic or preventative measures and, compared to an individual who has not received the treatment, exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, condition, symptom, and / or symptoms. In some embodiments, this invention relates to the treatment of a disease or condition; in other embodiments, this invention relates to the prevention of a disease or condition.

[0459] In some embodiments, the antibody of the present invention used in the method of the present invention comprises an Fc region that induces immune effector function and induces immune effects, such as ADCC and / or CDC activity, upon binding to cells (e.g., cancer cells) expressing CLDN18.2 on their surface. Therefore, in one embodiment of the therapeutic method of the present invention, the antibody of the present invention, or its fusion, conjugate, or composition thereof, mediates cancer cell killing by inducing CDC-mediated cell lysis, or ADCC-mediated cell lysis, or both.

[0460] The antibodies of the present invention (and pharmaceutical compositions or immunoconjugates comprising them, and any other therapeutic agents) may be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it may be administered via any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0461] Detection Application

[0462] This invention also provides methods and kits for detecting Claudin18.2 in samples, wherein the method comprises: (a) contacting the sample with an antibody of the present invention or an antigen-binding fragment or immunoconjugate thereof; and (b) detecting the formation of a complex between the antibody or its antigen-binding fragment or immunoconjugate and the Claudin18.2 protein. In some embodiments, the sample is obtained from a cancer patient, such as a patient with digestive system cancers, such as gastric cancer, pancreatic cancer, or esophageal cancer. The detection may be in vitro or in vivo.

[0463] When used herein, the term "detection" includes both quantitative and qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from hyperplastic or cancerous lesions. In some embodiments, the Claudin18.2 to be detected is human Claudin18.2.

[0464] In one embodiment, the anti-Claudin18.2 antibody is used to select subjects suitable for treatment with the anti-Claudin18.2 antibody. In yet another embodiment, the antibodies of the present invention can be used to diagnose cancer or tumors, such as to evaluate (e.g., monitor) the treatment or progression, diagnosis, and / or staging of the disease described herein (e.g., hyperplastic or cancerous disease) in a subject.

[0465] In some embodiments, a labeled anti-Claudin18.2 antibody is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0466] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features described above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are for illustrative purposes only and should not be construed as constituting any limitation. Example

[0467] Example I: Preparation and Characterization of Anti-Claudin 18.2

[0468] Example 1: Construction and Identification of Claudin18.2 and Claudin18.1 Expression Cell Lines

[0469] The amino acid sequences of human Claudin 18.2 were referenced from NP_001002026.1 in the NCBI protein database. The amino acid sequences of human Claudin 18.1 were referenced from NP_057453.1 in the NCBI protein database. The amino acid sequences of cynomolgus monkey Claudin 18.2 were referenced from XP_015300615.1 in the NCBI protein database. The amino acid sequences of mouse Claudin 18.2 were referenced from NP_001181850.1 in the NCBI protein database. The amino acid sequences of rat Claudin 18.2 were referenced from NP_001014118.1 in the NCBI protein database. These amino acid sequences were codon-optimized by GenScript and synthesized on the lentiviral vector pLVX or pCDNA3.4.

[0470] Viruses were prepared using a lentiviral packaging system and then used to infect HEK293T, L929, and CHO cells. Stable cell lines were obtained through puromycin screening: CHO-Claudin18.2 (human), HEK293T-Claudin18.1 (human), HEK293T-Claudin18.2 (human), L929-Claudin18.2 (human), HEK293T-Claudin18.2 (cynomolgus monkey), HEK293T-Claudin18.2 (rat), and HEK293T-Claudin18.2 (mouse).

[0471] NUGC4 is a gastric cancer cell line that endogenously expresses Claudin18.2. NUGC4 was selected for subsequent experiments (purchased from Nanjing Kebai, CBP60493); at the same time, a stable NUGC4-Claudin18.2 (human) cell line that overexpresses recombinant human Claudin18.2 (Beijing Kangyuan Bochuang, KC-1471) was purchased for subsequent experiments.

[0472] The positivity rate of Claudin 18.2 was determined by flow cytometry (FACS). The experimental procedure was as follows: adherent cells were digested, collected by centrifugation, and washed three times with pre-cooled PBS; the cells were resuspended in 1% BSA (in PBS), and 3 × 10⁻⁶ cells were added to each well of a 96-well conical plate. 5 50 μl of cells were collected; IMAB362 antibody (sequence derived from patent: CN 101312989 B, the amino acid sequence of the heavy chain variable region of antibody 175D10 SEQ ID NO: 135 and the amino acid sequence of the light chain variable region SEQ ID NO: 142, synthesized by Anhui General Biotechnology and expressed and purified in 293E cells) was diluted with 1% BSA (in PBS) to a final concentration of 40 μg / mL. 50 μl of this solution was added to each well to bring the final antibody concentration to 20 μg / mL. The cells were mixed and incubated at 4°C for 1 hour. The cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 50 μl of 1% BSA (in PBS). 1 μl of anti-human fluorescent secondary antibody (APC: Biolegend-410712 or PE: BioLegend-410708) was added to each well, mixed, and incubated at 4°C for 0.5 hours. The cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 200 μl of PBS. Cells were resuspended in PBS and analyzed using a flow cytometer (Sony-LE-SA3800GA or Beckman-Cytoflex) to obtain APC fluorescence signal values ​​and the percentage of positively stained cells (positive rate %).

[0473] The positivity rate of Claudin18.1 cells was determined using serum from immunized mice using the same method.

[0474] Flow cytometry positive rate results as follows Figure 1A-1I As shown, the positive rates of all the above stable cell lines were high (>95%), while the positive rate of Claudin18.2 expression in NUGC4 cells was 23.8%.

[0475] Example 2: Preparation of anti-human Claudin 18.2 murine monoclonal antibody

[0476] Twenty-four wild-type mice aged 5-6 weeks from three strains (Balb / C, KM, and CD01) were selected. Immunization was performed using pCDNA3.4-Claudin18.2 plasmid, CHO-Claudin18.2 (human) cells, and L929-Claudin18.2 (human) cells. After 3-4 immunizations, serum titers were detected by flow cytometry. The experimental procedure was as follows: HEK293T-Claudin18.1 (human) and HEK293T-Claudin18.2 (human) cells were collected by digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in PBS (containing 1% BSA) at 3 × 10⁶ cells per well. 5 Cells were seeded into 96-well conical plates, serially diluted serum was added, and the cells were incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 1 μl of anti-mouse IgG fluorescent secondary antibody was added to each well, and the cells were incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry. Mice with high affinity for HEK293T-Claudin18.2 (human) and low affinity for HEK293T-Claudin18.1 (human) were selected as candidates for fusion. The candidate mouse numbers were 61, 64, 79, 89, and 91.

[0477] Mouse spleen and lymph nodes were used to prepare cell suspensions, which were then mixed with SP2 / 0 mouse myeloma cells at a 1:1 ratio. The cells were resuspended in cell electrofusion buffer and electrofusion reactions were performed using a BTX-ECM2001 cell electrofusion instrument. After electrofusion, the cells were resuspended in complete fusion medium (RPMI 1640 + 20% FBS + 1×HAT) and aliquoted into 96-well cell culture plates at 20,000-25,000 cells per well, and cultured at 37°C and 5% CO2. After culture, hybridoma clones with high affinity for human Claudin18.2 were screened using an L929-Claudin18.2 (human) cell ELISA method. Flow cytometry (FACS) was used to exclude clones that could bind to Claudin18.1, which were then used for subcloning. After two rounds of fusion screening, 52 clones were selected for subcloning. Seventy-five cells from each parent clone were aliquoted into a 96-well cell culture plate and cultured as subclones using selection medium (RPMI 1640 + 20% FBS + 1×HT). The subclonal supernatant was collected, and single clones with high affinity for Claudin 18.1 and not binding to Claudin 18.1 were selected for scale-up culture using cell ELISA and flow cytometry. Hybridoma cells were cultured in serum-free medium, and the collected single-clonal supernatant was purified using ProA (GE, Mabselect XL) antibody purification media to obtain mouse antibodies.

[0478] Example 3: Evaluation of anti-human Claudin 18.2 murine antibody

[0479] The affinity of the mouse monoclonal antibody against human Claudin18.2 was evaluated by cell ELISA. The experimental procedure was as follows: One day before the experiment, HEK293T-Claudin18.2 (human) cells were seeded into 96-well plates, 5 × 10⁶ cells per well. 4 Cells; On the day of the experiment, remove the supernatant, wash each well once with 300 μl PBS, add 100 μl 4% paraformaldehyde to each well and fix at room temperature for 20 minutes; remove paraformaldehyde, wash each well twice with 300 μl PBS, add 100 μl 2% BSA (in PBS) to each well and block at 37°C for 2 hours; remove the blocking buffer, add 100 μl of serially diluted antibody (diluted with 2% BSA (in PBS), starting at 10 μg / mL, 3-fold serial dilution, for a total of 11 concentration points) to each well and incubate at 37°C for 2 hours; remove the supernatant, wash each well three times with 300 μl PBST, add 100 μl anti-mouse IgG HRP secondary antibody (Jackson, catalog number: 115-035-003) in 2% BSA to each well and incubate at 37°C for 1 hour; remove the secondary antibody, add 300 μl of paraformaldehyde to each well and fix at room temperature for 2 hours. Wash five times with PBST, then add 100 μl of TMB substrate (Huzhou Yingchuang) to each well and incubate for 5-20 minutes. Stop the reaction by adding 50 μl of 2N HCl to each well. Read the OD values ​​using a microplate reader (manufacturer: MD, model: SpectraMax). 450nm The numerical values ​​are then substituted into GraphPad Prism for plotting, and the EC50 value is calculated.

[0480] The specificity of the anti-human Claudin18.2 murine monoclonal antibody was evaluated by flow cytometry (FACS). The experimental procedure was as follows: HEK293T-Claudin18.1 (human) cells were digested and collected by centrifugation, and washed three times with pre-chilled PBS; the cells were resuspended in 1% BSA (in PBS), and 3 × 10⁻⁶ cells were added to each well of a 96-well conical plate. 5 50 μl of cells were collected; the antibody to be tested was diluted to 100 μg / mL with 1% BSA (in PBS), and 50 μl was added to each well to make the final antibody concentration 50 μg / mL. The mixture was then incubated at 4°C for 1 hour. The cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 50 μl of 1% BSA (in PBS). 1 μl of fluorescent secondary antibody (Biolegend, catalog number: 405308) was added to each well, and the mixture was incubated at 4°C for 0.5 hours. The cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 200 μl of PBS. The cells were then analyzed by flow cytometer (Sony-LE-SA3800GA) to obtain the APC fluorescence signal value. The percentage of positively stained cells (positive rate %) was obtained by using an isotype control antibody as a negative control antibody.

[0481] The experimental results are shown in Table 1. Clones with high affinity for HEK293T-Claudin18.2 and no non-specific binding to HEK293T-Claudin18.1 were selected for sequencing. The clone numbers are as follows: 1A6G10, 2D3A11, 25C7A5, 40G11H2, 8H11A10, 23F2F2, 32F7A6.

[0482] Table 1 Summary of Evaluation Results of Murine Antibodies

[0483]

[0484]

[0485] Example 4: Sequencing of anti-human Claudin18.2 murine monoclonal antibody

[0486] The above-mentioned candidate hybridoma cells were collected in approximately 1×10-1 5 RNA was extracted from samples using Trizol (Invitrogen, 15596026), and cDNA was obtained by reverse transcription using PrimeScript RT reagent (TAKARA, RR047A) via PolyA. Upstream primers were designed for the heavy and light chains, and downstream primers were designed for the CH1 region of the heavy chain and the CL region of the light chain. The products were amplified by PCR (Gold Medal MIX, Qingke Biotechnology, TSE101), and fragments were recovered using an agarose gel extraction kit. Samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the murine antibody sequences were determined using IMGT website analysis. Candidate murine antibody sequences are shown in Table 2.

[0487] Table 2. Candidate Murine Antibody Sequence List

[0488]

[0489]

[0490] Patent analysis and retrieval of the obtained sequences confirmed 25C7A5 and 2D3A11 as candidate antibodies with unique sequences, and then humanized design was carried out on them.

[0491] Example 5: Humanization of anti-human Claudin 18.2 murine antibody

[0492] Using the CDR-grafting method, the human germline sequence with the highest homology to the original mouse sequence was first identified using the conventional BLAST method and used as a template. The CDR of the mouse antibody was then grafted onto the human template to construct a chimera. Based on structural analysis, the FR amino acids in the mouse antibody that retained their original conformation were identified, and the corresponding amino acids in the chimera were reverted to mouse amino acids to maintain the original affinity. The constructed humanized antibodies were then subjected to computational and immunogenicity analysis to identify high-immunogenic fragments and replace low-immunogenic fragments. Humanized antibodies 25C7A5-HZ1 and 25C7A5-HZ2, as well as 2D3A11-HZ1 and 2D3A11-HZ2, were obtained, and their humanized sequences are shown in Table 3.

[0493] Table 3 Candidate Derivative Antibody Sequence List

[0494]

[0495] Example 6: Preparation of anti-human Claudin 18.2 humanized antibody

[0496] Humanized antibodies 25C7A5-HZ1 (heavy chain variable region sequence SEQ ID NO: 1 and light chain variable region sequence SEQ ID NO: 2), 25C7A5-HZ2 (heavy chain variable region sequence SEQ ID NO: 3 and light chain variable region sequence SEQ ID NO: 2), and the variable region amino acid sequence of the reference antibody IMAB362 (sequence from patent: CN 101312989B, heavy chain variable region amino acid sequence SEQ ID NO: 135 and light chain variable region amino acid sequence SEQ ID NO: 142 of antibody 175D10) were codon-optimized and then gene-synthesized (Anhui General Biotechnology), and constructed into the PTT5 vector. After plasmid synthesis, it was directly extracted by shaking culture and transfected into HEK293E cells using PEImax (Polysciences, 24765-1). After expression for about 7 days, the supernatant was collected by centrifugation. The supernatant was purified using MabSelectSure protein A affinity packing material (GE). The expressed reference antibody and the humanized antibody share the same human IgG1 heavy chain constant region (SEQ ID NO: 4) and human Kappa light chain constant region (SEQ ID NO: 5). All purified antibodies were ultrafiltered into PBS buffer, their concentrations were determined, and they were stored at -20°C.

[0497] Example 7: Cellular ELISA Affinity Detection of Humanized Anti-Human Claudin 18.2 Antibody

[0498] The affinity of the humanized monoclonal antibody against human Claudin18.2 was determined by cellular ELISA. The cellular ELISA procedure was as described in Examples I-3. The cells used were L929-Claudin18.2 (human), HEK293T-Claudin18.2 (human), NUGC4-Claudin18.2 (human), and NUGC4 cells endogenously expressing human Claudin18.2, all of which were transfected and stably expressed human Claudin18.2.

[0499] 1) For the two cell lines L929-Claudin18.2 and HEK293T-Claudin18.2, the experimental results are shown in Table 4. 2D3A11-HZ1, 2D3A11-HZ2, 25C7A5-HZ1 and 25C7A5-HZ2 all have high affinity for both.

[0500] Table 4. Cell ELISA Affinity of Anti-Claudin 18.2 Humanized Antibody

[0501]

[0502] Note: N=2 indicates two independent and repeated experiments.

[0503] 2) For NUGC4-Claudin18.2 cells, the experimental results are shown in Table 5 and... Figure 2A As shown, the EC50 value of 25C7A5-HZ1 binding to NUGC4-Claudin18.2 was 107.6 ng / mL, while the EC50 value of IMAB362 was 109.1 ng / mL. The maximum signal value of 25C7A5-HZ1 binding to NUGC4-Claudin18.2 was 1.284, which was significantly higher than the maximum signal value of IMAB362 (0.591), indicating that 25C7A5-HZ1 has a better affinity for NUGC4-Claudin18.2 than IMAB362.

[0504] 3) For NUGC4 cells, the experimental results are shown in Table 5 and... Figure 2B As shown, 2D3A11-HZ1, 25C7A5-HZ1, and 25C7A5-HZ2 have a high affinity for NUGC4, while IMAB362 has a weaker affinity for NUGC4 cells (not reaching the maximum signal value), indicating that 2D3A11-HZ1, 25C7A5-HZ1, and 25C7A5-HZ2 have a better affinity for NUGC4 than IMAB362.

[0505] Table 5. Cellular ELISA Affinity of Anti-Claudin 18.2 Humanized Antibody

[0506]

[0507] Example 8: Detection of cytosolic FACS and power of anti-human Claudin 18.2 humanized antibody

[0508] The affinity of the humanized antibody against human Claudin18.2 was detected by flow cytometry. The experimental procedure was as follows: HEK293T-Claudin18.2 (human), NUGC4-Claudin18.2 (human), and NUGC4 cells were digested and collected by centrifugation. Cells were washed three times with pre-chilled PBS. Cells were resuspended in 1% BSA (in PBS) and added to 96-well conical plates at a density of 3 × 10⁶ cells per well. 5 50 μl of cells were collected. The test antibody was diluted with 1% BSA (in PBS) starting at 100 μg / mL, followed by 3-fold serial dilutions to 10 concentration points. 50 μl of the serially diluted test antibody was added to each well of the cells, mixed, and incubated at 4°C for 1 hour. The cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 50 μl of 1% BSA (in PBS). 1 μl of fluorescent secondary antibody (Biolegend-410712) was added to each well, mixed, and incubated at 4°C for 0.5 hours. The cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 200 μl of PBS for analysis.

[0509] Experimental results are as follows Figures 3A-3C And as shown in Table 6. For HEK293T-Claudin18.2 cells ( Figure 3A ), 2D3A11-HZ1, 25C7A5-HZ1, and 25C7A5-HZ2 all have high affinity for it; for NUGC4-Claudin18.2 cells ( Figure 3B The affinity EC50 value of 25C7A5-HZ1 was 3192 ng / mL, while that of IMAB362 was 6668 ng / mL. Furthermore, the maximum signal value of 25C7A5-HZ1 was significantly higher than that of IMAB362, indicating that 25C7A5-HZ1 has a superior affinity for IMAB362. For NUGC4 cells (…), Figure 3C 25C7A5-HZ1 showed a significant binding affinity to NUGC4, while IMAB362 showed a very weak binding affinity to NUGC4, indicating that 25C7A5-HZ1 had a better affinity than IMAB362.

[0510] Table 6. Cellular FACS Affinity of Anti-Claudin18.2 Humanized Antibody

[0511]

[0512] Example 9: Evaluation of Species Affinity of Anti-human Claudin 18.2 Humanized Monoclonal Antibody

[0513] The affinity of the humanized monoclonal antibody against human Claudin18.2 for different species of Claudin18.2 was determined by flow cytometry (FACS). The experimental procedure was as follows: HEK293T-Claudin18.2 (cynomolgus monkey), HEK293T-Claudin18.2 (mouse), and HEK293T-Claudin18.2 (rat) cells were collected by digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in 1% BSA (in PBS). Cells were cultured in 3 × 10⁶ cells per well. 5 Cells were seeded into 96-well conical plates, serially diluted antibodies were added and incubated at 4°C for 1 h. After washing three times with pre-chilled PBS, 1 μl of fluorescent secondary antibody (APC: Biolegend-410712) was added to each well and incubated at 4°C for 0.5 h. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry.

[0514] Experimental results are as follows Figures 4A-4C As shown in Table 7, 2D3A11-HZ1, 25C7A5-HZ1, 25C7A5-HZ2, and IMAB362 all exhibit high affinity for Claudin18.2, a cynomolgus monkey. Figure 4A ); 25C7A5-HZ1 and IMAB362 and rat Claudin18.2 ( Figure 4B ), mouse Claudin18.2 ( Figure 4C It also has a good combination of species and genera.

[0515] Table 7. Cellular FACS Affinity of Anti-Claudin18.2 Humanized Antibody

[0516]

[0517] Example 10: Specificity evaluation of the anti-human Claudin 18.2 humanized monoclonal antibody

[0518] The specificity of the humanized monoclonal antibody against human Claudin 18.2 was determined by flow cytometry (FACS). The experimental procedure was as follows: HEK293T-Claudin 18.1 (human) cells were collected by digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in 1% BSA (in PBS) at 3 × 10⁶ cells per well. 5Cells were seeded into 96-well conical plates, and the antibody to be tested was added to a final concentration of 50 μg / mL. The cells were incubated at 4°C for 1 h, washed three times with pre-chilled PBS, and then 1 μl of fluorescent secondary antibody (APC: Biolegend-410712; or PE: BioLegend-410708) was added to each well. The cells were incubated at 4°C for 0.5 h, washed three times with pre-chilled PBS, and resuspended. The cells were then analyzed by flow cytometry. As a reference, positive control antibodies against human Claudin18.1, positive control antibody-1, and positive control antibody-2 were included in the experiment. The positive control antibodies were those from Example I-3 that showed significant binding to HEK293T-Claudin18.1 (human) cells. The antibody clone numbers were 13A6D2 (positive control antibody-1) and 15C12A2 (positive control antibody-2), respectively.

[0519] The experimental results are shown in Table 8. 2D3A11-HZ1, 2D3A11-HZ2, 25C7A5-HZ1 and 25C7A5-HZ2 do not non-specifically recognize human Claudin18.1.

[0520] Table 8 Non-specific detection of anti-Claudin 18.2 humanized antibodies

[0521] Antibody name Median APC fluorescence signal value Average APC fluorescence signal value Positive rate (%) 2D3A11-HZ1 233 281 0.69 2D3A11-HZ2 329 382 1 Positive control antibody-1 726 979 16.2 Antibody name Median PE fluorescence signal value Average PE fluorescence signal value Positive rate (%) 25C7A5-HZ1 504 583 0.54 25C7A5-HZ2 471 559 0.55 Positive control antibody-2 1055 1231 6.78 Negative control antibody (NC) 385 448 0.32

[0522] Example 11 Evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-human Claudin 18.2 humanized monoclonal antibody

[0523] ADCC activity of the humanized monoclonal antibody against Claudin18.2 was detected using a fluorescein reporter system. The Bio-Glory One-Step Firefly Luciferase Assay kit (Suzhou Ruian, RA-GL04) was used according to the manufacturer's instructions. The experimental steps were as follows: HEK293T-Claudin18.2 (human) cells were collected by digestion and centrifugation, resuspended in DMEM + 1% FBS, and seeded at 50,000 cells per well; Jurkat-NFAT-CD16a cells were collected by centrifugation, resuspended in 1640 + 1% FBS, and seeded at 100,000 cells per well; serially diluted antibody was added and incubated at 37°C for 5 hours; 50 μl of Bio-Glory One-Step luciferase substrate was added to each well, and the readings were taken using a microplate reader (MD, SpectraMax i3x).

[0524] Experimental results are as follows Figure 5As shown, 2D3A11-HZ1, 25C7A5-HZ1 and 25C7A5-HZ2 all have strong ADCC activity, with EC50 values ​​of 47.91 ng / mL, 46.49 ng / mL and 48.75 ng / mL, respectively.

[0525] Example 12 Evaluation of complement-dependent cytotoxic (CDC) activity of anti-human Claudin 18.2 humanized monoclonal antibody

[0526] The CDC activity of the anti-human Claudin18.2 humanized monoclonal antibody was detected by guinea pig serum killing assay. The experimental procedure was as follows: HEK293T-Claudin18.2 (human) cells were collected by digestion and centrifugation, and 50,000 cells were seeded per well overnight; the supernatant was discarded, and 100 μl of the antibody to be tested, serially diluted with DMEM + 20% guinea pig serum (Beijing Bosi, BM361Y), was added to each well. The cells were incubated at 37℃ for 5 h, and 20 μl of CCK8 reagent (Suzhou Ruian, QDY-003-D) was added to each well. The reaction was carried out for 1-4 hours, and the readings were taken at 450 nm using a microplate reader.

[0527] Experimental results are as follows Figure 6 As shown, 2D3A11-HZ1, 25C7A5-HZ1 and 25C7A5-HZ2 all have strong CDC activity, with EC50 values ​​of 1904 ng / mL, 3137 ng / mL and 3283 ng / mL, respectively.

[0528] Example 13 Evaluation of endocytic activity of humanized monoclonal antibody against human Claudin 18.2

[0529] The endocytic activity of the humanized monoclonal antibody against human Claudin18.2 was detected by flow cytometry (FACS). The experimental procedure was as follows: HEK293T-Claudin18.2 cells were collected by digestion and centrifugation, incubated with the antibody at a concentration of 10 μg / mL at 4°C for 1 hour, washed three times with PBS, resuspended in DMEM + 10% FBS, divided into four aliquots, and incubated at 37°C for 0, 1, 2, and 4 hours respectively. After washing three times with PBS, 1 μl of fluorescent secondary antibody (Biolegend, 410712) was added, and the cells were incubated at 4°C for 0.5 hours. After washing three times with PBS, the cells were resuspended and loaded onto the flow cytometry. The endocytosis rate was calculated using the following formula: Endocytosis rate (%) = [1 - (average fluorescence value of the sample detected at this time point - average fluorescence value of the negative control sample at this time point) / (average fluorescence value of the sample detected at 0 hours - average fluorescence value of the negative control sample at 0 hours)] * 100. The negative antibody was an isotype control antibody.

[0530] Experimental results showed that both 25C7A5-HZ1 and 25C7A5-HZ2 had endocytic activity, and the endocytosis rate of both was above 12% after 4 hours.

[0531] Example 14: Accelerated stability assay of anti-human Claudin 18.2 humanized antibody

[0532] The accelerated stability of the humanized monoclonal antibody against human Claudin 18.2 was determined using the following method: The antibody was ultrafiltered into PBS (pH = 7.4) at a concentration of 4.7 mg / mL, sterilized by filtration, and then incubated at 37°C for 0, 3, 7, and 14 days, or subjected to 4 and 8 freeze-thaw cycles at -80°C. The purity of the samples was then determined by SEC-HPLC and CE-SDS. A 0-day incubation at 37°C was used as a control to examine the stability changes of the antibody in the accelerated stability test.

[0533] The SEC-HPLC detection method is as follows:

[0534] Instrument: Waters Alliance e2695 HPLC;

[0535] Column: Thermo MabPac SEC-1, 5µm, 7.8*300mm;

[0536] Mobile phase: 61 mmol / L Na2HPO4, 39 mmol / L NaH2PO4, 200 mmol / L NaCl, 5% IPA;

[0537] Instrument parameters: Sample chamber temperature: 8℃; Column temperature: 30℃; Flow rate: 0.5ml / min; Injection volume: 20μg; Detection wavelength: 280nm; Isocratic run: 30min.

[0538] The CE-SDS detection method is as follows: Dilute the sample to 4 mg / ml with ultrapure water. Take 25 μl into a centrifuge tube, add 75 μl of premixed solution (SDS Sample buffer + iodoacetamide), mix well, and centrifuge at 10000g for 1 minute. Incubate at 70℃ for 5 minutes. After removal, cool to room temperature, centrifuge at 10000g for 1 minute, mix well, and transfer to a sample vial.

[0539] The analysis was performed using a SCIEX PA800 plus capillary electrophoresis system, with the instrument parameters set as follows:

[0540] Capillary tube: 30.2cm*50μm (bare tube), effective length: 20cm

[0541] Capillary column temperature: 25℃, sample tray temperature: 15℃

[0542] Detection wavelength setting: 220nm; Acquisition frequency: 4Hz.

[0543] Experimental results show that the 25C7A5 humanized antibody of this invention has good stability under accelerated and repeated freeze-thaw conditions, with SEC purity above 95%, CE-SDS non-reduced purity above 90%, and CE-SDS reduced purity above 95%.

[0544] Example 15: Pharmacokinetic assay of anti-human Claudin 18.2 humanized antibody in rats

[0545] The metabolism of the humanized anti-human Claudin18.2 antibody in vivo was examined as follows: The test sample, the humanized antibody, and the IMAB362 antibody were administered to male rats (Chengdu Dashuo) via tail vein injection, with three rats per group, at a dose of 10 mg / kg. Serum samples were collected at 30 min, 1 h, 2 h, 6 h, 24 h, 48 h, 96 h, and 168 h post-administration. Serum drug concentrations were detected using an L929-Claudin18.2 cell ELISA (method as described in Example 3). The results showed that the humanized 25C7A5 antibody of this invention had a comparable half-life in rats to the reference antibody IMAB362.

[0546] Example II: Preparation and Characterization of Antibody-Drug Conjugates

[0547] Example 1: Preparation of Antibody-Drug Conjugates (ADCs)

[0548] Example 1.1: Synthesis of intermediates used in the synthesis of "drug-linker compounds"

[0549] Example 1.1.1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (A1)

[0550]

[0551] Step 1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (A1B)

[0552] Under ice bath conditions, ferrous sulfate heptahydrate (570 mg dissolved in 1 mL of water) and 4,4-dimethoxychlorobutane (3.89 g) were added to a 75% sulfuric acid solution (5 mL) of compound (S)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (A1A, Cas No.: 151636-76-9, 500 mg). After stirring the reaction solution for three minutes, hydrogen peroxide (29%, 2.5 mL) was added dropwise. The reaction solution was stirred at 0 °C for 5 minutes, then brought to room temperature and stirred for 3 hours. The reaction solution was diluted with water (50 mL), extracted with ethyl acetate (80 mL × 2), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was further purified using a C18 column (acetonitrile / 0.05% formic acid aqueous solution: 5%-60%) to give the target compound A1B (yellow solid, 400 mg, yield: 67%).

[0553] LCMS(ESI)[M+H] + 468.9;

[0554] 1 H NMR (400MHz, DMSO-d6) δ7.65(s, 1H), 7.51(s, 1H), 7.24(s, 1H), 6.50(s, 1H), 6.30(s, 2H), 5.42(s, 2H), 5.26 (s, 2H), 3.81 (d, J=5.9Hz, 2H), 3.22 (s, 2H), 1.98 (d, J=6.7Hz, 4H), 0.88 (t, J=7.2Hz, 3H).

[0555] Step 2: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (A1)

[0556] The compound (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (100 mg, 0.213 mmol) was dissolved in 10% sulfuric acid (5 mL) solution, and the reaction was carried out at 110 °C for 48 hours. Saturated sodium bicarbonate solution (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (10 mL × 5). The extract was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The product was prepared and purified by high performance liquid chromatography (acetonitrile / water containing 0.05% formic acid) to obtain (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (A1, 1.78 mg).

[0557] LCMS(ESI)[M+H] + :451.0;

[0558] 1 H NMR (400MHz, DMSO-d6) δ7.63 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.28 (s, 2H), 5.47-5.37 ( m, 2H), 5.32-5.19 (m, 2H), 3.51-3.46 (m, 2H), 3.17-3.13 (m, 2H), 1.92-1.76 (m, 4H), 0.90-0.84 (m, 3H).

[0559] Example 1.1.2: Synthesis of (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)propoxy)methyl)acetamide (B1)

[0560]

[0561] Step 1: Compound (B1A) (Cas No.: 1599440-06-8, 368 mg), compound (A1) (440 mg), and pyridinium p-toluenesulfonate (PPTS) (25 mg) were refluxed in dichloromethane (20 ml) for 20 hours, then washed with aqueous sodium bicarbonate solution and aqueous hydrochloric acid solution, respectively. The organic solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound B1B (240 mg).

[0562] LCMS(ESI)[M+H] + : 759.5.

[0563] Step 2: Dissolve BIB (240 mg) in DMF (5 ml), add piperidine (1 ml), stir the compound for 20 minutes, remove low-boiling components under reduced pressure, and use the residue directly for the next synthesis. A small amount of crude product was purified by reversed-phase chromatography (acetonitrile / 0.05% FA aqueous solution: 5% to 50%) to obtain the target compound B1.

[0564] ESI-MS (m / z): 537.4 [M+H] + ;

[0565] 1 H NMR (400MHz, DMSO-d6) δ9.13 (t, 1H), 8.04 (br, 2H), 7.58 (s, 1H), 7.51 (s, 1H), 7.25 (s, 1H), 6.29 (s, 2H), 5.43 (S, 2H), 5.21 (s, 2H), 4.65 (d, 2H), 3.63 (m, 2H), 3.53 (m, 2H), 3.11 (m, 2H), 1.87 (m, 4H), 0.88 (t, 3H).

[0566] Example 1.1.3: N 6 N 6 -dimethyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1)

[0567]

[0568] 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid (268 mg, Cas No. 2356229-58-6), compound C1A (328 mg), and triethylamine (322 mg) were dissolved in N,N-dimethylformamide (5 mL). Then, 1-hydroxybenzotriazole (HOBT, 162 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 229 mg) were added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then purified directly through a C18 column in reverse phase (acetonitrile and 0.05% formic acid aqueous solution) to obtain the target compound N. 6 N 6 -dimethyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1, white solid, 327 mg).

[0569] LCMS(ESI)[M+H] + :524.4.

[0570] 1 H NMR (400MHz,) δ9.13 (s, 2H), 7.95 (t, J=8.8Hz, 2H), 4.21 (dd, J=8.8, 6.9Hz, 1H), 4.08-4.03(m, 1H), 3.41(s, 3H), 2.55(t, J=7.0Hz, 2H), 2.42-2.32(m, 4H ), 2.27 (s, 6H), 1.98 (dd, J=13.6, 6.8Hz, 1H), 1.86-1.77 (m, 2H), 1.74-1.55 (m, 2H), 1.47-1.37 (m, 2H), 1.31-1.23 (m, 2H), 0.85 (dd, J=12.8, 6.8Hz, 6H).

[0571] Example 1.1.4: N 6 N 6 -Diethyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C2)

[0572]

[0573] Step 1:

[0574] Compound C2A (5.0 g, 12.05 mmol, Cas No.: 1872-06-8) was dissolved in dichloromethane (100 mL). Acetaldehyde (3.2 g, 72.3 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes. Then, sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. LC-MS showed that the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction solution, and the mixture was stirred for one hour. The solution was then evaporated to dryness, filtered, and the filtrate was purified by reverse separation using a C18 column (acetonitrile to 0.05% formic acid aqueous solution: 5% to 55%) to give the target compound C2B (4.57 g, yield 82.0%) as a white solid.

[0575] LCMS(ESI)[M+H] + =436.4;

[0576] 1 H NMR (400MHz, DMSO-d6) δ7.70 (d, J=7.0Hz, 1H), 7.41 (d, J=9.0Hz, 1H), 7.38-7.26 (m , 5H), 5.08-4.99 (m, 2H), 4.00 (dd, J=12.6, 6.5Hz, 1H), 3.86 (dd, J=8.6, 6.8Hz, 1H), 2.74(dd, J=14.0, 6.9Hz, 4H), 2.64-2.54(m, 2H), 2.05-1.94(m, 1H), 1.72-1.52(m, 2 H), 1.52-1.38 (m, 2H), 1.38-1.18 (m, 2H), 1.04 (t, J=7.1Hz, 6H), 0.87-0.81 (m, 6H).

[0577] Step Two:

[0578] Compound C2B (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL) at room temperature. Pd / C (0.16 g) was then added to the reaction solution, and the mixture was stirred at room temperature under hydrogen atmosphere for 12 hours. LC-MS showed the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound C2C (900 mg, 82% yield) as a milky-white solid.

[0579] 1H NMR(400MHz, DMSO-d6)δ8.04(br s, 1H), 4.02-3.99 (m, 1H), 3.10 (d, J=4.5Hz, 1H), 2.65 (q, J=7.1Hz, 4H), 2.55-2.51 (m, 2H), 2.06-1.93 (m, 1H), 1.73-1 .54 (m, 2H), 1.47-1.38 (m, 2H), 1.30-1.21 (m, 2H), 1.01 (t, J=7.1Hz, 6H), 0.89 (d, J=6.9Hz, 3H), 0.79 (d, J=6.8Hz, 3H).

[0580] Step 3:

[0581] Compound 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (268 mg, 1 mmol) was dissolved in DMF (8 mL), followed by the addition of HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol). After stirring at room temperature for 20 minutes, compound C2C (301 mg, 1 mmol) was added, and stirring continued at room temperature for another 30 minutes. After LCMS analysis, the reaction solution was purified directly by reverse-phase C18 column chromatography (acetonitrile and 0.05% formic acid aqueous solution) to obtain the target compound C2 (280 mg, yield 51%) as a white solid.

[0582] LCMS(ESI)[M+H] + =552.3;

[0583] 1 H NMR (400MHz, DMSO-d6) δ9.13 (s, 2H), 7.95 (d, J = 8.9Hz, 1H), 7.86 (d, J = 7.2Hz, 1H), 4.18 (dd, J=8.8, 6.8Hz, 1H), 4.02 (dd, J=12.8, 7.2Hz, 1H), 3.41 (s, 3H), 2.74-2.69 (m, 4H), 2 .62-2.52(m, 4H), 2.44-2.29(m, 2H), 2.04-1.94(m, 1H), 1.86-1.77(m, 2H), 1.72-1.54( m, 2H), 1.51-1.39 (m, 2H), 1.33-1.23 (m, 2H), 1.02 (t, J=7.2Hz, 6H), 0.87-0.82 (m, 6H).

[0584] Example 1.1.5: N 6 N 6 -di-n-propyl-N 2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C3)

[0585]

[0586] Step 1:

[0587] Compound C2A (5.0 g, 12 mmol) was dissolved in dichloromethane (100 mL). Propanal (4.2 g, 72.3 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes. Then, sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was added, and the reaction was stirred at room temperature for 1 hour. LC-MS showed the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction solution, and the mixture was stirred for one hour. The solution was then evaporated to dryness, filtered, and the filtrate was purified by reverse-phase separation using a C18 column (acetonitrile to 0.05% formic acid aqueous solution: 5% to 55%) to give the target compound C3A (4.57 g, yield 82.0%) as a white solid.

[0588] LCMS(ESI)[M+H] + =464.0;

[0589] 1 H NMR (400MHz, DMSO-d6) δ7.81 (d, J=7.3Hz, 1H), 7.38-7.28 (m, 5H), 5.04 (d, J=1.7Hz, 2H), 4.12-4.02 (m, 1H), 3.94-3.82 (m, 1H), 2.6 5-2.52 (m, 6H), 2.06-1.94 (m, 1H), 1.76-1.64 (m, 1H), 1.64-1.53 ​​(m, 1H), 1.52-1.40 (m, 6H), 1.34-1.18 (m, 2H), 0.92-0.80 (m, 12H).

[0590] Step Two:

[0591] Compound C3A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL) at room temperature. Pd / C (0.16 g) was then added to the reaction solution, and the mixture was stirred at room temperature under hydrogen atmosphere for 12 hours. LC-MS showed the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the target compound C3B (1.2 g, 85.5% yield) as a white solid.

[0592] Step 3:

[0593] Compound 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ethynic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the addition of HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol). The mixture was stirred for 30 minutes, and then compound C3B (122 mg, 0.371 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed as determined by LCMS, the reaction solution was directly purified by reverse-phase C18 column chromatography (acetonitrile and 0.05% formic acid aqueous solution) to obtain the target compound N. 6 N 6 -di-n-propyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C3, 50 mg, yield 28%) is a pale yellow solid.

[0594] LCMS(ESI)[M+H] + =580.0;

[0595] 1 H NMR (400MHz, DMSO-d6) δ8.24 (s, 2H), 7.98-7.93 (m, 2H), 4.24-4.16 (m, 1H), 4 .10(d, J=5.2Hz, 1H), 3.41(s, 3H), 2.79-2.64(m, 6H), 2.55(t, J=7.1Hz, 2H), 2.45-2.26(m, 2H), 2.06-1.91(m, 1H), 1.89-1.78(m, 2H), 1.76-1.66(m, 1H), 1.64-1.57(m, 1H), 1.57-1.42(m, 6H), 1.37-1.24(m, 2H), 0.93-0.78(m, 12H).

[0596] Example 1.2 Synthesis of drug-linker compounds

[0597] Example 1.2.1: N-((11S,14S)-11-(4-(di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecane-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (DL-01)

[0598]

[0599] Compound C3 (43 mg, 0.074 mmol) and compound B1 (40 mg, 0.075 mmol) were dissolved in N,N-dimethylformamide (1 mL), followed by the sequential addition of HBTU (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (24 mg, 0.187 mmol). The reaction mixture was stirred at room temperature for 1 hour. After the reaction was completed as detected by LCMS, the reaction solution was directly purified by preparative chromatography (0.01% trifluoroacetic acid aqueous solution, acetonitrile) to obtain the target compound (DL-01, 8.5 mg, yield 10%) as a yellow solid.

[0600] LCMS(ESI)[M+H] + =1098.6;

[0601] 1 H NMR (400MHz, DMSO-d6) δ9.10 (s, 2H), 9.03 (s, 1H), 8.64 (t, J = 6.4Hz, 1H), 8.19 (t, J = 5.9Hz, 1H), 8.08 (d, J = 7.4Hz, 1H), 7.92 (d, J = 8.5Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.24 (s, 2H), 4.66-4.52 (m, 2H), 4.31-4 .21 (m, 1H), 4.19-4.10 (m, 1H), 3.74 (d, J=5.5Hz, 2H), 3.49-3.48 (m, 2H), 3.40 (s, 3H), 3.15-3.06 (m, 2H), 3.02-2.95 (m, 6H), 2.59 -2.52 (m, 3H), 2.41-2.29 (m, 2H), 2.05-1.90 (m, 2H), 1.91-1.77 (m, 6H), 1.63-1.57 (m, 6H), 1.31-1.29 (m, 2H), 0.92-0.80 (m, 15H).

[0602] Example 1.2.2: N-((11S,14S)-11-(4-(diethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecane-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (DL-02)

[0603]

[0604] Compound C2 (40 mg, 0.075 mmol) and compound B1 (41 mg, 0.075 mmol) were dissolved in DMF (1 mL), followed by the addition of HOBt (15.2 mg, 0.113 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.5 mg, 0.113 mmol), and then triethylamine (23 mg, 0.225 mmol). The reaction mixture was stirred at room temperature for 16 hours after the addition was complete. After LCMS analysis, the reaction mixture was concentrated to obtain the crude product. The crude product was purified by preparative chromatography (0.01% TFA aqueous solution, acetonitrile) to obtain the target compound (DL-02, 16 mg, yield 20%) as a yellow solid.

[0605] LCMS(ESI)[M+H] + =1070.6;

[0606] 1H NMR (400MHz, DMSO-d6) δ9.13-9.08 (m, 2H), 9.01 (s, 1H), 8.64 (t, J=6.5Hz, 1H), 8.20 (t, J=5.7Hz, 1H), 8.09 (d, J=7.4Hz, 1H), 7.92 (d, J=8 .4Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.50 (s, 1H), 6.29 (s, 2H), 5.43 (s, 2H), 5.24 (s, 2H), 4.65-4.53 (m, 2H), 4.26 (d, J=6.5 Hz, 1H), 4.20-4.10 (m, 1H), 3.74 (d, J=5.5Hz, 2H), 3.50 (t, J=5.8Hz, 2H), 3.41 (s, 3H), 3.14-3.07 (m, 6H), 2.98 (s, 2H), 2.55 (d, J=7.3Hz, 2H), 2.41-2.29(m, 2H), 2.03-1.89(m, 2H), 1.89-1.77(m, 7H), 1.61-1.56(m, 2H), 1.32(s, 2H), 1.16(t, J=7.2Hz, 6H), 0.91-0.78(m, 9H).

[0607] Example 1.2.3: N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentano[4,5-g]pyrano[3′,4′:6,7]indolazano[1,2-b]quinoline-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecano-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (DL-03)

[0608]

[0609] Compound B1 (100 mg, 0.186 mmol) and compound N 6 N 6 -dimethyl-N 2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (C1, 100 mg, 0.191 mmol) was dissolved in DMF (2 mL). Then, 1-hydroxybenzotriazole (38 mg, 0.280 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (53 mg, 0.280 mmol) were added, followed by triethylamine (56 mg, 0.559 mmol). After the addition was complete, the reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed by LCMS, the reaction mixture was purified by preparative chromatography (0.01% TFA aqueous solution, acetonitrile) to obtain the target compound (DL-03, 20 mg, yield 10%) as a yellow solid.

[0610] LCMS(ESI)[M+H] + =1042.5;

[0611] 1 H NMR (400MHz, DMSO-d6) δ9.28 (s, 1H, TFA), 9.10 (s, 2H), 8.64 (br s, 1H), 8.19 (br s, 1H), 8.09 (d, J=6.4Hz, 1H), 7.92 (d, J=7.8Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.5 0(s, 1H), 6.29(s, 2H), 5.43(s, 2H), 5.24(s, 2H), 4.67-4.52(m, 2H), 4.30-4.10(m, 2H), 3.74(br s, 2H), 3.50(br s, 2H), 3.41 (s, 3H), 3.17-3.07 (m, 2H), 3.04-2.91 (m, 2H), 2.75 (s, 6H), 2.46-2.24 ( m, 3H), 2.04-1.66 (m, 9H), 1.63-1.46 (m, 3H), 1.32-1.29 (m, 2H), 0.87-0.82 (m, 9H).

[0612] Example 1.3 Preparation of anti-CLDN18.2 antibody-drug conjugate

[0613]

[0614] Ab stands for antibody.

[0615] 1.3.1 Sample Preparation for CLDN18.2-ADC (DAR8)

[0616] 30 mg of anti-CLDN18.2 antibody was diluted with diluent (20 mM PB, pH 7.5), and a final concentration of 5 mM sodium edetate solution was added and mixed. 6.0 equivalents of the antibody in TCEP solution were added, mixed, and incubated at room temperature for 60 minutes. 12 equivalents of the antibody in DL-01 dissolved in dimethyl sulfoxide were added to the above solution, mixed, and incubated at room temperature for 1 hour to obtain the conjugated sample. After the reaction, the sample was replaced with 10 mM histidine buffer (pH 5.5) using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-CLDN18.2 antibody-ADC composition, CLDN18.2-ADC (DAR8). The DAR value was determined to be 8.0 using the mass spectrometry method described in Example 3.4.

[0617] 1.3.2 CLDN18.2-ADC(DAR2) Sample Preparation

[0618] 30 mg of anti-CLDN18.2 antibody was diluted with diluent (20 mM PB, pH 7.5), and a final concentration of 5 mM sodium edetate solution was added and mixed. Then, 6.0 equivalents of TCEP solution were added, mixed, and incubated at room temperature for 60 minutes. Next, 2.5 equivalents of DL-01 dissolved in dimethyl sulfoxide were added to the above solution, mixed, and incubated at room temperature for 1 hour to obtain the conjugated sample. After the reaction, the sample was replaced with 10 mM histidine buffer (pH 5.5) using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-CLDN18.2 antibody-ADC composition, CLDN18.2-ADC (DAR2). The DAR value was determined to be 1.7 by mass spectrometry.

[0619] 1.3.3 CLDN18.2-ADC(DAR4) Sample Preparation

[0620] 30 mg of anti-CLDN18.2 antibody was diluted with diluent (20 mM PB, pH 7.5), and a final concentration of 5 mM sodium edetate solution was added and mixed. Then, 6.0 equivalents of TCEP solution were added, mixed, and incubated at room temperature for 60 minutes. Finally, 4.5 equivalents of DL-01 dissolved in dimethyl sulfoxide were added to the above solution, mixed, and incubated at room temperature for 1 hour to obtain the conjugated sample. After the reaction, the sample was replaced with 10 mM histidine buffer (pH 5.5) using a 30 kDa ultrafiltration tube to remove low molecular weight substances. The sample was then concentrated to obtain a solution containing the anti-CLDN18.2 antibody-ADC composition, CLDN18.2-ADC (DAR4). The DAR value was determined to be 4.0 by mass spectrometry.

[0621] 1.3.4 CLDN18.2-ADC(DAR6) Sample Preparation

[0622] 30 mg of anti-CLDN18.2 antibody was diluted with diluent (20 mM PB, pH 7.5), and a final concentration of 5 mM sodium edetate solution was added and mixed. Then, 6.0 equivalents of TCEP solution were added, mixed, and incubated at room temperature for 60 minutes. Finally, 7.0 equivalents of DL-01 dissolved in dimethyl sulfoxide were added to the above solution, mixed, and incubated at room temperature for 1 hour to obtain the conjugated sample. After the reaction, the sample was replaced with 10 mM histidine buffer (pH 5.5) using a 30 kDa ultrafiltration tube to remove low molecular weight substances. The sample was then concentrated to obtain a solution containing the anti-CLDN18.2 antibody-ADC composition, CLDN18.2-ADC-07 (DAR6). The DAR value was determined to be 5.6 by mass spectrometry.

[0623] Example 1.4 Determination of DAR value of coupled sample by mass spectrometry

[0624] LC-MS analysis of molecular weight and DAR value of CLDN18.2-ADC-07(DAR8) was performed under the following conditions:

[0625] Chromatographic conditions:

[0626] Column: ACQUITY UPLC BEH200 SEC 1.7μm, 4.6×150mm

[0627] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN

[0628] Column temperature: 30℃

[0629] Sample chamber temperature: 8℃

[0630] Flow rate: 0.3 ml / min

[0631] Injection volume: 1 μl

[0632] Time (min) 1 5 7 7.1 10 Mobile phase A 90 40 10 90 90 Mobile phase B 10 80 80 10 10

[0633] Sample preparation: Take 50 μg of sample, add 2 μl of 1M DTT, add ultrapure water to 50 μl to dilute to about 1.0 mg / ml concentration, mix well, and reduce at room temperature for 30 min.

[0634] LC / MS Model: AB SCIEX X500 Q-TOF

[0635] Mass spectrometry conditions: Gas1: 45; Gas2: 45; CUR: 30; TEM: 450; ISVF: 5000; DP: 120; CE: 12; Mass number range: 600-4000

[0636] The results are shown in Table 9 below:

[0637] Table 9: Theoretical Molecular Weight and Measured Molecular Weight

[0638]

[0639] In Table 9, mAb represents unconjugated monoclonal antibodies; LC represents antibody light chains; HC represents antibody heavy chains; DAR1 represents conjugates containing one toxin molecule conjugated to either a light or heavy chain; DAR2 represents conjugates containing two toxin molecules conjugated to either a light or heavy chain; and DAR3 represents conjugates containing three toxin molecules conjugated to either a light or heavy chain. The theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycoform. mAb, LC, HC, DAR1, DAR2, and DAR3 will be explained as above in the following text.

[0640] The test results showed that the antibody light chain on CLDN18.2-ADC(DAR8) was conjugated with 0 to 1 toxin molecule (LC, DAR1 ratios were 0% and 100%, respectively), and the heavy chain was conjugated with 0 to 3 toxin molecules (mAb, DAR1, DAR2, and DAR3 ratios were 0%, 0%, 0%, and 100%, respectively). Therefore, the drug-antibody conjugation ratio (DAR value) of CLDN18.2-ADC(DAR8) was calculated to be 8.0.

[0641] Similarly, the drug-antibody conjugation ratio (DAR value) of other conjugated samples can be determined using mass spectrometry.

[0642] Example 2: Anti-Claudin18.2 antibody ADC conjugation and evaluation

[0643] Example 2.1 Anti-Claudin18.2 antibody ADC conjugation

[0644] Following the ADC preparation method described in Example 1, an ADC was constructed using 25C7A5-HZ1, 25C7A5-HZ2, and isotype control IgG antibodies.

[0645] 1) Antibody pretreatment: Add 0.1M EDTA stock solution at 1% of the antibody volume, and then adjust the sample pH to 7.7±0.2 with 1M disodium hydrogen phosphate stock solution.

[0646] 2) Preparation of TCEP stock solution: Weigh a certain mass of TCEP-HCl, add 80% TCEP solvent, then adjust the pH to 7.7±0.2 with 0.5M sodium hydroxide, and finally make up the volume with TCEP solvent to make the TCEP concentration 20mM.

[0647] 3) Reduction: Add the pretreated antibody to the TCEP stock solution at a ratio of 1:4.4 (antibody:TCEP molar ratio), mix well and react at room temperature for 0.5 h.

[0648] 4) Coupling: Add toxin linker to the reduced antibody at a ratio of 1:12 (antibody:toxin molar ratio), mix well and react at room temperature for ≥2h.

[0649] 5) Ultrafiltration: Use an ultrafiltration tube to replace the reaction solution with ultrafiltration buffer (10mM HCl-His, pH 5.5) (after concentration, add more than 4 times the volume of buffer each time, and change the solution more than 3 times), and then rinse the ultrafiltration membrane with a small amount of buffer to recover the sample.

[0650] The concentration, purity, and DAR of the sample were measured after conjugation. The results are shown in Table 10. No obvious aggregation was found after the antibody was conjugated with the ADC, and the purity determined by SEC-HPLC was higher than 98%.

[0651] Table 10. Results of ADC detection of anti-Claudin18.2 humanized antibody

[0652] No.* Sample Name SEC (%) DAR 1 25C7A5-HZ1-B82 98.85 6.82 2 25C7A5-HZ2-B82 99.34 7.10 3 IgG-B81 98.4 8.00 4 25C7A5-HZ1-B81 98.4 7.97

[0653] *: Samples 1-2 and 3-4 are two batches of preparations prepared separately.

[0654] Example 2.2 Anti-Claudin18.2 Humanized Antibody and ADC Affinity Detection

[0655] Following a method similar to that described in Example 2.2, the cell-binding affinity of the antibody before and after conjugation with the toxin was determined. L929-claudin 18.2 (human) or NUGC4 cells were collected by trypsin digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in 5 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates and incubated overnight. Cells were fixed with 4% paraformaldehyde and blocked with 2% BSA (in PBS) at 37°C for 2 hours. Serially diluted ADC drug or antibody was added and incubated at 37°C for 2 hours. HRP-labeled anti-human secondary antibody (Jackson, 109-035-088) was added and incubated at 37°C for 1 hour. TMB substrate was added for color development. After stopping with 2M HCl, the absorbance was read at 450 nM using a microplate reader.

[0656] Experimental results are as follows Figure 8A and 8B As shown, the affinity of 25C7A5-HZ1 and 25C7A5-HZ2 for cells did not change significantly before and after coupling.

[0657] Example 2.3 Detection of ADC in vitro killing activity

[0658] HEK293T-claudin 18.2 (human), HEK293T-claudin 18.1 (human), and NUGC4-Claudin18.2 cells were collected by trypsin digestion and resuspended in DEME + 2% FBS, with 10,000 cells per well. The antibody ADC drug to be tested was serially diluted with DEME + 2% FBS and added to the plate, and incubated at 37°C for 72 hours. After incubation, CCK8 reagent was added at 20 μL / well, and the reaction was carried out for 2-4 hours. The readings were taken at 450 nm using a microplate reader and imported into Graphpad Prism for curve fitting.

[0659] Experimental results Figures 9A-9E As shown, the ADCs formed by conjugation of 25C7A5-HZ1 and 25C7A5-HZ2 can effectively kill HEK293T-claudin18.2 cells and NUGC-4-Claudin18.2 cells; at the same time, the conjugated ADCs of 25C7A5-HZ1 and 25C7A5-HZ2 have basically no effect on HEK293T-claudin18.1 cells, indicating that the conjugated ADCs do not non-specifically recognize claudin18.1 and have good stability.

[0660] Example 2.4 In vivo efficacy of ADC

[0661] To verify the in vivo efficacy of the anti-Claudin18.2 humanized ADC drug, the antitumor effect of the test drug in a female Balb / c Nude mouse model with subcutaneous xenograft of human gastric cancer cells NUGC-4 (Nanjing Kebai) was evaluated.

[0662] Purchase 5-6 week old female Balb / c Nude mice (Vitollife). NUGC4 cells that have reached the logarithmic growth phase were digested with EDTA and resuspended in PBS. 5 x 103 cells were subcutaneously injected into each mouse. 6 Cells. Wait until the tumor grows to 100-200m². 3The ADC drug was administered intravenously once a week at a dose of 10 mg / kg, as detailed in Table 7 below. The main observation indicators for this experiment were: 1) Relative tumor proliferation rate, T / C (%), which is the percentage of tumor volume or weight in the treatment group and the control group at a specific time point. The calculation formula is: T / C (%) = TRTV / CRTV × 100% (TRTV: average RTV in the treatment group; CRTV: average RTV in the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment); or T / C% = TTW / CTW × 100% (TTW: average tumor weight in the treatment group at the end of the experiment; CTW: average tumor weight in the control group at the end of the experiment). 2) Relative tumor inhibition rate, TGI (%), calculated as: TGI (%) = (1-T / C) × 100% (T and C are the relative tumor volume (RTV) or tumor weight (TW) in the treatment group and the control group at a specific time point, respectively). 3) Photographs of tumor volume and weight at the experimental endpoint. 4) Effect of ADC drugs on mouse body weight.

[0663] Table 11. In vivo efficacy assay protocol for anti-Claudin18.2 humanized antibody ADC

[0664]

[0665] Experimental results are as follows Figure 10 and Figure 11 This indicates that 25C7A5-hz1-B81 has significant efficacy in the NUCC-4 CDX model. Figure 10 Moreover, the mice generally tolerated the condition well, with no adverse reactions. Figure 11 ).

[0666] Sequence List:

[0667]

[0668]

[0669]

[0670]

Claims

1. An antibody-drug conjugate (ADC) having the following formula (I) or a pharmaceutically acceptable salt thereof: Ab-[L-D] q (I) in, Ab indicates anti-Claudin18.2 antibody. L represents the connector. D represents cytotoxic drugs, and q=1-20， The Ab contains three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs), wherein: (i) According to the IMGT definition, HCDR1 consists of the amino acid sequence of SEQ ID NO:6, HCDR2 consists of the amino acid sequence of SEQ ID NO:7, HCDR3 consists of the amino acid sequence of SEQ ID NO:8, LCDR1 consists of the amino acid sequence of SEQ ID NO:9, LCDR2 consists of the amino acid sequence of SEQ ID NO:10, and LCDR3 consists of the amino acid sequence of SEQ ID NO:11; or (ii) According to Kabat’s definition, HCDR1 consists of the amino acid sequence of SEQ ID NO:12, HCDR2 consists of the amino acid sequence of SEQ ID NO:13, HCDR3 consists of the amino acid sequence of SEQ ID NO:14, LCDR1 consists of the amino acid sequence of SEQ ID NO:15, LCDR2 consists of the amino acid sequence of SEQ ID NO:16, and LCDR3 consists of the amino acid sequence of SEQ ID NO:

17.

2. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein D represents a topoisomerase I inhibitor.

3. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 1, wherein q = 1-10.

4. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 1, wherein q = 1-8.

5. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 1, wherein q = 2-8.

6. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 1, wherein q = 4-8.

7. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 1, wherein q = 6-8.

8. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The Ab includes: - A heavy chain variable region sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 3; or - A light chain variable region sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

9. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 1, wherein, The Ab includes: (1) The heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2; or (2) The heavy chain variable region shown in SEQ ID NO: 3 and the light chain variable region shown in SEQ ID NO:

2.

10. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The Ab is an IgG1 antibody.

11. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The Ab comprises heavy chains and light chains, wherein: (a) The heavy chain contains the amino acid sequence of SEQ ID NO:18, and the light chain contains the amino acid sequence of SEQ ID NO:20; or (b) The heavy chain contains the amino acid sequence of SEQ ID NO:19, and the light chain contains the amino acid sequence of SEQ ID NO:

20.

12. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The Ab is a full-length antibody composed of two heavy chains shown in SEQ ID NO:18 and two light chains shown in SEQ ID NO:

20.

13. The antibody-drug conjugate or its pharmaceutically acceptable salt according to any one of claims 1-12, wherein, D indicates camptothecin-based drugs.

14. The antibody-drug conjugate of claim 13 or a pharmaceutically acceptable salt thereof, wherein, D indicates camptothecin-based drugs containing the following structures: Among them, R a Selected from: hydrogen; C3-C8 cycloalkyl; Phenyl; C1-C8 alkyl groups optionally substituted with substituents selected from the following: halogen, hydroxyl, C3-C8 cycloalkyl, heterocyclic alkyl, phenyl, NR 1 R 2 Or, optionally, C1-C4 alkoxy groups substituted with NH2, NH (C1-C4 alkyl), and N (C1-C4 alkyl)2. Where R 1 and R 2 Selected independently from each other hydrogen; C1-C8 alkyl groups optionally substituted with substituents selected from the following: hydroxyl, amino, amino group substituted with one or two C1-C4 alkyl groups, amino group substituted with one or two C1-C4 hydroxyalkyl groups, amino group substituted with (C1-C4 hydroxyalkyl) and (C1-C4 alkyl) groups. By 1 or 2 Cs 3- C 10 cycloalkyl, C 3- C 10 Heterocyclic alkyl, phenyl, or heteroaryl-substituted C1-C4 alkyl groups; C3-C 10 cycloalkyl; C 3- C 10 Heterocyclic alkyl C2-C6 heteroalkyl groups; Mixed aromatics; Optional halogenated phenyl groups; C can be substituted with hydroxyl or amino groups. 1- C8 alkyl-C(=O)-; Or, R 1 and R 2 These atoms combine with their respective nitrogen atoms to form 5-, 6-, or 7-membered heterocycles having 0 to 3 substituents selected from halogens, C1-C4 alkyl, OH, C1-C4 alkoxy, NH2, NH(C1-C4 alkyl), and N(C1-C4 alkyl)2. Each of the cycloalkyl, heterocycloalkyl, phenyl, and heteroaryl groups is independently and optionally substituted by 0 to 3 substituents selected from the following: OH, C1-C4 alkyl, C1-C4 alkoxy, NH2, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2.

15. The antibody-drug conjugate of claim 14 or a pharmaceutically acceptable salt thereof, wherein R a It can be -C1-C4 alkyl-OH, -C1-C4 alkyl-O-C1-C4 alkyl-NH2, or -C1-C4 alkyl-NH2.

16. The antibody-drug conjugate of claim 14 or a pharmaceutically acceptable salt thereof, wherein, The drug unit D is connected to the linker unit L via hydroxyl or amino groups present thereon.

17. The antibody-drug conjugate or its pharmaceutically acceptable salt according to any one of claims 1-12, wherein, The LD unit in equation (I) has the following structure: or .

18. The antibody-drug conjugate of claim 17 or a pharmaceutically acceptable salt thereof, wherein, The LD unit in equation (I) has the following structure: 。 19. The antibody-drug conjugate or its pharmaceutically acceptable salt according to any one of claims 1-12, wherein, L is a peptide linker and comprises the structure of formula (II): -ZYM- in Z is the linker base connected to Ab. Y is a peptide with 2-5 amino acids. M is absent, or it is a spacer group used to link with drug D.

20. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein, Y is a peptide with the following amino acid sequence from the N-terminus to the C-terminus; Xaa1-Xaa2-Xaa3-Xaa4-Xaa5, Xaa1 is absent, or it is an amino acid selected from valine, glycine, alanine, and glutamic acid. Xaa2 is an amino acid selected from phenylalanine, leucine, and valine; Xaa3 is either unsubstituted or substituted lysine; Xaa4 is an amino acid selected from leucine, glycine, and alanine. Xaa5 is absent, or it may be an amino acid selected from glycine and alanine. The N-terminus of the amino acid sequence is connected to Z, and the C-terminus is connected to M when M is present or directly connected to drug D when M is absent.

21. The antibody-drug conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein Xaa2 is valine.

22. The antibody-drug conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein Xaa3 is a lysine with an ε-amino group mono- or di-substituted by a C1-C3 alkyl group.

23. The antibody-drug conjugate of claim 20 or a pharmaceutically acceptable salt thereof, wherein Y is a peptide selected from: Phe-Lys-Gly, Leu-lys-Gly, Gly-Val-Lys-Gly, Val-Lys-Gly-Gly, Val-Lys-Gly, Val-Lys-Ala, Val-Lys-Leu, wherein the Lys residue is an unsubstituted or C1-C3 alkyl monosubstituted or disubstituted lysine.

24. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein, Y is R3 and R4 are each independently selected from methyl, ethyl, and propyl. The wavy line on the left indicates the position connected to Z; the wavy line on the right indicates the position connected to M.

25. The antibody-drug conjugate of claim 24 or a pharmaceutically acceptable salt thereof, wherein, R3 and R4 are the same.

26. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 25, wherein, R3 and R4 are each independently propyl.

27. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein, Z has the following structure: -Z1-Z2-Z3-Z4-, Z1 represents the sulfur atom in Ab. Z2 is a 5-10 membered heterocyclic group containing one or two heteroatoms selected from N, S and O; Z3 is selected from key, -C(=O)-, -C1-C 10 Alkylene -C(=O)-, -C3-C 10 Ethyne-C(=O)-, -C3-C 10 alkenyl-C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 cycloalkyl-C(=O)-, -O-C1-C8 alkylene-C(=O)-, -arylene-C(=O)-, -C1-C 10 alkylene-arylene-C(=O)-, -arylene-C 1- C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-C3-C8 cycloalkylene-C(=O)-, -C3-C8 cycloalkylene-C1-C 10 Alkylene -C(=O)-, -C 3- C8 heterocyclic group -C(=O)-, -C1-C 10 Alkylene-C3-C8 heterocyclic-C(=O)-, -C3-C8 heterocyclic-C1-C 10 Alkylene-C(=O)-, Z4 is a bond or a PEG unit represented by the following formula. R5 is selected from C 1-4 Alkylene, -NH-, -NH-C 1-4 alkylene-heteroaryl-, wherein the heteroaryl group is a 5- or 6-membered nitrogen-containing heteroaryl group; R6 is -C(=O)-, C 1-4 Alkylene, C 1-4 Alkylene -C(=O)-, -NH-C(=O)-(CH2OCH2)-C(=O)-, C 1-4 Alkylene -NH-C(=O)-(CH2OCH2)-C(=O)-, where m is an integer from 2 to 12.

28. The antibody-drug conjugate of claim 27 or a pharmaceutically acceptable salt thereof, wherein, R5 is -NH-C 1-4 Alkylene-heteroaryl-, wherein the heteroaryl group is a triazolyl group.

29. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 27, wherein, m can be 2, 4, 6 or 8.

30. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein Z has the following structure: ， Where, R b It is either ynyl-C(=O)- or alkenyl-C-(=O)-. in, The asterisk on the left indicates the position connected to Ab; the asterisk on the right indicates the position connected to Y.

31. The antibody-drug conjugate of claim 30 or a pharmaceutically acceptable salt thereof, wherein, R b yes 。 32. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 31, wherein, Z has a structure: ， The asterisk on the left indicates the position connected to Ab, and the asterisk on the right indicates the position connected to Y.

33. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein, M is absent, or is either amino-C1-C3 alkylene or amino-phenyl-C1-C3 alkylene-OC(=O)-.

34. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein, M is -NH-CH2-.

35. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein, M is: or , The wavy line on the left indicates a connection to Y, and the wavy line on the right indicates a connection to drug unit D.

36. The antibody-drug conjugate of claim 19 or a pharmaceutically acceptable salt thereof, wherein, L is a connector containing the following structure: R3 and R4 are each independently selected from methyl, ethyl, and propyl groups. The asterisk on the left indicates the position connected to Ab, and the asterisk on the right indicates the position connected to drug D.

37. The antibody-drug conjugate or its pharmaceutically acceptable salt according to claim 36, wherein, R3 and R4 are both methyl; or R3 and R4 are both ethyl; or R3 and R4 are both propyl.

38. The antibody-drug conjugate or its pharmaceutically acceptable salt according to any one of claims 1-12, wherein, The LD unit of Formula I is linked to the antibody by forming a thioether bond with the thiol group of cysteine ​​in the light chain and / or heavy chain of the antibody.

39. The antibody-drug conjugate or its pharmaceutically acceptable salt according to any one of claims 1-12, wherein, The ADC is selected from: or or 。 40. The antibody-drug conjugate of claim 39 or a pharmaceutically acceptable salt thereof, wherein, q is the average DAR value from 1 to 8.

41. The antibody-drug conjugate of claim 40 or a pharmaceutically acceptable salt thereof, wherein, The average DAR value is approximately 2, approximately 4, approximately 6, approximately 7, or approximately 8.

42. A pharmaceutical composition comprising an antibody-drug conjugate of any one of claims 1-41 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

43. Use of the antibody-drug conjugate of any one of claims 1-41 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating Claudin 18.2 positive tumors, wherein the tumor is gastric cancer or gastroesophageal junction cancer (EGJA).

44. The use of claim 43, wherein the tumor is advanced or metastatic gastric cancer.

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