Dual-load antibody drug conjugates and uses thereof

Abstract

The present disclosure relates to dual loaded antibody-drug conjugates (ADCs) as well as compositions containing the ADC molecules and therapeutic applications thereof.

Description

Technical Field

[0001] This disclosure relates to dual-load ligand-conjugated pharmaceutical products, and more specifically to ligand-conjugated pharmaceutical products comprising a bifunctional cyclic dinucleotide (CDN) STING agonist and one or more other payloads, compositions comprising the thereof, and their use in the treatment or prevention of diseases. Background Technology

[0002] The cGAS-STING signaling pathway is an important mechanism for cytoplasmic DNA sensing, playing a crucial role in regulating pathogen infection, tumor immunity, and autoimmune diseases. When cGAS detects dsDNA from various sources, such as viral, bacterial, and mitochondrial DNA, it catalyzes the synthesis of cGAMP (cyclic dinucleotide 2'3'-cyclic AMP-GMP), activating STING. This, in turn, regulates the expression of type I IFN (interferon) and NF-κB-transforming factor-driven gene expression (inflammatory gene expression, including cytokines and chemokines). These substances can limit viral replication and induce apoptosis, thereby regulating pathogen infection and tumor immunity.

[0003] Given that endogenous cGAMP is a key mediator in the innate immune system's response to tumors, ultimately promoting the production of interferon or pro-inflammatory cytokines and thus achieving therapeutic benefits, a series of CDN-type STING agonists are being developed for antiviral or cancer immunotherapy. However, clinical development faces challenges, including: being easily destroyed and inactive by ENPP1 and ENPP3 phosphodiesterases in the body; having a short half-life in the circulation system; low bioavailability; and relying on intratumoral administration in clinical use. If injected by other means, they may induce systemic cytokine responses. Even with intratumoral administration, their convenience and patient acceptability are limited. In addition, transient exposure of these compounds to immune cells in the tumor environment can reduce their efficacy.

[0004] The inventors previously developed a bifunctional CDN-type STING agonist (WO2022083584, the entire contents of which are incorporated herein by reference). This bifunctional CDN compound is the first to integrate cytotoxic molecules into the CDN STING agonist structure. Upon entering the body, the resulting bifunctional CDN compound first activates the STING innate immune pathway, killing tumor cells. After drug decomposition, it releases the cytotoxic sub-structural units, further killing tumor cells and increasing the antigen-presenting activity of innate immune cells. This provides tumor marker fragments for the acquired immune system, endowing the body with immune memory and consolidating long-term immunosuppressive effects. Studies have shown that this bifunctional CDN-type STING agonist exhibits a more effective inhibitory effect on tumor cell proliferation than the pure STING agonist marker compound ADU-S100 in multiple tumor cell lines. Furthermore, it demonstrates significantly superior antitumor effects compared to ADU-S100 in in vivo tumor pharmacological models (CT26, 4T1, A20, B16F10, H22, etc.). However, the common problems of CDN-type immune agonist drugs still exist, including easy degradation by enzymes, short half-life, and limited intratumoral administration, which restricts their use as systemic immunotherapy.

[0005] Ligand-targeted drug-conjugates (ADCs), especially antibody-targeted drug-conjugates (ADCs), represent a highly efficient and low-toxicity drug delivery technology that can, to some extent, address the shortcomings of CDN-based STING agonists in application. However, ADCs also face numerous challenges, such as uncontrolled or insufficient release of the payload, off-target toxicity, tendency to aggregate leading to defects like short half-life, rapid clearance and immunogenicity, complex pharmacokinetics, and drug resistance. In practice, developing specific cytotoxic molecules into ideal ligand-conjugate drugs like ADCs—which remain stable in the bloodstream, precisely reach the therapeutic target, and ultimately release the cytotoxic payload within or near the target (e.g., cancer cells) with high efficacy and low toxicity—is far from easy.

[0006] Therefore, there is an urgent need for novel therapies that can precisely deliver CDN STING agonists to the tumor environment, replace intratumoral administration with more user-friendly dosing methods, prolong and improve efficacy, and reduce toxic side effects. Furthermore, given the ongoing demand for safe and effective therapeutic drugs in the field of cancer treatment, other drug design strategies are needed to further enhance the efficacy and safety of STING agonists. Summary of the Invention

[0007] To meet the aforementioned needs in the field, the inventors, through in-depth research, adopted an ADC drug delivery system, based on the mechanism of action of STING agonists, and further employed the idea of ​​intramolecular drug co-action. They coupled the previously developed bifunctional CDN-type STING agonists with other payload drugs and specific guide molecules through linker units, thereby fusing them into a single ADC molecule, and obtained the dual-load-bifunctional cyclic dinucleotide conjugate drug as shown in this paper.

[0008] This drug conjugate targets and delivers immune-activating and cytotoxic molecules with different mechanisms of action to the same cell. The synergistic effect of these two mechanisms achieves safe and efficient treatment, addressing the aforementioned needs. The obtained dual-load conjugate exhibits favorable physical properties, with no significant antibody aggregation, and can be rapidly and effectively endocytosed by tumor cells. In animal models, it not only demonstrates significantly improved tumor growth-suppressing activity and good tolerability compared to single CDN STING agonists and single-load bifunctional CDN STING ADCs, but also shows a synergistic tumor-suppressing effect exceeding the sum of the effect of single-load bifunctional CDN STING ADCs and other single-load ADCs. Therefore, the dual-load ADC disclosed herein holds promise for using more traditional, mature, and user-friendly administration methods (such as intravenous, subcutaneous, intramuscular, and topical administration) to replace intratumoral administration, expanding indications and the beneficiary population, and providing highly effective and safe treatment.

[0009] Therefore, in a first aspect, this disclosure provides ligand-drug conjugates having the following formula (X) or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels thereof:

[0010]

[0011] in,

[0012] Q represents the targeted ligand;

[0013] D1 represents the first payload, which is a cyclic dinucleotide (a compound of formula (II), (II-a), (II-a'), (II-b), (II-b')) as defined herein;

[0014] D2 represents the second payload, as defined below;

[0015] L represents the connecting body unit that connects D1 and D2 to Ab;

[0016] q indicates connection to Q. The quantity, for example, q is an integer or non-integer from about 1 to 20, such as 2-18, 4-16, 5-12, 1-10, 1-8, 2-8, 3-8, 2-6, 4-6, 4-8, 6-8, 6-10.

[0017] In the ligand drug conjugates disclosed herein, the targeting ligand is selected from antibody or antigen-binding fragments, peptide ligands, and any other molecule capable of targeting cancer cells.

[0018] In some embodiments, the targeting ligand in the ligand-drug conjugate of this disclosure is an antibody or antigen-binding fragment, and formula X can be represented as follows:

[0019]

[0020] In some embodiments, the targeting ligand in the ligand drug conjugate of this disclosure is a peptide ligand.

[0021] The following description of ligand-drug conjugates in this disclosure uses antibody or antigen-binding fragments as examples; however, it should be understood that the antibody or antigen-binding fragments may be replaced with peptide ligands or any other molecules capable of targeting cancer cells.

[0022] In a second aspect, this disclosure provides pharmaceutical compositions comprising the ADC of this disclosure or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotopic label thereof, optionally at least one other therapeutic agent, and optionally one or more pharmaceutically acceptable excipients.

[0023] In a third aspect, this disclosure provides the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels or pharmaceutical compositions containing the thereof for use as therapeutic agents for treating or preventing diseases, specifically as therapeutic agents for STING-related or mediated diseases, more specifically for treating or preventing inflammatory, allergic or autoimmune diseases, infectious diseases or hyperproliferative diseases, especially as therapeutic agents for antitumor or antiviral diseases, or as vaccine adjuvants; specifically, as cytotoxic agents for treating or preventing hyperproliferative diseases, especially tumors, or as cytotoxic agents for treating or preventing viral infections.

[0024] In a fourth aspect, this disclosure provides for the use of the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs or isotopic markers or pharmaceutical compositions comprising the thereof in the prevention or treatment of diseases, specifically diseases related to or mediated by STING, more specifically inflammatory, allergic or autoimmune diseases, infectious or proliferative diseases, especially tumors or viral infections.

[0025] In a fifth aspect, this disclosure provides the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled derivatives, or pharmaceutical compositions comprising thereof, as multifunctional active agents, possessing both immunotherapeutic and cytotoxic therapeutic activities, including the ability to activate the immune system to exert antitumor and antiviral replication functions by stimulating the STING signaling pathway, to induce tumor cell death or inhibit viral replication by releasing at least two cytotoxic agents, to continuously activate STING to kill tumor cells by releasing tumor DNA, and to provide "immune memory" or durable immunity against tumors by releasing tumor neoantigens to generate antibody-antigen responses. In this regard, this disclosure also provides uses of the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled derivatives, or pharmaceutical compositions comprising thereof, for achieving the above-mentioned multiple functions.

[0026] In a sixth aspect, this disclosure provides methods for treating or preventing diseases in subjects, specifically diseases related to or mediated by STING, more specifically inflammatory, allergic, or autoimmune diseases, infectious diseases, or hyperproliferative diseases, especially tumors or viral infections, comprising administering to a human or animal an ADC of the present disclosure or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled substance thereof, or a pharmaceutical composition comprising thereunder. Specifically, this disclosure provides methods for treating or preventing hyperproliferative diseases, especially tumors, in subjects, or methods for treating or preventing viral infections in subjects, said methods comprising administering to a human or animal an ADC of the present disclosure or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotope-labeled substance thereof, or a pharmaceutical composition comprising thereunder.

[0027] In a seventh aspect, this disclosure provides the use of the ADC of this disclosure or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels, or pharmaceutical compositions comprising thereof, in the preparation of medicaments for the prevention or treatment of diseases, specifically those related to or mediated by STING, more specifically inflammatory, allergic, or autoimmune diseases, infectious diseases, or hyperproliferative diseases, particularly tumors or viral infections. Specifically, this disclosure provides the use of the ADC of this disclosure or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels, or pharmaceutical compositions comprising thereof, in the preparation of medicaments for the treatment or prevention of hyperproliferative diseases, particularly tumors, and the use of the ADC of this disclosure or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels, or pharmaceutical compositions comprising thereof, in the preparation of medicaments for the treatment or prevention of viral infections.

[0028] In an eighth aspect, this disclosure provides a pharmaceutical combination comprising the disclosed ADC or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotopic label thereof, and at least one other therapeutic agent; the combination being used for the prevention or treatment of a disease, specifically a disease associated with or mediated by STING, more specifically for the treatment or prevention of inflammation, allergic or autoimmune diseases, infectious or proliferative diseases, especially tumors or viral infections; and a method of treating or preventing a disease, specifically a disease associated with or mediated by STING, more specifically for inflammation, allergic or autoimmune diseases, infectious or proliferative diseases, especially tumors or viral infections, in a subject, the method comprising administering the disclosed pharmaceutical combination to a human or animal.

[0029] In a ninth aspect, this disclosure also provides a method for preparing the ADC of the present disclosure or a pharmaceutically acceptable salt or solvate thereof by conjugating a CDN (e.g., a compound of formula (II), (II-a), (II-a'), (II-b), (II-b')) as defined herein and a second payload to an antibody or an antigen-binding fragment thereof via a linker unit.

[0030] The following figures and specific embodiments further illustrate the invention. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims.

[0031] definition

[0032] 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 disclosure, the following terms are defined below.

[0033] 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.

[0034] In this article, the term “about” used to modify numerical values, numerical ranges, or parameters means that the numerical value or parameter used with it fluctuates by ±10%, such as ±5%, ±2%, or ±1%.

[0035] In this document, the term “substantially” means the vast majority, i.e., >50% of the population, mixture, or sample, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0036] In this document, the term "and / or" should be understood as meaning any one of the options or any combination of two or more of the options.

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

[0038] In this document, the term “immune system” has the common meaning understood by those skilled in the art, referring to the whole or any one or more components of molecules, substances (such as body fluids), anatomical structures (such as cells, tissues or organs) and physiological processes that are associated with preventing infection in the body, protecting the body during infection or disease, and / or helping the body recover health after infection or disease.

[0039] In this article, the term "ADC" refers to antibody-drug conjugate.

[0040] In this document, the terms "drug" or "payload" refer to one or more substances administered to humans or animals to achieve a therapeutic effect, including substances that prevent, cure, or mitigate the effects of a disease or improve health. In this disclosure, "drug" or "payload" is selected from small molecule compounds, nucleotides, peptides, and proteins, and particularly from small molecule compounds or nucleotides.

[0041] In some embodiments, the payload of this disclosure includes, but is not limited to, anticancer drugs, radioactive substances, vitamins, anti-AIDS drugs, antibiotics, immunosuppressants, antiviral drugs, enzyme inhibitors, neurotoxins, opioids, cell-extracellular matrix interaction modulators, vasodilators, antihypertensive drugs, hypnotics, antihistamines, anticonvulsants, muscle relaxants, anti-Parkinson's drugs, antispasmodics and muscle contraction agents, antiparasitic and antiprotozoal drugs, analgesics, antipyretics, steroidal or nonsteroidal anti-inflammatory drugs, anti-angiogenic factors, antisecretory factors, anticoagulants and antithrombotic agents, local anesthetics, prostaglandins, antipsychotics, antiemetics, or imaging agents.

[0042] In some embodiments, the payload in the dual-load antibody-drug conjugate of this disclosure has a free amino, hydroxyl, or carboxyl group to be acylated with the corresponding portion of the conjugate to be linked to the conjugate.

[0043] In this document, the term "small molecule compound" refers to a compound having a molecular weight of less than or equal to about 2 kDa, for example, less than or equal to about 1.5 kDa. In some embodiments, the small molecule compound has a molecular weight of less than or equal to about 1 kDa, 800 Da, 700 Da, 600 Da, or 500 Da.

[0044] In this paper, the term "derivative" refers to a compound obtained by replacing one or more atoms or groups of atoms in a parent compound molecule with other atoms or groups of atoms, and that the compound has biological activity equivalent to or improved upon that of the parent compound molecule.

[0045] In this paper, the term "STING" is an abbreviation for stimulator of interferon genes. STING is a transmembrane protein receptor in human cells. Activation of STING by cyclic dinucleotides (CDNs) leads to activation of the IRF3 and NF-κB pathways, thereby inducing type I interferon and pro-inflammatory cytokines, respectively. The term "STING agonist" refers to any substance that activates STING in vitro or in vivo to elicit a corresponding physiological response, particularly CDN STING agonists as defined herein.

[0046] In this document, the term "diseases associated with or mediated by STING" means diseases in which an immune response induced by the STING pathway can be alleviated, i.e., diseases in which activating STING will reduce the incidence of the disease, reduce or eliminate disease symptoms, including but not limited to inflammatory, allergic or autoimmune diseases, infectious diseases or cancer. For the purposes of this disclosure, "diseases associated with or mediated by STING" are preferably selected from tumors or cancer.

[0047] In this document, the terms “hyperproliferative disorder,” “tumor,” or “cancer” refer to a physiological condition in a subject characterized by uncontrolled or disordered cell growth or death, including solid tumors and hematogenous tumors, whether malignant or benign, and all precancerous and cancerous cells and tissues. For all aspects of this disclosure, the cancers or tumors include, but are not limited to, brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, hematologic malignancies, lung cancer (small cell lung cancer, non-small cell lung cancer), and bone cancer. Examples of the aforementioned cancer types include neuroblastoma, colorectal cancer such as rectal cancer, colon cancer, colorectal cancer, familial adenomatous polyposis carcinoma and hereditary non-lymphatic colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal carcinoma, pharyngeal squamous cell carcinoma, oral cancer, head and neck cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urothelial carcinoma, sweat gland cancer, gastric cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, pancreatic cancer, prostate cancer, testicular cancer, breast cancer (including HER2-negative breast cancer), urinary tract cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, and lymphoma. Examples of tumor types include Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL), lymphocytic carcinoma, acute myeloid leukemia (AML), myeloid leukemia (chronic myeloid leukemia (CML), adult T-cell lymphoma, diffuse lymphoma (DLBCL), liver cancer, hepatocellular carcinoma, multiple myeloma, seminoma, osteosarcoma, chondrosarcoma, anal canal cancer, renal cell carcinoma, adrenocortical carcinoma, chordoma, fallopian tube cancer, gastrointestinal stromal tumor, myeloproliferative disorders, mesothelioma, biliary tract cancer, Ewing sarcoma, and other rare tumor types, as well as recurrent forms of the above tumors.

[0048] In this article, 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.

[0049] In this article, the term "cytotoxic activity" refers to the cell-killing effect of a drug or its conjugates or metabolites. Cytotoxic activity can be expressed as the IC50 value, which is the concentration of drug (molar or mass) per unit volume at which half of the cells are viable.

[0050] In this document, the term "cytotoxic agent" or similar expression refers to an active agent that has cytotoxic activity and causes cell damage, used to combat abnormal and uncontrolled cell growth. The cytotoxic agents involved in the antibody-drug conjugates disclosed herein include bifunctional CDN STING agonists and second payloads as defined herein, as well as substances with cytotoxic activity released from them in vivo.

[0051] As used herein, the term "multifunctional active agent" refers to the payload carried in the conjugated drug of this disclosure, which, based on its unique structural design, can perform multiple functions in the body of a subject, possessing both immunotherapeutic activity and multiple cytotoxic therapeutic activities, including but not limited to activating the immune system by stimulating the STING signaling pathway to exert anti-tumor and antiviral replication functions, synergistically killing tumor cells or inhibiting viral replication by releasing multiple cytotoxic agents, continuously activating STING to kill tumor cells by releasing tumor DNA, and providing the ability to provide "immune memory" or durable immunity against tumors by releasing tumor neoantigens to generate antibody-antigen responses.

[0052] In this document, the terms “inhibition” and “reduction” or any variations thereof refer to the ability of a bioactive agent to reduce the signal transduction activity of a target by interacting directly or indirectly with the target, and to any measurable reduction or complete inhibition of the target activity. For example, this reduction in activity compared to normal can be about, at most about, or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, or any range derived therefrom.

[0053] In this article, the term "antigen" refers to an entity that specifically binds to an antibody.

[0054] 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. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subtype.

[0055] 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. Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab')2, Fab'-SH, Fv, single-chain Fv, diabody, triabody, tetrabody, minibody, single-domain antibody (sdAb), heavy chain antibody fragment VHH; and monospecific, bispecific, and multispecific fusion proteins formed from antibody fragments.

[0056] In this document, the term IgG antibody refers to a heterotetrameric protein having the structure of an IgG-like immunoglobulin. 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 an immunoglobulin hinge region and two dimerized Fc regions. In some embodiments, the IgG antibody is, for example, an IgG1, IgG2, IgG3, or IgG4 antibody. In other embodiments, the IgG antibody is an IgGκ or IgGλ antibody, such as an IgG1κ or IgG1λ antibody.

[0057] 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, its CDR sequence can be determined using various schemes known in the art, including CDR sequences based on Kabat, AbM, Chothia, Contact, and IMGT definitions. Furthermore, CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence.

[0058] In this paper, "variable region" or "variable domain" refers to the 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 respects, the antibody variable region 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, CDR sequences from known antibodies are transplanted into the framework regions of different antibodies with different properties. The properties of mutated and / or modified antibodies or ADC conjugates containing them 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.

[0059] In this document, the term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. For example, the antibody portion of an ADC according to this disclosure may be an IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibody, and has a heavy chain constant region of said immunoglobulin type. Furthermore, this disclosure contemplates not only antibodies employing native sequence constant regions but also antibodies containing variant sequence constant regions.

[0060] In this paper, the term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins or otherwise interacting with molecules. Epitope determinants typically consist of chemically active surface groups of a molecule, such as amino acid or carbohydrate or sugar side chains, and may possess specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be "linear" or "conformal." The distinction between conformational and linear epitopes lies in the loss of binding with the former, but not the latter, in the presence of denaturing solvents.

[0061] In this paper, the term "receptor-mediated endocytosis" refers to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment, triggered by the binding of a ligand to the corresponding receptor on the cell surface. The receptor-mediated endocytic activity of an antibody can be characterized by measuring the endocytosis rate.

[0062] In this document, "sequence identity" refers to the degree of sequence similarity on a nucleotide-by-nucleotide or amino-by-amino acid basis within a comparison window, and the percentage of sequence identity can be calculated using various methods known in the art. The optimal alignment for determining the percentage of sequence identity 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 sequence being compared or within the target sequence region.

[0063] In this document, the term "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). 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. The term "monoclonal antibody" or "monoclonal antibody composition" refers to polypeptides, including antibodies, bispecific antibodies, etc., that have substantially the same amino acid sequence or are derived from the same genetic source; this term also includes antibody molecular products of single molecular compositions. The term "humanized antibody" refers to an antibody for which a CDR sequence derived from another mammalian species, such as a mouse line, has been grafted onto a human scaffold sequence. The term "human antibody" includes antibodies with a variable region, wherein both the scaffold and the CDR region are derived from human sequences. The term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means.

[0064] 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 90%, 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).

[0065] In this paper, the term "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair, such as the strength of the interaction between an antibody and an antigen at a single antigenic site; the stronger the interaction, the stronger the affinity. The affinity of a molecule for its partner can generally be determined by the equilibrium dissociation constant (Kp). D The equilibrium dissociation constant is represented by the dissociation rate constant and the binding rate constant (k, k ... dis and k onThe ratio of affinity to antigen protein or cell-based assays. Affinity can be measured by common methods known in the art, such as ELISA, flow cytometry, biofilm layer optical interference (BLI), and surface plasma resonance.

[0066] In this article, the term "K" D "(M) in this paper refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, K." D The measurement of the value is known in the art. Affinity and K D The values ​​are inversely correlated; that is, the higher the affinity, the higher the K value. D The smaller the value, the lower the affinity; conversely, the larger the value, the lower the affinity. D The larger the value, the better. Generally, K... D The value depends on the dissociation rate constant (Kd or Kdis, sec) between the interacting antibody-antigen pairs. -1 ) and binding rate constant (Ka, M -1 x sec -1 ).

[0067] In this paper, the term "binding" or "specific binding" refers to the ability of a single antibody binding site to react with an antigenic determinant. In the context of antibody-associated antigen binding, it is used to refer to binding with approximately 10... -6 M or smaller K D Value affinity combination, for example, K D The value is approximately 10 -7 M or smaller, or about 10 -8 M or smaller. Binding to non-specific antigens (e.g., irrelevant antigens such as BSA) K D Compared to the value, the binding K of the antibody to its associated antigen D The value is preferably at least 100 times lower or, for example, at least 1000 times lower.

[0068] In this document, the term "effective function" refers to those biological activities attributable to the Fc region of an antibody, which vary depending on the antibody class. It is known that the IgG Fc region can mediate several important effector functions. In one embodiment, this disclosure provides an antibody having an Fc region that induces effector functions such as ADCC or CDC, thereby inducing tumor cell apoptosis, cell lysis, and / or inhibiting the proliferation, dissemination, and / or metastasis of tumor cells carrying the associated antigen in tumor cells. In other embodiments, this disclosure also provides antibodies with Fc regions having altered effector functions. Effector functions can be altered by sequence modification of the Fc region of the antibody. Alternatively, antibodies with altered types of glycosylation in the Fc region can be prepared. Modification of the Fc region glycosylation pattern can be conveniently achieved by altering the amino acid sequence of the Fc region to create or remove one or more glycosylation sites.

[0069] In this paper, the term "HER2" (also referred to as ERBB2; NEU; NGL; TKR1; CD340; p185; MLN19; HER-2 / neu) refers to the transmembrane tyrosine kinase receptor of the epidermal growth factor (EGF) receptor family. HER2 comprises an extracellular binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. HER2 does not possess its own ligand-binding domain and therefore cannot bind growth factors. However, HER2 binds tightly to other ligand-binding EGF receptor family members such as HER1 or HER3 to form a heterodimer, thereby stabilizing ligand binding and enhancing the activation of kinase-mediated downstream signaling pathways. The human HER2 / NEU gene is located at chromosome 17q12, and its genomic sequence can be found in GenBank at NG_007503.1. In humans, there are five HER2 isotypes: A, B, C, D, and E; the term "HER2" is used in this paper to collectively represent all HER2 isotypes. As used herein, human HER2 protein also includes proteins that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity in their full length with HER2 isotypes A, B, C, D, and E, wherein such proteins still possess at least one function of HER2.

[0070] In this paper, the term "HER2-positive cancer" or "HER2-expressing cancer" refers to cancer in which the HER2 protein is present on the surface of the cells, and the presence of HER2 on cancer cells can be detected or determined by methods known in the art.

[0071] In this document, the terms “epidermal growth factor receptor” or “EGFR” are used interchangeably and, unless otherwise stated, include any variant of human EGFR, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and encompass its species homologs. Furthermore, it should be understood that the term covers EGFR expressed on natural or recombinant cells. An example of EGFR is the human EGFR protein containing the amino acid sequence UniProtKB-P00533. In this document, unless otherwise specified, the term “epidermal growth factor receptor” or “EGFR” refers to the human epidermal growth factor receptor.

[0072] In this article, the term "EGFR-positive" tumor cells refer to tumor cells that express EGFR on their cell surface, including but not limited to EGFR-positive breast cancer cells and lung cancer cells.

[0073] In this document, the term "linker unit" or "linker" refers to a bifunctional or multifunctional portion in an antibody-drug conjugate that covalently links a drug to an antibody. It can be cleavable or non-cleavable. The linker unit of this disclosure has several components, such as a self-disintegrating linker; a cleavable linker; an optional property-regulating unit; and an antibody linker head.

[0074] In this document, the term "self-decomposing linker" refers to a temporary elongator, spacer, or placeholder unit that links two or more molecules together by chemical bonds, which break under defined conditions to release the two molecules. A self-decomposing linker can be defined as a bifunctional chemical group capable of covalently linking two spaced-apart chemical moieties together to form a generally stable molecule, releasing one of the spaced-apart chemical moieties from the molecule via enzymatic cleavage; and, following the enzymatic cleavage, spontaneously cleaving from the remaining portion of the bifunctional chemical group to release the other of the spaced-apart chemical moieties. In some embodiments, a self-decomposing linker refers to a heterocyclic self-decomposing moiety. Typical self-decomposing linkers include p-aminobenzyl, p-hydroxybenzyl, p-aminobenzyloxycarbonyl (PABC), p-hydroxybenzyloxycarbonyl, 2,4-bis(hydroxymethyl)aniline, 2-aminomethyl-1-carbonylpiperidine, 2-aminomethyl-1-carbonylpiperazine, -NH-(CH2)4-C(O)-, and -NH-(CH2)3-C(O)-, etc.

[0075] In this document, the term "cleavable linker" refers to a portion of the linker unit of an ADC that is unstable in vivo and cleavable by the biological environment. Preferably, the "cleavable linker" allows activation of a marker or therapeutic agent by cleaving the marker or agent from the remainder of the conjugate. Cleavage can originate from any process without limitation, such as enzymatic, reduction, pH, etc. Preferably, the cleavable group is selected so that activation occurs at the desired site of action, which can be a site in or near the target cell (e.g., cancer cell) or tissue, such as the site of therapeutic action or marker activity. This cleavage can be enzymatic, and exemplary cleavable groups include, but are not limited to, natural amino acids or peptide sequences ending with natural amino acids, and are linked to a linker unit or self-degrading linker at their carboxyl terminus.

[0076] In this paper, the term "antibody linker" refers to any chemical group designed to facilitate the attachment of a drug conjugate to an antibody, for example, to overcome steric hindrance.

[0077] In this paper, the term "property regulation unit" refers to the functional portion connected in series or branched manner within the connective unit of an ADC, designed to regulate the properties of the ADC, such as stability in blood circulation and improved hydrophilicity. Commonly used property regulation units for ADCs include, but are not limited to, polyethylene glycol (PEG), hydrophilic peptides, monosaccharides, oligosaccharides, polysaccharides, cyclodextrin units, aminosulfonamide fragments, sulfonic acid groups, phosphate groups, polyamines, polyamides, dendritic polymers, and bifunctional hydrocarbon chains.

[0078] In this document, the term "solubilizing unit" refers to a functional portion connected in series or branched manner to the linker unit of an ADC to increase solubility. Specific examples include "solubilizing sugar unit," which refers to a sugar unit directly or indirectly connected to the linker unit, such as the self-decomposing linker portion of the linker. In the ADC of this disclosure, the introduction of glycosyl groups (e.g., monosaccharides, disaccharides, oligosaccharides, polysaccharides, or derivatives thereof) into the linker unit helps improve the hydrophilicity of the conjugated chain, increase the loading rate to the theoretical maximum, while improving the solubility and purity of the conjugate, reducing aggregation, improving the drug-likeness of the conjugate, and ultimately enhancing the inhibitory effect on tumor cell proliferation.

[0079] In this document, "monosaccharide" as a solubilizing unit or part thereof refers to polyhydroxy aldehydes (aldoses) or polyhydroxy ketones (ketooses) and their derivatives containing three or more carbon atoms. These are the basic structural units constituting sugars and their complexes, which cannot be further hydrolyzed and are not linked to other similar units by glycosidic bonds. Based on the number of carbon atoms in the monosaccharide, monosaccharides can also be classified as trioses (trioses), tetraoses (tetraoses), pentoses (pentoses), hexoses (hexoses), heptoses (heptoses), etc. The ADC of this disclosure preferably carries pentoses or hexoses. Derivatives of the monosaccharide include, for example, phosphate esters of monosaccharides, sugar alcohols, sugar acids, deoxy sugars, amino sugars, acylated amino sugars, and glycosides. Examples of monosaccharides or their derivatives include, but are not limited to, glyceraldehyde, glyceraldehyde phosphate, dihydroxyacetone phosphate, erythrosine, erythropoietin, thulose, arabinose, ribose, ribulose, deoxyribose, ribitol, ribose phosphate, xylose, xylulose, xylitol, lysose, glucose, glucosamine, acetylglucosamine, glucuronic acid, acetylglucosamine, glucose phosphate, mannose, mannitol, aminomannitol, acetylglucosamine, fructose, fructose phosphate, galactose, galactitol, galactosamine, acetylglucosamine, allose, deoxyallose, adroose, deoxyadroose, cinchonaose, rhamnose, allulose, sorbose, sorbitol, tagaise, gulose, deoxygulose, iduroose, taloose, fucose, deoxytaloose, etc.

[0080] It should be noted that the monosaccharides and their derivatives described in this disclosure include their D configuration, L configuration, racemic (DL) and meso configuration, as well as forms with arbitrary optical activity ((+), (-), (±)). The monosaccharides and their derivatives described in this disclosure include their chain isomers, cyclic isomers, or mixtures thereof, wherein the cyclic forms include α-terminal isomers and β-terminal isomers, and, for example, include pyranose or furanose forms.

[0081] In this paper, "disaccharide" as a property regulating unit or solubilizing unit refers to a compound and its derivatives in which two monosaccharides are linked by a glycosidic bond. Based on the different linkage methods, it is divided into two types: one is a non-reducing sugar formed by the dehydration of the hemiacetal (hemiketal) hydroxyl groups of two sugars to form a glycoside, which is a glycosyl glycoside; the other is a reducing sugar formed by the glycosidic bond between the hemiacetal (hemiketal) hydroxyl group of one sugar and the non-hemiacetal (hemiketal) hydroxyl group of another sugar to form a glycoside, which is a glycosyl sugar. The glycosidic bond can be an α-1,4 glycosidic bond, an α-1,6 glycosidic bond, a β-1,4 glycosidic bond, an α-1-2β glycosidic bond, a β-1,6 glycosidic bond, an α-1,1 glycosidic bond, a β-1,3 glycosidic bond, or a β-2,1 glycosidic bond. Specific examples of disaccharides include, but are not limited to, maltose, isomaltose, lactose, sucrose, chitobiose, rutinose, trehalose, xylobiose, gentiobiose, etc., wherein the monosaccharide unit constituting the disaccharide can be in the form of various monosaccharide derivatives as described above.

[0082] In this article, the term "oligosaccharide" refers to a low-degree polymeric sugar and its derivatives consisting of 3 to 9 monosaccharides linked by glycosidic bonds. The monosaccharides, glycosidic bonds and derivatives are as defined above, such as, but not limited to, mannotriose, selaginella triose, gentian triose, plantain, stachyose, raffinose, etc.

[0083] In this document, the term "polysaccharide" refers to a polysaccharide composed of 10 or more monosaccharide groups linked by glycosidic bonds, which can be α-type, β-type, or a mixture of α / β. Structurally, it can be linear, branched, or cyclic. A polysaccharide can be a homopolysaccharide composed of a single monosaccharide, wherein the sugar unit is selected from trioses, tetraoses, pentoses, hexoses, heptaoses, and octaoses, or deoxyglucose units, such as dextran, xylan, and polysialic acid; or a heteropolysaccharide composed of two or more monosaccharides, such as hyaluronic acid and heparin. Examples of polysaccharides include, but are not limited to, dextran, levtran, hyaluronic acid, cyclodextrins (α, β, and γ, etc.), hydroxyethyl starch, xylan, water-soluble starch, water-soluble cellulose, carboxymethyl cellulose, galactosamine, polysialic acid, rhamnose, Ganoderma lucidum polysaccharide, lentinan, chitin, deacetylated chitosan, alginate, carrageenan, glucoside, pullulan, stearyl glucan, xanthan gum, xyloglucan, amylose, etc. Structurally, the polysaccharides may have one or more substituents selected from the following groups, either naturally or after structural modification: carboxylic acid group, carboxylate group, amino group, sulfonic acid group, sulfonate group, phosphate group, phosphate group, hydroxyethyl group, hydroxypropyl group, methyl group, acyl group, carboxymethyl group, natural amino acid group, non-natural amino acid group, etc.

[0084] In this paper, the term "derivative" used to describe sugar units primarily refers to sugar phosphate esters (sugar compounds formed by the esterification of one or more hydroxyl groups of a sugar with phosphate), sugar alcohols (sugar compounds formed by the reduction of the carbonyl group of a sugar to OH), sugar acids (aldonic acids formed by the oxidation of the aldehyde group of an aldose to a carboxyl group, uronic acids formed by the oxidation of the primary alcohol group of an aldose to a carboxyl group, and glycosidic acids formed by the oxidation of both the aldehyde and primary alcohol groups of an aldose to carboxyl groups), deoxysugars (sugars in which one or two hydroxyl groups of a sugar molecule are replaced by hydrogen atoms), amino sugars (sugar derivatives in which one or more hydroxyl groups of a sugar molecule are replaced by amino groups), acylated amino sugars (sugar derivatives in which the amino group of an amino sugar is acylated), and glycosides (sugar derivatives in which the hemiacetal hydroxyl group of a sugar reacts with another molecule such as an alcohol, sugar, purine, or...). Pyrimidine Sugar derivatives formed by the condensation of hydroxyl, amino, or thiol groups, i.e., derivatives consisting of sugar residues (sugars with the hemiacetal hydroxyl group removed) and ligands linked by glycosidic bonds. This term also includes compounds in which the sugar unit is derived from more than one derivative form.

[0085] The sugar derivatives applicable to the ADC of this disclosure can also be derivatives formed by derivatizing sugar molecules in one or more of the above ways, such as acylated aminouronic acids, such as 2-acetamido-2-deoxy-D-galacturonic acid, 2-acetamido-2-deoxy-D-glucuronic acid, and 2-acetamido-2-deoxy-D-mannuronic acid.

[0086] In this document, specific examples of the terms "property regulation unit" or "solubilizing unit" also include "hydrophilic peptide". The term "hydrophilic peptide" can be attached, for example, to a self-decomposing linker of a linker unit, or to an antibody linker head, which can be directly attached to the linker unit or attached to the linker unit via a suitable structural fragment (such as the (S) fragment defined in this disclosure for S1 or S2). Specifically, a "hydrophilic peptide" can be an organic portion comprising a repeating subunit -CO-CR′R″-NR-, wherein one or more amino acids, identical or different from each other, are linked by amide bonds in a linear, branched, or star configuration. Each hydrophilic peptide generally contains 1-20 amino acids, preferably 4-14 amino acids, more preferably 6-12 or 5-10 amino acids.

[0087] In this paper, the amino acid monomers constituting the hydrophilic peptides can be natural amino acids, such as alanine (Ala), arginine (Arg), aspartic acid (Asp), asparagine (Asn), histidine (His), glycine (Gly), glutamic acid (Glu), glutamine (Gln), phenylalanine (Phe), lysine (Lys), leucine (Leu), serine (Ser), tyrosine (Tyr), threonine (Thr), isoleucine (Ile), proline (Pro), tryptophan (Trp), valine (Val), cysteine ​​(Cys), and methionine (Met). Correspondingly, for example, in the repeating subunit -CO-CR'R”-NR-, R is H and one of R' and R” is H, and the other corresponds to the corresponding group or fragment in each natural amino acid. Polar natural amino acids are preferred, such as arginine, serine, threonine, tyrosine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, histidine, glycine, and tryptophan.

[0088] In this paper, the amino acid monomers constituting the hydrophilic peptides may also be amino acids other than the twenty natural amino acids mentioned above, such as ornithine (Orn), β-alanine (β-Ala), citrulline (Cit), sarcosine (Sar), and those amino acids in the repeating subunit -CO-CR'R”-NR- where R, R', and R” are different from the corresponding groups or segments in natural amino acids. For example, R, R', and R” are selected from alkyl, aryl, acyl, amide, ketone, azide, hydroxyl, mercapto, hydrazine, cyano, quaternary ammonium, halogen, acylhydrazine, alkenyl, alkynyl, ether, thiol, selenyl, sulfonic acid, sulfate, borate ester, phosphonyl, phosphate, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, etc., or any of them. Combinations, or groups comprising alkyl, aryl, acyl, amide, ketone, azide, hydroxyl, mercapto, hydrazine, cyano, quaternary ammonium, halogen, acylhydrazine, alkenyl, alkynyl, ether, thiol, selenyl, sulfonic acid, sulfate, borate ester, phosphonoyl, phosphoric acid, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, etc.; preferably, R, R', and / or R" contain hydrophilic amino acids, for example, R, R', and R" are each independently carboxyl, sulfonic acid, sulfate, phosphoric acid, amino, amide, quaternary ammonium, oxygen-containing group, ether, mercapto, or hydroxyl groups, or groups comprising carboxyl, sulfonic acid, sulfate, phosphoric acid, amino, amide, quaternary ammonium, oxygen-containing group, ether, mercapto, and / or hydroxyl groups, such as alkyl, such as C 1-6 alkyl.

[0089] For the ADC of this disclosure, when the linker unit carries a hydrophilic peptide, at least 50% to 100% of the amino acids constituting the hydrophilic peptide are hydrophilic amino acids, for example 80%-100%, such as 60%, 70%, 80%, 90%, or 100%, and the amino acids are preferably arginine, serine, threonine, tyrosine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, histidine, glycine, tryptophan, ornithine, citrulline (Cit), and sarcosine (Sar).

[0090] For the ADC disclosed herein, the hydrophilic peptide carried by the linker unit is preferably polysarcosine, polyarginine, or polyglycine with 4-14 units, more preferably polysarcosine with 6-12 units or 5-10 units.

[0091] In this document, the term "PEG unit" refers to an organic portion containing repeating ethylene-oxy subunits (PEG or PEG subunits), which may be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy subunits).

[0092] The PEG units provided herein can be linked together, for example, in linear, branched, or star configurations. Typically, prior to incorporation into the ADC, at least one polyethylene glycol chain is derived at one end with an alkyl portion substituted with an electrophilic group, such as a urethane nitrogen covalently linked to a methylene urethane unit. Terminal ethoxylates (if present) in the polyethylene glycol chain that do not participate in covalently linking to the remainder of the linker unit are modified with PEG end-capping units, typically optionally substituted alkyl groups such as -CH3, CH2CH3, or CH2CH2CO2H. Preferred PEG units have a single polyethylene glycol chain having 2 to 24 -CH2CH2O- subunits covalently linked in tandem.

[0093] In this article, the term "base" refers to the basic building block of nitrogenous biological compounds linked to sugars within nucleosides, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Naturally occurring nucleobases include cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA), and uracil (RNA), abbreviated as C, G, A, T, and U, respectively.

[0094] In this document, the term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) coupled to the Ab portion as described herein to the Ab portion in an ADC conjugate. In some embodiments described herein, the DAR may be determined by q in the general formula, for example, the DAR may be an integer or non-integer of at least 1, such as about 1 to 20, such as about 2-18, 4-16, 5-12, 1-10, 1-8, 2-8, 3-8, 2-6, 4-6, 4-8, 6-8, 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 as described herein to the Ab portion in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is about 1 to 20, for example about 2-18, 4-16, 5-12, 1-10, 1-8, 2-8, 3-8, 2-6, 4-6, 4-8, 6-8, 6-10, for example 1.0-8.0, 2.0-6.0, for example about 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.

[0095] In this article, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, for example, fluorine and chlorine.

[0096] In this document, the term "alkyl" refers to a straight-chain or branched aliphatic saturated hydrocarbon group having a specified number of carbon atoms. Specifically, alkyl groups may have 1 to 20, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. A suitable C... 1-14 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, dimethylmethyl, dipropylmethyl, ethylbutylmethyl, diethylmethyl, methylethylmethyl, ethylpropylmethyl, diethylethyl, diethylpropyl, dipropylethyl, etc. A particular alkyl group has 1 to 7 carbon atoms, for example, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.

[0097] In this document, 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, alkylene groups 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 "C 1-5 "Alkylene" refers to a straight-chain or branched alkylene with 1 to 5 carbon atoms, including but not limited to methylene, ethylene, propylene, butylene, etc.

[0098] In this document, the term "-O-alkyl" or "alkoxy" means an alkyl group as defined herein, which is attached to the remainder of the molecule by an oxygen atom. Specifically, -O-alkyl has 1 to 10, for example, 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 "-OC"... 1-6 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group with 1 to 6 carbon atoms that is attached to the rest of the molecule by an oxygen atom. Examples include -O-methyl, -O-ethyl, -O-propyl (including -O-n-propyl and -O-isopropyl), -O-butyl (including -O-n-butyl, -O-isobutyl, -O-sec-butyl or -O-tert-butyl), -O-pentyl (including -O-n-pentyl, -O-isopentyl, -O-neopentyl), -O-n-hexyl, 2-methylpentyl-O-, etc.

[0099] In this document, the term "optionally halogen-substituted alkyl" refers to the alkyl group described herein, wherein one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms are optionally replaced by a halogen. Those skilled in the art will understand that when there is more than one halogen substituent, the halogens can be the same or different, and can be located on the same or different C atoms. "Halogen-substituted C..." 1-6Examples of "alkyl" include -CH2F, -CHF2, -CF3, -CCl3, -C2F5, -C2Cl5, -CH2CF3, -CH2Cl, -CH2CH2CF3, or -CF(CF3)2, etc.

[0100] In this document, 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 2-20, 2-10, 2-8, for example, 2-6, 2-5, 2-4, or 2-3 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. The carbon atoms in the alkenyl group that are bonded to the remainder of the molecule can be saturated or alkene-bonded carbon atoms.

[0101] In this document, the term "cycloalkyl" refers to a monocyclic, fused polycyclic, bridged polycyclic, or spirocyclic non-aromatic monovalent hydrocarbon ring structure having a specified number of ring atoms. It may be saturated or unsaturated, for example, containing one or more double bonds, preferably saturated. The cycloalkyl group may contain three or more carbon atoms in the ring, for example, 3-10 or 3-8 carbon atoms, such as C64-C ... 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.

[0102] In this document, the term "heterocycle" or "heterocyclic group" refers to a 5-20 membered (e.g., 5-14, 5-10, 5-8, 5-6 membered) aromatic or non-aromatic monocyclic, bicyclic, or polycyclic ring system having 1-4 independent cyclic heteroatoms 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, such as a heteroaryl or heterocyclic alkyl group, each of which may be a monocyclic or fused bicyclic ring containing 1 or 2 heteroatoms, more preferably a 5-6 membered monocyclic heteroaryl or heterocyclic alkyl group. Representative examples include, but are not limited to, pyrrolidine, succinimide (2,5-dioxopyrrolidine), piperidine, morpholine, tetrahydrofuran, tetrahydropyran, benzofuran, benzothiophene, indole, benzopyrazole, pyrrole, thiophene (thiophene), furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazine, and isoxazole.

[0103] In this document, the term "heterocyclic alkyl" means a monocyclic, fused polycyclic, spirocyclic, or bridged polycyclic saturated ring structure comprising 1-4 heteroatoms independently selected from O, N, and S, and a specified number of ring atoms, or its N-oxide, S-oxide, or C-oxide. Heterocyclic alkyls may have, for example, 5 to 12 ring members (which may be referred to as 5-12-membered heterocyclic alkyls), such as 5 to 10 ring members, 5 to 8 ring members, 5 to 7 ring members, or 5 to 6 ring members. Heterocyclic alkyls typically contain, for example, 1-4 heteroatoms, such as 5-6-membered heterocyclic alkyls containing 1 to 2 heteroatoms selected from N, O, and S. The atoms in the heterocyclic alkyl group attached to the remainder of the compound may be carbon atoms or heteroatoms, provided it is chemically feasible.

[0104] In this document, the term "heteroaryl" refers to a 5-20 membered (e.g., 5-14, 5-10, 5-8, 5-6) aromatic monocyclic or polycyclic system containing 1-4 heteroatoms independently 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 containing 1-2 heteroatoms, more preferably a 5-6 membered heteroaryl.

[0105] For clarity, the term "guanine" is used in this article. " can also be expressed as The thiophosphate groups used in the structural formulas of the compounds disclosed herein can be drawn as follows:

[0106] Unless otherwise specified, C in the definition of compounds in this invention n-n+m Or C n -C m This includes various cases with n to n+m carbons, such as C 1-6 This includes C1, C2, C3, C4, C5, and C6, as well as any range from n to n+m, such as C 0-6 Including C1, C2, C3, C4, C5, C6, C 0-1 C 0-2 C 0-3 C 0-4 C 0-5 C 1-2 C 1-3 C 1-4 C 2-3 C 1-6 Including C 1-2 C 1-3 C 1-4 C 2-6 C 3-6 wait.

[0107] In this document, the term "pharmaceutically acceptable salt" means a salt that retains the biological effects and properties of the ADC of this disclosure, and that such salt is not biologically or otherwise undesirable. The ADCs of this disclosure may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In this disclosure, a pharmaceutically acceptable non-toxic acid addition salt means a salt formed by the ADC of this disclosure 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. A pharmaceutically acceptable non-toxic base addition salt means a salt formed by the ADC of this disclosure with an organic or inorganic base, 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.

[0108] In this document, the term "solvent" refers to an association formed by one or more solvent molecules with the ADC of this disclosure. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc. It should be understood that such solvates of the compounds of this disclosure also include solvates of pharmaceutically acceptable salts of the compounds of this disclosure.

[0109] In this document, the term "isotope variant" refers to a compound in which one or more atoms constituting the compound are replaced by atoms having an atomic mass or mass number different from those normally found in nature. Examples of isotopes that may be incorporated into one or more atoms of the compounds disclosed herein include, for example... 2 H, 3 H, 13 C 14 C 15 N、 17 O、 18 O、 31 P, 32 P, 35 S and 18 F, thereby forming isotopic variations of the compounds disclosed herein, whether or not they are radioactive, are intended to be included within the scope of this disclosure. In some embodiments, the incorporated isotope is 2 H (deuterium); in other embodiments, the incorporated isotope is 3 H (tritium).

[0110] In this document, the term "prodrug" refers to a chemical derivative of the ADC of this disclosure, which is converted into the ADC of this disclosure in vivo through a chemical reaction.

[0111] In this document, the term "isomer" means any stereoisomer, enantiomer mixture, including racemic mixtures, diastereomer mixtures, geometric isomers, blocked isomers, and / or tautomers that a compound may exist structurally. Methods for determining and isolating the stereochemistry of such isomers are well known to those skilled in the art (SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994). This disclosure covers all possible isomeric forms of the compounds as defined herein, and their pharmaceutically acceptable salts or solvates, unless otherwise indicated. Furthermore, the compounds of this disclosure may exist as mixtures of two or more different structural forms in rapid equilibrium (commonly referred to as tautomers). It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0112] In this document, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers can be produced. Similarly, the compounds of this disclosure can exist as mixtures of two or more different structural forms in rapid equilibrium (commonly referred to as tautomers). It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0113] The compounds disclosed herein may have one or more asymmetric centers, and therefore can be prepared as (R)- or (S)- stereoisomers, or as mixtures thereof. The structural formulas or structural fragments of the compounds used herein... or The configuration of the asymmetric center, i.e., the chiral center, is indicated by R and S in the nomenclature of the compounds or intermediates provided in this disclosure. Those skilled in the art will recognize that the thiophosphate bonds in the compounds of this disclosure are inherently chiral and can each exist in either an R or S configuration; therefore, it is possible for the two thiophosphate bonds to be in the forms of R,R, S,S, S,R, and R,S. This disclosure covers the compounds of this disclosure and specific embodiments thereof in substantially pure or mixed forms. Compounds containing two thiophosphate bonds are preferably in substantially pure forms of R,R, S,S, S,R, and R,S stereoisomers, and particularly preferably substantially pure R,R stereoisomers, i.e., both phosphorus atoms have an R configuration. Their absolute configuration assignments can be made according to the literature method (Zhao et al. Nucleosides, Nucleotides and Nucleic Acids 2009, 289, 352-378; Knouse et al. Science 2018, 361, 1234). It should be noted that errors in the specification of configuration due to methodological mistakes in the literature do not affect the actual configuration of the compounds disclosed herein.

[0114] In this document, the term "substantially pure" as used with respect to CDN means that a certain stereoconfiguration is at least 75% pure relative to other possible stereochemical configurations at the chiral center. In preferred embodiments, substantially pure CDN is at least 85% pure, at least 90% pure, at least 95% pure, at least 97% pure, and at least 99% pure. The term "stereochemically pure" for substantially pure CDN formulations in this disclosure means that all CDNs within the formulation have a specific stereochemical configuration at these chiral centers, and is not intended to indicate that all CDNs within the formulation having a specific stereochemical configuration at these chiral centers are otherwise identical. For example, a substantially pure CDN R,R cGAMP thiophosphate formulation may comprise a combination of R,R c-di-GMP thiophosphate and R,R c-di-AMP thiophosphate, and is still a substantially pure cyclic purine dinucleotide formulation.

[0115] It should be understood that when a person skilled in the art can determine, based on the structure of the compound shown herein, that the compound has a chiral isomer and can be easily separated using conventional methods in the art, then the disclosure of the racemic form of the compound herein (whether in terms of structural formula or chemical name) should be regarded as having disclosed each isomer of the compound separately.

[0116] In this document, the number of groups attached to each atom in a compound definition, structural formula, or structural fragment depends on the chemical valence of that atom and need not all be shown. Generally, only non-hydrogen groups are shown in the group definition, structural formula, or structural fragment; groups not shown generally represent H, and those skilled in the art can easily determine whether and how many of the not shown groups exist.

[0117] The structural fragments discussed in this article use The bonds that cross it are the bonds that connect the structural segment to the rest of the molecule.

[0118] In this document, the terms “pharmaceutical-acceptable excipient,” “pharmaceutical-acceptable carrier,” and “therapeutic-inert excipient” are used interchangeably and refer to any pharmaceutically acceptable component in a pharmaceutical composition that is not therapeutically active and is non-toxic to the subject to administration, such as disintegrants, binders, fillers, solvents, buffers, tensioning agents, stabilizers, antioxidants, surfactants, carriers, diluents, or lubricants used to formulate pharmaceutical products.

[0119] In this document, the term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients, such as (i) the ADC of this disclosure, and (ii) other therapeutic agents are administered to a patient simultaneously, without a specific time limit, or 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 and other therapeutic agents of this disclosure 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.

[0120] 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.

[0121] In this article, the term "treatment" refers to slowing down, interrupting, blocking, relieving, stopping, reducing, or reversing the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0122] In this document, the term "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 programs. 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.

[0123] In this document, the term "therapeutic or preventive effective amount" refers to such an amount or dose of the ADC of this disclosure or a combination thereof, which, when administered to a patient in a single or multiple doses, produces the intended therapeutic or preventive effect in a patient requiring treatment or prevention.

[0124] I: Dual-loaded antibody-drug conjugate

[0125] In one aspect, this disclosure provides antibody-drug conjugates (ADCs) having the following formula (X-1), or pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels thereof:

[0126]

[0127] in,

[0128] Ab represents an antibody or antigen-binding fragment;

[0129] D1 represents the first payload, which is a cyclic dinucleotide (a compound of formula (II), (II-a), (II-a'), (II-b), (II-b')) as defined herein;

[0130] D2 represents the second payload, as defined below;

[0131] L represents the connecting body unit that connects D1 and D2 to Ab;

[0132] q indicates connection to Ab. The quantity, for example, q is an integer or non-integer from about 1 to 20, such as 1-10, 1-8, 2-8, 3-10, 3-8, 4-10, 4-8, 6-8, or 6-10.

[0133] In some embodiments of formula X-1, q represents an integer selected from 1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, the range of q is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, or a range consisting of any two values ​​between 1 and 20, such as 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 8, 3 to 10, 4 to 6, 4 to 8, 4 to 10, 6 to 8, or 6 to 10.

[0134] In some embodiments, Formula X-1 describes the ADC in the ADC mixture such that the q value of more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ADC in the mixture is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, Formula X-1 describes the ADC in the ADC mixture such that the q value of more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ADC in the mixture ranges from about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, or is a range of any two values ​​between 1 and 20, such as about 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 8, 3 to 10, 4 to 6, 4 to 8, 4 to 10, 6 to 8, or 6 to 10.

[0135] In other embodiments, equation X-1 describes the ADC mixture, where q is "q 平均 ", " represents the average q-value of the mixture, i.e., the average number of linker units (L) in the mixture that are linked to a given antibody (Ab). In such an implementation, q 平均 Represents an integer or non-integer value from approximately 1 to 20, such as approximately 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, or an integer or decimal within the range of any two values ​​between 1 and 20, such as approximately 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 8, 3 to 10, 4 to 6, 4 to 8, 4 to 10, 6 to 8, or 6 to 10.

[0136] In a preferred embodiment, q in this disclosure X-1 is an integer or non-integer from about 1 to 10, or a range of any two values ​​from 1 to 10, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 8, 3 to 10, 4 to 6, 4 to 8, 4 to 10, 6 to 8, or 6 to 10.

[0137] In some implementations, q represents the average DAR of approximately 1-10, 1-8, 2-8, 3-10, 3-8, 4-10, 4-8, 6-8, or 6-10.

[0138] In some implementations, q represents an average DAR of approximately 3. In some implementations, q represents an average DAR of approximately 6. In some implementations, q represents an average DAR of approximately 8.

[0139] Those skilled in the art should also understand that the various ADCs described herein can be pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled substances, and in some specific embodiments, are pharmaceutically acceptable salts.

[0140] In specific implementations, the dual-load antibody-drug conjugate (ADC) of formula (X-1) disclosed herein has the following general formula:

[0141]

[0142] in:

[0143] Ab represents an antibody or antigen-binding fragment;

[0144] D1 represents the first payload, which is a cyclic dinucleotide (a compound of formula (II), (II-a), (II-a'), (II-b), (II-b')) as defined herein;

[0145] D2 represents the second payload, as defined below;

[0146] G indicates the antibody linker that is connected to Ab;

[0147] E represents an optional property adjustment unit;

[0148] W indicates a branch connector;

[0149] P1 and P2 are each independently cleavable linkers, selected from peptide residues of 2-8 amino acids, preferably dipeptides, tripeptides or tetrapeptides;

[0150] A1 and A2 are each independently optional self-decomposing connectors;

[0151] S1 and S2 are each an independently arbitrarily existing solubilizing unit;

[0152] q represents the number of connector units—payload segments—connected to Ab. For example, q is an integer or non-integer number from about 1 to 20, such as about 1-10, 1-8, 3-8, 4-8, or 6-8.

[0153] In some implementations, E does not exist, and equation (I) is:

[0154]

[0155] In some implementations, neither E nor S1 exists, and equation (I) is:

[0156]

[0157] In some implementations, E, S1, and S2 are all absent, and equation (I) is:

[0158]

[0159] In some implementations, S1 does not exist, and equation (I) is:

[0160]

[0161] In some implementations, S2 is absent, and equation (I) is:

[0162]

[0163] In some implementations, neither S1 nor S2 exists, and equation (I) is:

[0164]

[0165] The number of D1 or D2 of the antibody or its antigen-binding fragment linked to an ADC can vary (referred to as the "drug-antibody ratio" or "DAR") and will be limited by the number of available linker sites on the antibody or its antigen-binding fragment and the number of D1 or D2 linked to a single linker. In an ADC containing multiple D1 or D2, each D1 or D2 may be the same or different. An ADC with a DAR of 10 or even higher can be considered, provided that the D1 or D2 does not exhibit unacceptable levels of polymerization under the conditions of use and / or storage. In some embodiments, the ADCs described herein may have a DAR in the range of about 1 to 10, about 1 to 9, about 1 to 8, about 1 to 7, about 1 to 6, about 1 to 5, or about 1 to 4. In some embodiments, the ADC described herein may have a DAR in the range of about 2 to 10, about 2 to 9, about 2 to 8, about 2 to 7, about 2 to 6, about 2 to 5, about 2 to 4, about 3 to 10, about 3 to 8, about 4 to 10, about 4 to 8, about 4 to 6, about 6 to 8, or about 6 to 10. In some specific embodiments, the DAR of the ADC may be about 1, 2, 3, or 4. In other specific embodiments, the DAR of the ADC may be about 5, 6, 7, or 8.

[0166] The following provides a detailed description of each component of the ADC disclosed herein, and the ADC composed of them. Those skilled in the art will understand that the ADC disclosed herein is inherently "modular," as each has the aforementioned modular components Ab, L, and D. Throughout this disclosure, various specific, non-limiting embodiments and examples of these modular components are described, and this disclosure covers specific combinations of particular implementations of all modules as if each specific combination were described separately and explicitly.

[0167] Ab—Antibody or antigen-binding fragment

[0168] In the CDN-ADC disclosed herein, the antibody is an antibody or antigen-binding fragment that specifically binds to a target antigen. Its function is to target and present the CDN drug component to a specific target cell population. Due to the presence of its targeting component or molecule, the antibody or antigen-binding fragment interacts with the specific target cell population, subsequently releasing free drug within the target cells (intracellular mode) or releasing free drug near the target cells (extracellular mode).

[0169] Antibody or antigen-binding fragments are bonded to linker units via their heteroatoms. Heteratoms present on the antibody or antigen-binding fragment for this bonding include sulfur (in one embodiment, from a thiol group of the targeting ligand), oxygen (in one embodiment, from a carboxyl or hydroxyl group of the targeting ligand), and optionally substituted nitrogen (in one embodiment, from a primary or secondary amine functional group of the targeting ligand, or in another embodiment, from an optionally substituted amide nitrogen). These heteroatoms may be present on the antibody unit in the native state of the ligand, such as in naturally occurring antibodies, or may be introduced into the antibody unit through chemical modification or bioengineering.

[0170] In some embodiments, the antibody or antigen-binding fragment can form a covalent bond between the linker unit and the antibody or antigen-binding fragment corresponding to the antibody unit by interacting with a reactive functional group on the linker unit. The functional group capable of interacting with the antibody unit will depend on the nature of the antibody or antigen-binding fragment. In some embodiments, the reactive group is maleimide. The covalent linking of the antibody or antigen-binding fragment to the linker unit is achieved by the interaction of the thiol functional group of the antibody or antigen-binding fragment with the maleimide functional group of the linker unit, forming a sulfur-substituted succinimide. The thiol functional group can be present in the native state of the antibody or antigen-binding fragment, such as in naturally occurring residues, or can be introduced into the antibody or antigen-binding fragment through reduction, chemical modification, or bioengineering.

[0171] In some embodiments, the antibody or antigen-binding fragment has a thiol functional group, such that it is bonded to a linker unit via the sulfur atom of the thiol functional group. In some embodiments, the thiol group is generated by reducing the interchain disulfide of the antibody, and the linker unit is conjugated to a cysteine ​​residue from the reduced interchain disulfide. In other embodiments, the thiol group is chemically introduced into the antibody, for example, by introducing a cysteine ​​residue, and the linker unit is conjugated to the antibody or antigen-binding fragment via the cysteine ​​residue introduced into the antibody or antigen-binding fragment.

[0172] In some embodiments, the antibody or antigen-binding fragment has one or more lysine residues that are capable of reacting with an activated ester (including, but not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl ester) in the linker unit, thereby providing an amide bond consisting of a nitrogen atom of the antibody or antigen-binding fragment and a C=O atom of the linker unit.

[0173] In other embodiments, the antibody or antigen-binding fragment has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. In these embodiments, the antibody or antigen-binding fragment is covalently attached to the linker unit via the sulfur atom of the thiol functional group. Reagents that can be used to modify lysine in this manner include, but are not limited to, N-succinimide-S-acetylthioacetate (SATA) and 2-iminothiacyclopentane hydrochloride (Traut reagent).

[0174] In other embodiments, the antibody or antigen-binding fragment has one or more carbohydrate groups that can be modified to provide one or more thiol functional groups. The chemically modified antibody or antigen-binding fragment in the ADC is bonded to the linker unit via the sulfur atom of the thiol functional group.

[0175] In other embodiments, the antibody or antigen-binding fragment has one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) functional group. In these embodiments, the corresponding aldehyde interacts with reactive sites on the linker unit to form a chemical bond between the linker unit and the antibody unit.

[0176] Other schemes for modifying proteins to link to linker units or related substances can be found in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002), which are incorporated herein by reference.

[0177] The antibodies constituting the ADC of this disclosure may be polyclonal, monoclonal, genetically engineered and / or otherwise modified, and are preferably suitable for human administration, such as humanized antibodies or fully human antibodies.

[0178] In some embodiments, the Ab unit of the ADC of this disclosure is a monospecific antibody. In some embodiments, the Ab unit of the ADC of this disclosure is a multispecific antibody. In some embodiments, the Ab unit, upon binding to an antigen receptor expressed on the surface of tumor cells, triggers antigen receptor-mediated endocytosis, thereby effectively delivering the antitumor drug of the ADC into the tumor cells.

[0179] In some implementations, the Ab unit of the ADC disclosed herein can be a bispecific antibody, a dual variable domain antibody, a multi-chain or single-chain antibody, a single domain antibody, a camelified antibody, an scFv-Fc antibody, a substitute antibody, etc.

[0180] This disclosure considers not only the use of monospecific antibody molecules as Ab units, but also the use of bispecific antibodies as Ab units. One specificity of the antibody can target tumor-associated antigens to promote the specific binding of ADCs to tumor cells; while the other specificity of the antibody can target receptors on the surface of tumor cells to further promote the internalization and degradation of ADCs. Examples of such combinations of bispecific targets include, but are not limited to, the combination of HER2 and PRLR dual targets on breast cancer cells. The two specificities of the antibody can also target different epitopes of the same tumor-associated antigen to increase the selectivity of the antibody for cancer cells, and / or enhance internalization and transport to lysosomes by inducing the clustering and cross-linking of antigens on the surface of tumor cells. Examples of such tumor-associated antigens include, but are not limited to, HER2 on breast cancer cells.

[0181] The antibody portion constituting the ADC of this disclosure may be in the form of a full-length antibody. The antibody portion constituting the ADC of this disclosure may have or be derived from any antibody isotype, including, for example, IgA, IgD, IgE, IgG, IgM, or IgY. In some embodiments, the antibody portion constituting the ADC of this disclosure is an antibody of the IgG isotype (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibody portion constituting the ADC of this disclosure comprises all or part of the constant region of IgG immunoglobulin.

[0182] The antibody portion constituting the ADC of this disclosure may be an antibody fragment or in the form of an antibody fragment. For example, the antibody includes an alpaca single-domain antibody fragment VHH and a single-specific, bispecific, or multispecific fusion protein of its Fc domain with any class (e.g., IgG, IgE, IgM, IgD, and IgA), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subtype of a human antibody.

[0183] The antibody portion constituting the ADC of this disclosure may be a functionally active fragment, derivative, or analog of an antibody that specifically binds to target cells (e.g., cancer cell antigens, immune cell antigens, viral antigens, or microbial antigens). In this respect, "functionally active" means that the fragment, derivative, or analog is capable of specifically binding to target cells.

[0184] Useful antibody fragments include, but are not limited to, F(ab')2 fragments, Fab fragments, Fvs, VHH fragments, single-chain antibodies, bispecific antibodies, triple-chain antibodies, quadruple-chain antibodies, scFv, scFv-FV, or any other molecule with the same specificity as the antibody. These fragments can be obtained through molecular engineering, chemical or enzymatic treatment of the intact antibody or antibody chain, or recombinant methods.

[0185] Useful modified antibody analogs and derivatives include, but are not limited to, antibody derivatives and analogs obtained through glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, and linking to cellular antibody units or other proteins. Any of the numerous chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and metabolic synthesis in the presence of tunicamycin.

[0186] Target antigens and antibodies

[0187] The antibody portion of the ADC disclosed herein can target any suitable target molecule presented on the surface of target cells, such as peptides, proteins, polysaccharides, or lipid molecules. The binding of the antibody to the target molecule should be highly specific to ensure that the ADC specifically binds to the target cell and reduce off-target toxicity. In some embodiments, the binding affinity of the antibody to the target molecule can be selected at the nanomolar or sub-nanomolar level.

[0188] Suitable target antigens can be selected by searching for cell surface proteins that are highly expressed in tumors but poorly expressed or even barely expressed in non-malignant tissues. In some embodiments, such target molecules are membrane antigens expressed on the surface of target tumor cells, such as tumor-specific antigens or tumor-associated antigens, including hematologic malignancies and solid tumors, including primary and metastatic tumors. In a particularly preferred embodiment, the antibody or antigen-binding fragment in the ADC of this disclosure specifically binds to one or more tumor-specific antigens or tumor-associated antigens, or immune cell-associated antigens.

[0189] In some embodiments, the tumor-specific antigens or tumor-associated antigens targeted by the ADC antibody portion of this disclosure are selected from: HER2, Her3, HER1 (ErbB1), HER4 (ErbB4), TROP2, Nectin4, tissue factor, PD-L1, PD-1, PD-L1 / PD-L2, MET, CLDN18.2, KIT, CTLA-4, RPR1, adrenaline A2 receptor (EphA2), folate receptor (FR) a) Mesothelin, endothelin receptor, GCPII, IL-13Ra, BCMA, GD2, CLL-1, CA-IX, MUC1, 5T4, AOC3, ALK, AXL, C242, CA-125, CCL11, CCR5, CD2, CD3, CD4, CDS, CD15, CA15-3, CD16, CD18, CD19, CD20, CD21, CD22, CD25, CD30, CD32, C D33, CD37, CD38, CD44, CD52, CD56, CD64, CD66e, CD70, CD72, CD74, CD79a, CD79b, CD123, CD138, CD14 2. CD174, CD276, CDH3, CDH6, CDH17, CCD79b, CLDN9 / CLDN6, CA19-9, DPEP3, AGS-16, IGF1R, IGF2R, VEG FR1, VEGFR2, VEGFR3, PDGFR-α, PDGFR-β, EGFR, EGFRvIII, ENPP3, FcRH5, FRα, KAAG1, LIV-1, Mesothelin, cMet, ROR1, SLTRK6, TF, BMPR1B, E16, TOP1, STEAP1, Steap2, 0772P, MUC16, Napi2b, Napi3b, Sema 5b. PSCAhIg, ETBR, RNF124, prostate cancer-associated gene 1, TrpM4, teratoma-derived growth factor 1, C3DR, FcRH2, NCA, MDP, IL20R-α, Brevican, EphB2R, ASLG659, prostate stem cell antigen precursor, GEDA, BAFF-R, CXCR5, CCR2, CCR8, HLA-DOB, P2X5, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, integrin α4β7, FGF2, FGFR1, FGFR2, FGFR3, FGFR4, PSMA, somatostatin receptor, RANK, SLAMF7, ITGB6, CEACAM5, CA9, EGFRv IL-11, IL-2RA, AXL receptor tyrosine kinase, TGF-βR, TNFRSF8, cancer / testis-associated antigen, CLEC14A, GRP78, stem cell-specific antigen, ASG-5, PRR4, GUCY2C, SLC39A6, TPBG, tumor-associated antigen CA242, FOLR1, GPNMB, HAVCR1, prostate tumor target Mindin, VTCN1, PTK7 protein tyrosine kinase 7, macrophage stimulation 1 receptor, TACSTD2, CA6, DLL3, DLL4, EpCAM, FAP, Fibronectin-EDB, DKK-1, Endoglin, VCAM1, GPC3, DR5, ASCT2, B7H1, B7H3, B7H4.

[0190] In some embodiments, the tumor-specific antigen or tumor-associated antigen is selected from AXL, B7H1, B7H3, B7H4, BCMA, CD16, CD19, CD22, CD25, CD30, CD32, CD33, CD44, CD64, CD70, CD74, CD79, CD138, CD142, CD276, CDH3, CEACAM5, Claudin 18.2, CLDN9 / CLDN6, DPEP3, EGFR, ENPP3, EphA, FcRH5, FORR1, FRα, GCPII, HER2, HER3, KAAG1, KIT, LIV-1, Mesothelin, cMet, MUC1, NECTIN4, PD-L1, PD-L1 / PD-L2, PSMA, ROR1, RPR1, TF, TOP1, TROP2.

[0191] In some embodiments, the tumor-associated antigen is selected from KIT, FORR1, CD276, PD-L1, NECTIN4, Mesothelin, MUC1, GCPII, BCMA, cMet, RPR1, CD22, CD19, TOP1, Claudin 18.2, EGFR, TROP2, and HER2. The corresponding antibodies are commercially available or can be prepared using techniques known in the art, such as hybridoma technology, recombinant technology, phage display technology, transgenic animals, or combinations thereof.

[0192] In a preferred embodiment, the antibody is an antibody that binds to antigens preferentially expressed or overexpressed in cancer cells, such as HER2 (ErbB2), PD-1, PD-L1, EGFR, TROP2, Claudin 18.2, EphA-2, and Met, with HER2 (ErbB2), EGFR, TROP2, and Claudin 18.2 being more preferred.

[0193] The ADC compounds disclosed herein can treat tumors associated with the antigen expression mentioned above, and the specific list of tumors can be known or determined by those skilled in the art based on the prior art.

[0194] Antibodies with immune specificity against tumor-associated antigens can be commercially available or produced by any method known to those skilled in the art, such as recombinant expression techniques. The nucleotide sequences encoding immune-specific antibodies against cancer cell antigens can be obtained, for example, from GenBank or similar databases, literature publications, or through routine cloning and sequencing.

[0195] Antibodies applicable to the ADCs disclosed herein include, but are not limited to: abagovomab, abatacept acasunlimab,abciximab adalimumab adebrelimab,adecatumumab,alacizumab,alemtuzumab altumomab, amivantamab, afelimomab, amostomig, anatumob, anbenitamab, anetumumab, anvatabart, anrukizumab, apolizumab, arcitum omab, aselizumab, atezolizumab, atlizumab, atorulimab, avelumab, azerutamig, azirkitug, bapineuzumab, barecetamab, basiliximab bavituximab, becotatug, bectumomab belimumab benmelstobart, bertilimab, betifosolimab, besilesomab, bevacizumab blinatumomab, biciromab, bintrafusp, brallobarbital, brentuximab, bivatuzumab, cafelkibart, calotatug, camidanlumab, cantuzumab, canakinumab (ACZ885), cantuzumab, capromab catumaxomab oral, cedelizumab cemavafusp, certolizumab, cetuximab ciletatug, citatuzumab,clenoliximab, clivatuzumab,conatumumab, coprelotamab, cosibelimab, dacetuzumab, dacliximab, daclizumab danburstotug,daratumumab,datopotamab,demupitamab,denikitug,denosumab(AMG162),depatuxizumab,detumomab,disitamab,dorlimomab,dorlixizumab,duligotuzumab,duntumumab,durlimumab,durmulumab,durvalumab,edrecolomab,ecromegab edobacomab,edrecolomab(Mabl7-lA, ), efalizumab efungumab elgemtumab,elotuxumab,elsilimomab,emphyzatamab2,enlimomab,enoblituzumab,enristomig,enzelkitug,envafolimab,epitumomab cituxetan,efalizumab,epitumomab,epratusumab,erfonrilimab,erlizumab,ertumaxomab etanercept etevritamab,etaracizumab(etaratuzumab, ),exbivirumab,fanolesomab faralimomab,farletuzumab,felvizumab,figtumumab,fontolizumab futuximab, galiximab, gantenerumab, gavilimomab Garetatug, Garivulimab, Gevastomig, Gresonitamab, Glembatumab, Golimumab (CNTO148), Gomiliximab, Ibalizumab (TNX-355), Ibritumab, Finatamab, Igovomab, Intetumab, Dixim, Abkit Uglybarizumab, inolimomab, inotuzumab, ipilimumab (MDX-010, MDX-101), istratumab, iratumumab, itanistomig, izalontamab, keliximab, LAPR Tuximab, Labetuzumab, Lanerkitug, Lemalesomab, Lebrilizumab, Lesabelimab, Lerdelimumab, Lexatumumab (HGS-ETR2, ETR2-ST01). , lexitumumab, libivirumab, lintuzumab, lodapolymab, losatuxizumab, lucatumumab, lumiliximab, lumretuzumab, manilimab, MAP atumumab (HGSETRl, TRM-1), marlotamig, margetuximab, marstacimab, matuzumab, maslimomab, matuzumab (EMD72000), mepolizumab metelimumab, milatuzumab, minretumab, mirzotamab, mitumab, modotuximab, morolimumab, motavizwnab nacolomab, nebratamig1, naptumab, natalizumab nebacumab, necitumumab, nerelimomab, nezutatug, nimotuzumab (THERACIM THERA- ), nofetumomab,bintunzumab,obrindatamab, ocrelizumab, odulimomab,ofatumumab, olaratumab,omalizumab omburtamab, omectatug, oregovomab osemitamab, opamistomig, opucolimab, oportuzumab, otelixizumab, pacmilimab, pagibaximab, palivizumab Pamvatamig, panitumumab(ABX-EGF, ), panitumumab,pascolizumab, patritumab, pemtumab pertuzumab(2C4, ), petosemtamab, pexelizumab, pimurutamab, pintumomab, priliximab, pritumumab, pumitamig2, ragistomig, ranibizumab raxibacumab, regavirumab, reozalimab, retlirafusp, reslizumab, rilotumab, rituximab ( ), robotumab,rovalpituzumab,rovelizumab,ruplizumab,ruzaltatug,safimestomig,sacituzumab(IMMU-132),satumomab,sevirumab,serclutamab,seribantumab,sibrotuzumab,s imlukafusp, simridarlimab, splizumab (MEDI-507), socazolimab, solabafusp, sonestatug, sontuzumab, spevatamig, stamulumab (MY0-029), sudubrilimab, sugemalimab, sulesomab tacatuzumab,tadocizumab,tagitanlimab,talizumab,taplitumomab,tecotabart,tenatumomab,tefibazumab telimomab,teneliximab,teplizumab,ticilimumab,tigatuzumab,timigutuzumab,tizetatug,tocilizumab tomuzotuximab,toralizumab,tositumomab,trastuzumab tremelimumab,tositumomab,tucotuzumab,tuvirumab,umizortamig2,urtoxazumab,ustekinumab(CNTO1275),vapaliximab,veltuzumab,vepalimomab,vobramitamab,volociximab,vopikitug,votumumab xirestomig, zalutumumab, zanolimumab (HuMAX-CD4), zanidatamab, zenocutuzumab, ziralimumab, zolbetuximab, zolimomab.

[0196] Ab unit targeting HER2

[0197] In a particularly preferred embodiment, the ADC provided herein comprises an antibody or antigen-binding fragment (anti-HER2 antibody) that specifically binds to human HER2, i.e., the antibody partially and specifically targets the tumor-associated antigen HER2. Therefore, in some aspects, this disclosure provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to HER2 as the Ab unit of the ADC.

[0198] HER2 is the receptor tyrosine protein kinase ErbB2, and it is overexpressed or amplified in approximately 20-30% of breast cancers. Increased activation of HER2 triggers multiple downstream pathways, leading to abnormal proliferation of cancer cells (Treish I, Schwartz R, Lindley C: Pharmacology and therapeutic use of trastuzumab in breast cancer. Am J Health Syst Pharm. 2000, 57(22), 2063-76, quiz 2077-9). HER2 is also overexpressed in many other types of cancer, such as gastric cancer, esophageal cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, bladder cancer, pancreatic cancer, lung cancer, prostate cancer, osteosarcoma, neuroblastoma, or head and neck cancer.

[0199] In some implementations, the antibody that specifically binds to human HER2 or an antibody fragment thereof (e.g., an antigen-binding fragment) may be selected from trastuzumab, pertuzumab, margetuximab, or HT-19, or an antibody fragment thereof or a site-specific mutant thereof, or other anti-human HER2 antibodies that recognize the same epitope or competitively bind to human HER2.

[0200] Trastuzumab (trade names Herceptin or Herclon) is a humanized monoclonal antibody against human epidermal growth factor 2 (HER2) used to treat HER2-positive breast cancer, gastrointestinal cancer, and gastric cancer. It binds to the juxtamembranous portion of the extracellular domain of the HER2 receptor. The amino acid sequences of its heavy and light chain variable regions are described in US Patent 5,821,337. It interacts with three loop regions formed by human HER2 residues 557–561, 570–573, and 593–603 (Cho et al., Nature 2003, 421, 756-760), potentially interfering with HER2 signaling by preventing HER2 receptor dimerization, promoting HER2 receptor endocytosis, and inhibiting the shedding of the extracellular domain (Hudis CA, N Engl J Med. 2007, 357(1), 39-51). Another important mechanism of action for anti-HER2 antibodies is the mediation of antibody-dependent cytotoxicity (ADCC). In ADCC, anti-HER2 antibodies bind to tumor cells and then recruit immune cells, such as macrophages, through Fcε receptor (FcεR) interaction. Trastuzumab was approved by the U.S. FDA in September 1998 for the treatment of patients with metastatic breast cancer.

[0201] Pertuzumab (also known as 2C4, Omnitarg, Perjeta) is a humanized monoclonal antibody that binds to the extracellular domain of the HER2 receptor and inhibits the dimerization of HER2 with other HER receptors. The amino acid sequences of its heavy and light chains are described in US Patent 7,560,111. Pertuzumab primarily interacts with residues in the 245–333 region of human HER2, particularly residues His 245, Val 286, Ser 288, Leu 295, His 296, or Lys 311 (Franklin et al., Cancer Cell 2004, 5, 317–328). Pertuzumab has shown greater efficacy than trastuzumab in disrupting the formation of HER1-HER2 and HER3-HER2 complexes in breast and prostate cancer cell lines (Agus et al., J Clin Oncol. 2005, 23(11), 2534-43). In terms of efficacy, pertuzumab does not require antibody-dependent cytotoxicity because the complete Fc region is not required for its activity (Agus et al., J Clin Oncol. 2005, 23(11), 2534-43.). Pertuzumab is approved by the FDA for use in combination with trastuzumab and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer.

[0202] Margetuximab (also known as MGAH22) is another anti-HER2 monoclonal antibody (see http: / / www.macrogenics.com / products-margetuximab.html). The Fc region of margetuximab has been optimized to increase its binding to activating FcεRs. Margetuximab is currently undergoing clinical trials for the treatment of patients with recurrent or refractory advanced breast cancer whose tumors are found to express HER2 at a 2+ level by immunohistochemistry and who lack evidence of HER2 gene amplification via FISH.

[0203] HT-19 is another anti-HER2 monoclonal antibody that binds to epitopes in human HER2 that differ from those of trastuzumab or pertuzumab. It has been shown to inhibit HER2 signaling in a manner similar to that of trastuzumab, and when combined with trastuzumab and pertuzumab, it promotes HER2 degradation (Bergstrom DA et al., Cancer Res. 2015, 75, LB-231).

[0204] Exemplary antibodies targeting HER2 that can be used with the ADC disclosed herein may be antibodies or antigen-binding fragments comprising all six CDR sequences of antibodies selected from the group consisting of: trastuzumab (Herceptin, Genentech, US6,054,297); ATCC accession numbers PTA-10355, PTA-10356, PTA-10357, PTA 10358 (US20100119511); ATCC accession number CRL-10463 (Genentech); ATCC accession numbers HB-12215, HB-12216, CRL 10463, HB-12697; pertuzumab (Pertuzumab, Genentech, US20110117097); ATCC accession numbers HB-12215, HB-12216, CRL 10463, CRL 10463, HB-12697; 10463, HB-12698 (US20090202546); ATCC Registry Numbers HB-12215, HB-12216 (US20060088523); ATCC Registry Numbers (7C2)HB-12215, (7F3)HB-12216, (4D5)CRL-10463, (2C4)HB-12697 (US20060018899); TrasGEX (Glycotope: http: / / www.glycotope.conn / pipeline). This disclosure also considers anti-HER2 antibodies disclosed in the following documents: US20110177095, US20100119511, US20110117097, US20090285837, US20090202546, US20060088523, US20060018899, US2011 / 0159014, US20090187007, and US20110217305.

[0205] Trastuzumab light chain amino acid sequence (SEQ ID NO:1)

[0206] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS

[0207] RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP

[0208] SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

[0209] LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0210] >Amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO:2)

[0211] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY

[0212] ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0213] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0214] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0215] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0216] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE

[0217] MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0218] >Amino acid sequence of the light chain LCDR1 of trastuzumab (SEQ ID NO:3)

[0219] RASQDVNTAVA

[0220] >Amino acid sequence of the light chain LCDR2 of trastuzumab (SEQ ID NO:4)<o001019>

[0221] SASFLYS

[0222] Trastuzumab light chain LCDR3 sequence (SEQ ID NO:5)

[0223] QQHYTTPPT

[0224] Trastuzumab heavy chain HCDR1 sequence (SEQ ID NO:6)

[0225] DTYIH

[0226] Trastuzumab heavy chain HCDR2 sequence (SEQ ID NO:7)

[0227] RIYPTNGYTRYADSVKG

[0228] Trastuzumab heavy chain HCDR3 sequence (SEQ ID NO:8)

[0229] WGGDGFYAMDY

[0230] Trastuzumab light chain variable region VL (SEQ ID NO:9)

[0231] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS

[0232] RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0233] Trastuzumab heavy chain variable region VH (SEQ ID NO:10)

[0234] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY

[0235] ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0236] In one embodiment, the antibody portion for the ADC of this disclosure comprises all six CDR sequences of trastuzumab. In another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain variable region sequence and the light chain variable region sequence of trastuzumab. In yet another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain sequence and the light chain sequence of trastuzumab.

[0237] In some embodiments, the Ab unit of the ADC of this disclosure comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:10 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO:9, and preferably, wherein the CDRs are defined according to Kabat or IMGT or a combination thereof.

[0238] In some embodiments, the Ab unit of the ADC of this disclosure includes three heavy chain complementarity determination regions (HCDRs) and three light chain complementarity determination regions (LCDRs), wherein:

[0239] According to Kabat's definition, 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:3, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:4, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:5.

[0240] In one embodiment, the Ab unit of the ADC of this disclosure includes a heavy chain variable region, wherein the heavy chain variable region comprises: the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0241] In one embodiment, the Ab unit of the ADC of this disclosure includes a light chain variable region, wherein the light chain variable region comprises: the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0242] In some preferred embodiments, the Ab unit of the ADC of this disclosure includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence described in SEQ ID NO:10, and wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO:9.

[0243] In some embodiments, the Ab unit of the ADC of this disclosure 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 unit 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 unit can be a κ light chain constant region or a λ light chain constant region, especially the human κ light chain constant region.

[0244] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human IgG1 heavy chain constant region. Preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:11, 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:11, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:11.

[0245] An exemplary amino acid sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 11)

[0246] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0247] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human κ light chain constant region. Preferably, the light chain constant region comprises the amino acid sequence of SEQ ID NO:12, 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:12, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:12.

[0248] An exemplary amino acid sequence of the constant region of the human κ light chain (SEQ ID NO:12)

[0249] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0250] In some embodiments, the Ab unit of the ADC of this disclosure is a full-length antibody comprising a heavy chain constant region and a light chain constant region. In some embodiments, the Ab unit is a tetrameric structure having two light chains and two heavy chains. In still other embodiments, the Ab unit is an IgG antibody, particularly an IgG1 antibody.

[0251] In some preferred embodiments, the Ab unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the heavy chain comprises, or consists of, 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. In other preferred embodiments, the Ab unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the light chain comprises, or consists of, 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.

[0252] In some more preferred embodiments, the Ab unit of the ADC of this disclosure includes:

[0253] (a) A heavy chain containing the amino acid sequence of SEQ ID NO:2, and

[0254] (b) A light chain containing the amino acid sequence of SEQ ID NO:1.

[0255] Ab unit targeting TROP2

[0256] In a particularly preferred embodiment, the ADC provided herein comprises an antibody or antigen-binding fragment (anti-TROP2 antibody) that specifically binds to human TROP2, i.e., the antibody partially and specifically targets the tumor-associated antigen TROP2. Therefore, in some aspects, this disclosure provides an ADC comprising an antibody or antigen-binding fragment that specifically binds to TROP2 as the Ab unit of the ADC.

[0257] Tropoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a type I transmembrane cell surface glycoprotein. The sequence of human TROP2 is available from UniProtKB accession number P09758. TROP2 has been confirmed to be overexpressed in many solid tumors, including, but not limited to, various human epithelial cancers such as cervical cancer, endometrial cancer, breast cancer, urothelial carcinoma, lung cancer, gastric cancer, prostate cancer, colorectal cancer, and pancreatic cancer. Furthermore, TROP2 can play a role in tumor cell proliferation, invasion, migration, apoptosis, and treatment resistance by binding to or interacting with various molecules. These characteristics make TROP2 an attractive pan-cancer target for cancer therapy. Ying Wen et al., Aliterature review of the promising future of TROP2: a potential drug therapy target, Ann Transl Med. 2022, 10(24), 1403, doi:10.21037 / atm-22-5976.

[0258] Antibodies targeting human TROP2 that can be used in the ADCs of this disclosure can be prepared using antibody preparation processes known in the art. For example, anti-TROP2 antibodies can be obtained by immunizing animals with human TROP2 (UniProtKB accession number P09758) or a polypeptide containing the amino acid sequence of the TROP2 extracellular domain, harvesting antibodies from the immunized animals, and purifying and preferably humanizing them. Alternatively, fully human sequence anti-human TROP2 antibodies can be obtained using yeast display libraries expressing human immunoglobulin sequences or transgenic animals.

[0259] In some implementations, the antibody that specifically binds to human TROP2 or an antibody fragment thereof (e.g., an antigen-binding fragment) may be selected from Sacituzumab, Datopotamab, or an antibody fragment thereof, or other anti-human TROP2 antibodies that recognize the same epitope or competitively bind to human TROP2.

[0260] Sacituzumab, also known as cerutuzumab, is a humanized form of the mouse monoclonal antibody RS7 developed by Immunomedics. It is a humanized IgG1κ monoclonal antibody targeting TROP2. This antibody can directly bind to cancer cells expressing TROP2 and trigger antibody endocytosis. The sequence of Sacituzumab can be found in US10179171B2.

[0261] Datopotamab is an IgG1-type anti-TROP2 antibody. This antibody is produced by humanizing a mouse mAb that specifically binds to human TROP2. The sequence of Datopotamab can be found in Daisuke Okajima et al., Datopotamab Deruxtecan, a Novel TROP2-directed Antibody–drug Conjugate, Demonstrates Potent Antitumor Activity by Efficient Drug Delivery to Tumor Cells, Mol Cancer Ther 2021 20(12), 2329–2340.

[0262] Exemplary antibodies targeting human TROP2 that can be used with the ADC of this disclosure may be antibodies or antigen-binding fragments comprising all six CDR sequences of antibodies selected from the group consisting of Sacituzumab and Datopotamab. This disclosure also considers anti-human TROP2 antibodies disclosed in the following documents: WO2010089782A1; US ​​2021 / 0393792 A1; WO2008 / 144891, WO2011 / 145744, WO2011 / 155579, WO2013 / 077458, WO2003 / 074566, WO2011 / 068845, WO2013 / 068946, US2023 / 0270870A1.

[0263] >Sacituzumab light chain amino acid sequence (SEQ ID NO:13)

[0264] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0265] >Amino acid sequence of the heavy chain of Sacituzumab (SEQ ID NO:14)

[0266] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0267] >Amino acid sequence of the light chain LCDR1 of Sacituzumab (SEQ ID NO:15)

[0268] KASQDVSIAVA

[0269] >Amino acid sequence of the light chain LCDR2 of Sacituzumab (SEQ ID NO:16)

[0270] SASYRYT

[0271] >Amino acid sequence of the light chain LCDR3 of Sacituzumab (SEQ ID NO:17)

[0272] QQHYITPLT

[0273] >Sacituzumab heavy chain HCDR1 sequence (SEQ ID NO:18)

[0274] NYGMN

[0275] >Sacituzumab heavy chain HCDR2 sequence (SEQ ID NO:19)

[0276] WINTYTGEPTYTDDFKG

[0277] >Sacituzumab heavy chain HCDR3 sequence (SEQ ID NO:20)

[0278] GGFGSSYWYFDV

[0279] >Sacituzumab light chain variable region VL (SEQ ID NO:21)

[0280] DIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIK

[0281] >Sacituzumab heavy chain variable region VH (SEQ ID NO:22)

[0282] QVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSS

[0283] In one embodiment, the antibody portion for the ADC of this disclosure comprises all six CDR sequences of Sacituzumab or Datopotamab. In another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain variable region sequence and the light chain variable region sequence of Sacituzumab or Datopotamab. In yet another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain sequence and the light chain sequence of Sacituzumab or Datopotamab.

[0284] In some embodiments, the Ab unit of the ADC of this disclosure comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:22 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO:21, and preferably, wherein the CDRs are defined according to Kabat or IMGT or a combination thereof.

[0285] In some embodiments, the Ab unit of the ADC of this disclosure includes three heavy chain complementarity determination regions (HCDRs) and three light chain complementarity determination regions (LCDRs), wherein:

[0286] According to Kabat's definition, HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:18, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:19, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:20, 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.

[0287] In one embodiment, the Ab unit of the ADC of this disclosure includes a heavy chain variable region, wherein the heavy chain variable region comprises: the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0288] In one embodiment, the Ab unit of the ADC of this disclosure includes a light chain variable region, wherein the light chain variable region comprises: the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0289] In some preferred embodiments, the Ab unit of the ADC of this disclosure includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence described in SEQ ID NO:22, and wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO:21.

[0290] In some embodiments, the Ab unit of the ADC of this disclosure 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 unit 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 unit can be a κ light chain constant region or a λ light chain constant region, especially the human κ light chain constant region.

[0291] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human IgG1 heavy chain constant region. Preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:23, 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:23, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:23.

[0292] An exemplary amino acid sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 23)

[0293] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0294] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human κ light chain constant region. Preferably, the light chain constant region comprises the amino acid sequence of SEQ ID NO:12, 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:12, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:12.

[0295] In some embodiments, the Ab unit of the ADC of this disclosure is a full-length antibody comprising a heavy chain constant region and a light chain constant region. In some embodiments, the Ab unit is a tetrameric structure having two light chains and two heavy chains. In still other embodiments, the Ab unit is an IgG antibody, particularly an IgG1 antibody.

[0296] In some preferred embodiments, the Ab unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:14, 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 unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0297] In some more preferred embodiments, the Ab unit of the ADC of this disclosure includes:

[0298] (c) A heavy chain containing the amino acid sequence of SEQ ID NO:14, and

[0299] (d) A light chain containing the amino acid sequence of SEQ ID NO:13.

[0300] Cancers that can be treated with this disclosed ADC targeting TROP2 include, but are not limited to, adenocarcinoma, squamous cell carcinoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), colorectal cancer, gastric adenocarcinoma, esophageal cancer, hepatocellular carcinoma, ovarian epithelial cancer, breast cancer, metastatic breast cancer, triple-negative breast cancer (TNBC), prostate cancer, hormone-refractory prostate cancer, pancreatic ductal adenocarcinoma, head and neck cancer, renal cell carcinoma, bladder tumors, cervical cancer, endometrial cancer, uterine cancer, follicular thyroid cancer, and glioblastoma multiforme.

[0301] Ab unit targeting Claudin18.2

[0302] In a particularly preferred embodiment, the ADC provided herein comprises an antibody or antigen-binding fragment (anti-Claudin18.2 antibody) that specifically binds to human Claudin18.2, i.e., the antibody partially and specifically targets the tumor-associated antigen Claudin18.2. Therefore, in some aspects, this disclosure provides an ADC comprising an antibody or antigen-binding fragment thereof that specifically binds to Claudin18.2 as the Ab unit of the ADC.

[0303] Claudin 18.2 (also abbreviated as CLDN18.2) is a tight junction membrane protein belonging to the Claudin family. The Claudin 18.2 sequence in humans and many other mammals can be found in UniProtKB. For example, the human Claudin 18.2 sequence is available in UniProtKB accession number P56856-2. While expression of this protein in healthy tissues is primarily confined to differentiated gastric mucosal epithelial cells, it exhibits aberrant overexpression in a range of malignancies, particularly digestive system malignancies. Therefore, Claudin 18.2 has been proposed as a promising target for the development of antibody-drug conjugate (ADC) cancer therapeutics. Daisuke Kyuno et al., Claudin-18.2 as a therapeutic target in cancers:cumulativefindings from basic research and clinical trials, Tissue Barriers.2022,10(1),1967080.doi:10.1080 / 21688370.2021.1967080; Jinxia Chen, Targeting CLDN18.2 incancers of the gastrointestinal tract: New drugs and new indications, FrontOncol.2023,13,1132319, doi:10.3389 / fonc.2023.1132319.

[0304] Antibodies targeting human Claudin18.2 that can be used in the ADCs of this disclosure can be prepared using antibody preparation processes known in the art. For example, anti-Claudin18.2 antibodies can be obtained by immunizing animals with human Claudin18.2 (UniProtKB accession number P56856-2) or a polypeptide containing the amino acid sequence of the extracellular domain of Claudin18.2, harvesting antibodies from the immunized animals, and purifying and preferably humanizing them. Alternatively, antibodies against human Claudin18.2 with a complete human sequence can be obtained using a yeast display library expressing a human immunoglobulin sequence or transgenic animals.

[0305] In some embodiments, the antibody that specifically binds to human Claudin18.2 or an antibody fragment thereof (e.g., an antigen-binding fragment) may be selected from Zolbetuximab, Osemitamab (TST001), CMG901, ASKB589, ZL-1211, or an antibody fragment thereof, or other anti-human Claudin18.2 antibodies that recognize the same epitope or competitively bind to human Claudin18.2.

[0306] Zolbetuximab (also known as GC-182, IMAB-362, IMAB362, claudiximab) is an IgG1 antibody derived from a mouse monoclonal antibody and has been chimericized to display the human IgG1 constant region for clinical use. This antibody can directly bind to cancer cells expressing CLDN18.2 and trigger antibody endocytosis. The sequence of Zolbetuximab can be found in WO2007059997 and WO2016 / 165762.

[0307] Osemitamab (TST001) is a high-affinity humanized anti-Claudin18.2 antibody. This antibody exhibits enhanced antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), demonstrating strong antitumor activity in tumor xenograft models. The sequence of this antibody can be found in Inxight Drugs and INN (code 11927).

[0308] CMG901, ASKB589, and ZL-1211 are exemplary antibodies against human CLDN18.2 currently undergoing clinical trials. See DOI:10.1200 / JCO.2023.41.4_suppl.352; DOI:10.1200 / JCO.2023.41.4_suppl.397; DOI:10.1200 / JCO.2023.41.16_suppl.2537.

[0309] Exemplary antibodies that can be used in the ADC of this disclosure to target human CLDN18.2 may be antibodies or antigen-binding fragments comprising all six CDR sequences of antibodies selected from the group consisting of: Zolbetuximab, Osemitamab (TST001), and CMG901.

[0310] This disclosure also considers anti-human Claudin18.2 antibodies disclosed in the following documents: WO2007059997A1, CN107667118A, WO2016 / 166122, US11555070B2, WO2020 / 135674, WO2018 / 006882, CN109762067, WO2019 / 242505, WO2020 / 038404, WO2020 / 043044, WO2020 / 063988, WO2020 / 082209, WO2020 / 018852, WO2020 / 023679, WO2020 / 135674, WO2020 / 135201, WO2020 / 139956, WO2020 / 025792, WO2020160560, CN111808194, and WO2020200196.

[0311] >Zolbetuximab light chain amino acid sequence (SEQ ID NO:24)

[0312] DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0313] >Zolbetuximab heavy chain amino acid sequence (SEQ ID NO:25)

[0314] QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0315] >Zolbetuximab light chain LCDR1 sequence (SEQ ID NO:26)

[0316] KSSQSLLNSGNQKNYLT

[0317] >Zolbetuximab light chain LCDR2 sequence (SEQ ID NO:27)

[0318] WASTRES

[0319] >Zolbetuximab light chain LCDR3 sequence (SEQ ID NO:28)

[0320] QNDYSYPFT

[0321] >Zolbetuximab heavy chain HCDR1 sequence (SEQ ID NO:29)

[0322] SYWIN

[0323] >Zolbetuximab heavy chain HCDR2 sequence (SEQ ID NO:30)

[0324] NIYPSDSYTNYNQKFKD

[0325] >Zolbetuximab heavy chain HCDR3 sequence (SEQ ID NO:31)

[0326] SWRGNSFDY

[0327] >Zolbetuximab light chain variable region VL (SEQ ID NO:32)

[0328] DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDYSYPFT FGSGTKLEIK

[0329] Zolbetuximab heavy chain variable region VH (SEQ ID NO:33)

[0330] QVQLQQPGAELVRPGASVKLSCKASGYTFT SYWIN WVKQRPGQGLEWIG NIYPSDSYTNYNQKFKD KATLTVDKSSSTAYMQLSSPTSEDSAVYYCTR SWRGNSFDY WGQGTTLTVSS

[0331] In one embodiment, the antibody portion for the ADC of this disclosure comprises all six CDR sequences of Zolbetuximab. In another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain variable region sequence and the light chain variable region sequence of Zolbetuximab. In yet another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain sequence and the light chain sequence of Zolbetuximab.

[0332] In some embodiments, the Ab unit of the ADC of this disclosure comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:33 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO:32, and preferably, wherein the CDRs are defined according to Kabat or IMGT or a combination thereof.

[0333] In some embodiments, the Ab unit of the ADC of this disclosure includes three heavy chain complementarity determination regions (HCDRs) and three light chain complementarity determination regions (LCDRs), wherein:

[0334] 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:26, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:27, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:28.

[0335] In one embodiment, the Ab unit of the ADC of this disclosure includes a heavy chain variable region, wherein the heavy chain variable region comprises: the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0336] In one embodiment, the Ab unit of the ADC of this disclosure includes a light chain variable region, wherein the light chain variable region comprises: the amino acid sequence shown in SEQ ID NO:32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0337] In some preferred embodiments, the Ab unit of the ADC of this disclosure includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence described in SEQ ID NO:33, and wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO:32.

[0338] In some embodiments, the Ab unit of the ADC of this disclosure 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 unit 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 unit can be a κ light chain constant region or a λ light chain constant region, especially the human κ light chain constant region.

[0339] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human IgG1 heavy chain constant region. Preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:34, 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:34, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:34.

[0340] An exemplary amino acid sequence of the constant region of the human IgG1 heavy chain (SEQ ID NO: 34)

[0341] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0342] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human κ light chain constant region. Preferably, the light chain constant region comprises the amino acid sequence of SEQ ID NO:12, 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:12, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:12.

[0343] In some embodiments, the Ab unit of the ADC of this disclosure is a full-length antibody comprising a heavy chain constant region and a light chain constant region. In some embodiments, the Ab unit is a tetrameric structure having two light chains and two heavy chains. In still other embodiments, the Ab unit is an IgG antibody, particularly an IgG1 antibody.

[0344] In some preferred embodiments, the Ab unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:25, 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 unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0345] In some more preferred embodiments, the Ab unit of the ADC of this disclosure includes:

[0346] (e) A heavy chain containing the amino acid sequence of SEQ ID NO:25, and

[0347] (f) A light chain containing the amino acid sequence of SEQ ID NO:24.

[0348] Cancers that can be treated with the presently disclosed ADC targeting Claudin18.2 include, but are not limited to, various gastrointestinal cancers such as gastric cancer (GC), gastroesophageal junction (GEJ) cancer, esophageal cancer, and pancreatic cancer.

[0349] Ab units targeting EGFR

[0350] In a particularly preferred embodiment, the ADC provided herein comprises an antibody or antigen-binding fragment (anti-EGFR antibody) that specifically binds to human EGFR, i.e., the antibody partially and specifically targets the tumor-associated antigen EGFR. Therefore, in some aspects, this disclosure provides an ADC comprising an antibody or antigen-binding fragment that specifically binds to EGFR as the Ab unit of the ADC.

[0351] Epidermal growth factor receptor, abbreviated as EGFR in this article, is a member of the epidermal growth factor receptor (HER) family and is encoded by the c-erbB proto-oncogene (also known as HER-1 or Erb-B1). EGFR sequences in humans and many other mammals can be found in UniProtKB. For example, the human EGFR sequence is available in UniProtKB accession number P00533. EGFR is overexpressed in many solid tumors, including lung cancer, head and neck cancer, breast cancer, kidney cancer, gastric cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, and bladder cancer; and EGFR can induce tumor proliferation through homodimerization.

[0352] EGFR has been proposed as a promising target for antibody-based cancer therapies. To date, five EGFR-targeting monoclonal antibodies have been approved for clinical cancer treatment, including cetuximab. ), panitumumab ), nimotuzumab, ), necitumuma and amivantamab(amivantamab-vmjw); In addition, three EGFR-based ADCs are in clinical trials. However, no EGFR-based ADCs have yet been approved for treatment. See Jinfeng Yu et al., Antibody-Drug Conjugates Targeting the Human Epidermal Growth Factor Receptor Family in Cancers, Front Mol Biosci. 2022, 9, 847835, doi:10.3389 / fmolb.2022.847835.

[0353] Various anti-EGFR monoclonal antibodies have been developed to bind to the extracellular domain of this receptor to block receptor-ligand interaction or dimerization. These antibodies are all applicable to this disclosure. Furthermore, antibodies targeting human EGFR that can be used in the ADCs of this disclosure can also be prepared using antibody preparation processes known in the art. For example, anti-EGFR antibodies can be obtained by immunizing animals with human EGFR (UniProtKB accession number P00533) or a polypeptide containing the amino acid sequence of the EGFR extracellular domain, harvesting antibodies from the immunized animals, and purifying and preferably humanizing them. Alternatively, a yeast display library expressing a human immunoglobulin sequence or transgenic animals can be used to obtain antibodies against human EGFR with a complete human sequence.

[0354] The antibody targeting human EGFR used in the ADC of this disclosure can be a monospecific antibody that binds to EGFR. In some cases, the antibody targeting human EGFR used in the ADC of this disclosure can also be a multispecific antibody, especially a bispecific antibody, such as a bispecific antibody targeting EGFR and MET; or a bispecific antibody targeting MUC1 and EGFR; or a bispecific antibody targeting Her3 and EGFR.

[0355] In some embodiments, the anti-human EGFR antibody or its antibody fragment (e.g., antigen-binding fragment) used for the ADC of this disclosure may be selected from cetuximab. ), panitumumab ), nimotuzumab, ), necitumuma amivantamab(amivantamab-vmjw; The list includes depatuxizumab (ABT-806), NECITUMUMAB, IZALONTAMAB, BAFISONTAMAB, Petosemtamab, PIMURUTAMAB, FUTUXIMAB, MODOTUXIMAB (Zatuximab), or antibody fragments thereof, or other anti-human EGFR antibodies that recognize the same epitope or competitively bind to human EGFR. The sequences of these antibodies can be found in Inxight Drugs and International Nonproprietary Names (INN) codes 7906, 8499, 8545, 9083, 11030, 10263, 9083, 12022, 11851, 11136, 11309, 9612, and 9613.

[0356] Cetuximab Cetuximab is a recombinant chimeric human / mouse IgG1 monoclonal antibody. This antibody binds to the extracellular domain of inactivated EGFR with a much higher affinity than endogenous ligands, competitively blocking ligand-receptor binding, inhibiting ligand-receptor agonism, and promoting EGFR endocytosis, thus downregulating EGFR expression at the cell membrane. Furthermore, this antibody can activate antibody-dependent cell-mediated cytotoxicity (ADCC), producing further cytotoxic effects. The sequence of cetuximab can be found in WO2007092453 and INN code 7906.

[0357] Panitumumab Panitumab is a recombinant humanized IgG2 monoclonal antibody. Panitumab specifically binds to EGFR on tumor cells and competitively inhibits the binding of EGFR ligands. Preclinical studies have shown that the binding of panitumab to EGFR prevents ligand-induced receptor autophosphorylation and activation of receptor-associated kinases, thereby inhibiting cell growth, inducing apoptosis, reducing the production of pro-inflammatory cytokines and angiogenesis factors, and inducing EGFR internalization. The sequence of panitumab can be found in INN code 8499.

[0358] Amivantamab (also known as JNJ-61186372) is an anti-EGFR-MET bispecific antibody derived from Chinese hamster ovary cells and approved for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with epidermal growth restriction. This antibody binds to EGFR and MET, blocking the binding of these receptors to their ligands and inducing the internalization of EGFR and MET on the cell surface. Amivantamab significantly downregulates the expression of EGFR and MET on the surface of NSCLC cells, further reducing downstream signaling. The sequence of Amivantamab can be found in INN code 9083.

[0359] Exemplary antibodies targeting human EGFR that can be used with the ADC of this disclosure may be antibodies or antigen-binding fragments comprising all six CDR sequences of antibodies selected from the group consisting of: cetuximab, panitumumab, nimotuzumab, necitumuma, amivantamab, depatuxizumab, NECITUMUMAB, IZALONTAMAB, BAFISONTAMAB, Petosemtamab, PIMURUTAMAB, FUTUXIMAB, and MODOTUXIMAB.

[0360] This disclosure also considers anti-human EGFR antibodies disclosed in the following documents: WO2023040941A1,WO2022271722A1,WO2022159576A1,WO2022128716A1,WO2022105878A1,WO2021247798A1,WO2022104697A1,WO2021066869A1,WO2020233534A1,WO2020130125A1,WO2019046858A1,WO2019046859A1,WO2019035630A2,WO2019035630A3,WO2018098035A1,WO201721423 3A1,WO2017214282A1,WO2017214301A1,WO2017161206A1,WO2017139623A1,WO2017136581A1,WO2017076492A1,WO2017060322A3,WO2017025458A1,WO2017008169A1,WO2016065456A1,WO2015143382A1,WO2014143765A1,WO2014143765A8,WO2014152199A1,WO2014094355A1,WO2012143495A2, andWO2012143495A3.

[0361] >Cetuximab light chain amino acid sequence (SEQ ID NO:35)

[0362] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0363] >Cetuximab heavy chain amino acid sequence (SEQ ID NO:36)

[0364] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0365] >Cetuximab light chain LCDR1 sequence (SEQ ID NO:37)

[0366] RASQSIGTNIH

[0367] >Cetuximab light chain LCDR2 sequence (SEQ ID NO:38)

[0368] YASESIS

[0369] >Cetuximab light chain LCDR3 sequence (SEQ ID NO:39)

[0370] QQNNNWPTT

[0371] >Cetuximab heavy chain HCDR1 sequence (SEQ ID NO:40)

[0372] NYGVH

[0373] >Cetuximab heavy chain HCDR2 sequence (SEQ ID NO:41)

[0374] VIWSGGNTDYNTPFTS

[0375] >Cetuximab heavy chain HCDR3 sequence (SEQ ID NO:42)

[0376] ALTYYDYEFAY

[0377] >Cetuximab light chain variable region VL (SEQ ID NO:43)

[0378] DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK

[0379] >Cetuximab heavy chain variable region VH (SEQ ID NO:44)

[0380] QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSA

[0381] In one embodiment, the antibody portion for the ADC of this disclosure comprises all six CDR sequences of Cetuximab. In another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain variable region sequence and the light chain variable region sequence of Cetuximab. In yet another embodiment, the antibody portion for the ADC of this disclosure comprises the heavy chain sequence and the light chain sequence of Cetuximab.

[0382] In some embodiments, the Ab unit of the ADC of this disclosure comprises three CDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:44 and three CDRs of the light chain variable region (VL) sequence of SEQ ID NO:43, and preferably, wherein the CDRs are defined according to Kabat or IMGT or a combination thereof.

[0383] In some embodiments, the Ab unit of the ADC of this disclosure includes three heavy chain complementarity determination regions (HCDRs) and three light chain complementarity determination regions (LCDRs), wherein:

[0384] According to Kabat's definition, HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:40, HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:41, HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:42, LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:37, LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:38, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:39.

[0385] In one embodiment, the Ab unit of the ADC of this disclosure includes a heavy chain variable region, wherein the heavy chain variable region comprises: the amino acid sequence shown in SEQ ID NO:44, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0386] In one embodiment, the Ab unit of the ADC of this disclosure includes a light chain variable region, wherein the light chain variable region comprises: the amino acid sequence shown in SEQ ID NO:43, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0387] In some preferred embodiments, the Ab unit of the ADC of this disclosure includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence described in SEQ ID NO:44, and wherein the light chain variable region includes the amino acid sequence shown in SEQ ID NO:43.

[0388] In some embodiments, the Ab unit of the ADC of this disclosure 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 unit 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 unit can be a κ light chain constant region or a λ light chain constant region, especially the human κ light chain constant region.

[0389] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human IgG1 heavy chain constant region. Preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:34, 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:34, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:34.

[0390] In some embodiments, the Ab unit of the ADC of this disclosure comprises a human κ light chain constant region. Preferably, the light chain constant region comprises the amino acid sequence of SEQ ID NO:12, 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:12, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO:12.

[0391] In some embodiments, the Ab unit of the ADC of this disclosure is a full-length antibody comprising a heavy chain constant region and a light chain constant region. In some embodiments, the Ab unit is a tetrameric structure having two light chains and two heavy chains. In still other embodiments, the Ab unit is an IgG antibody, particularly an IgG1 antibody.

[0392] In some preferred embodiments, the Ab unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:36, 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 unit of the ADC of this disclosure comprises a heavy chain and a light chain, wherein: the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0393] In some more preferred embodiments, the Ab unit of the ADC of this disclosure includes:

[0394] (g) A heavy chain containing the amino acid sequence of SEQ ID NO:36, and

[0395] (h) A light chain containing the amino acid sequence of SEQ ID NO:35.

[0396] Cancers that can be treated with the EGFR-targeting ADCs of this disclosure include, but are not limited to, various primary and metastatic solid tumors, such as lung cancer (e.g., lung adenocarcinoma, lung squamous cell carcinoma and non-small cell lung cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), nasopharyngeal carcinoma, esophageal cancer, biliary tract cancer, colon cancer, colorectal cancer, pancreatic cancer, gastric cancer, and glioblastoma.

[0397] First effective payload – D1

[0398] The D1 that can be used in the ADC of this disclosure is a bifunctional cyclic dinucleotide (CDN) having the following formula:

[0399]

[0400] in

[0401] B1 is adenine that can be substituted by X. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl; or optionally R b Replaced guanine Where R b Selected from H or -C 1-6 alkyl;

[0402] R1 and R1' are each independently selected from H, F or -OH;

[0403] B2 is guanine. R b Selected from H or -C 1-6 alkyl;

[0404] This indicates that the phosphate bond can be attached to either the 2' or 3' position of the pentose sugar, where the site not cyclized with the phosphate is replaced by R2 and R2'; and

[0405] R2 and R2' are each independently selected from H, -OH, or F;

[0406] Or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled forms.

[0407] In a preferred embodiment, the CDN (D1) of formula (II) is covalently bonded to the connector unit (L) in the ADC of formula (X) via any one of its -SH groups.

[0408] In specific embodiments, the two thiophosphate bonds in D1 of the ADC of this disclosure exist in the form of R,R, S,S, S,R, or R,S configurations or mixtures thereof. In a preferred embodiment, the two thiophosphate bonds in the compound of this disclosure exist in substantially pure R,R, S,S, S,R, or R,S configurations, and are particularly preferred to exist in substantially pure R,R configurations.

[0409] In one embodiment of a compound of formula (II), B1 is an adenine optionally substituted with X. Where X is selected from H.

[0410] In one embodiment of a compound of formula (II), B1 is an adenine optionally substituted with X. Where X is selected from Cl or F.

[0411] In one embodiment of a compound of formula (II), B1 is an adenine substituted with X. Where X is -NHC 1-6 Alkyl groups, such as -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, preferably -NHCH3.

[0412] In one embodiment of a compound of formula (II), B1 is optionally subjected to R b Replaced guanine Where R b Selected from H or -C 1-6 Alkyl groups, such as R b The options are -CH3, -CH2CH3, and -CH2CH2CH3, with -CH3 being the preferred option.

[0413] In an embodiment of a compound of formula (II), R1 and R1' are both H, or one of them is H and the other is F, or one of them is H and the other is OH, or both are F.

[0414] In one embodiment of a compound of formula (II), B2 is guanine.

[0415] In an embodiment of a compound of formula (II), R2 and R2' are both H, or one of them is H and the other is F, or one of them is H and the other is OH, or both are F; preferably one of them is H and the other is OH.

[0416] In one embodiment of a compound of formula (II), B1 is adenine. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl; or guanine Where R b -C 1-6 Alkyl group; B2 is guanine. R1 and R1' are both H, or one of them is H and the other is F, or one of them is H and the other is OH; one of R2 and R2' is H and the other is OH.

[0417] The compounds of formula (II) disclosed herein also cover any combination of the above embodiments and their preferred or exemplary embodiments.

[0418] In a further specific embodiment, the drug D1 in the ADC of this disclosure is a bifunctional cyclic dinucleotide (CDN) having the following formula:

[0419]

[0420] B1, B2, R1, R1', R2, and R2' have the meanings defined above for the compound of formula (II) and its various specific embodiments.

[0421] In an embodiment of a compound of formula (II-a) or (II-b), B1 is an adenine optionally substituted with X. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl, or optionally R b Replaced guanine Where R b -C 1-6 Alkyl; for example In this specific embodiment, R1 and R1' are both H, or one of them is H and the other is F, or one of them is H and the other is OH; in this specific embodiment, F or OH and B1 are located on the same side of the ribose.

[0422] In an embodiment of a compound of formula (II-a) or (II-b), B2 is guanine. In this specific embodiment, R2 and R2' are both H, or one of them is H and the other is selected from -OH or F, preferably one of them is H and the other is -OH; in this specific embodiment, F or OH and B2 are located on opposite sides of the ribose.

[0423] In an embodiment of a compound of formula (II-a) or (II-b), B1 is an adenine optionally substituted with X. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl, or optionally R b Replaced guanine Where R b -C 1-6 Alkyl group; B2 is guanine. R1 and R1' are both H, or one of them is H and the other is F, or one of them is H and the other is OH; R2 and R2' are both H, or one of them is H and the other is OH, preferably one of them is H and the other is -OH.

[0424] In one embodiment of a compound of formula (II-a), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F. Further, in each of the specific embodiments, both R2 and R2' are H, or one is H and the other is OH, preferably one is H and the other is -OH.

[0425] In one embodiment of a compound of formula (II-a), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F. Further, in each of the specific embodiments, both R2 and R2' are H, or one is H and the other is OH, preferably one is H and the other is -OH.

[0426] In one embodiment of a compound of formula (II-b), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F; in yet another specific embodiment, one of R1 and R1' is H and the other is OH. Further, in each of these embodiments, both R2 and R2' are H, or one is H and the other is OH, preferably one is H and the other is -OH.

[0427] In one embodiment of a compound of formula (II-b), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F; in yet another specific embodiment, one of R1 and R1' is H and the other is OH. Further, in each of these embodiments, both R2 and R2' are H, or one is H and the other is OH, preferably one is H and the other is -OH.

[0428] In one embodiment of a compound of formula (II-b), B1 is Preferred And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F; in yet another specific embodiment, one of R1 and R1' is H and the other is OH. Further, in each of these embodiments, both R2 and R2' are H, or one is H and the other is OH, preferably one is H and the other is -OH.

[0429] In one embodiment of a compound of formula (II-b), B1 is Where R b -C 1-6 Alkyl group, such as -CH3, -CH2CH3, -CH2CH2CH3, preferably -CH3; and B2 is... In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F; in yet another specific embodiment, one of R1 and R1' is H and the other is OH. Further, in each of these embodiments, both R2 and R2' are H, or one is H and the other is OH, preferably one is H and the other is -OH.

[0430] In one embodiment of a compound of formula (II-b), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, one of R1 and R1' is H and the other is F; in yet another specific embodiment, one of R1 and R1' is H and the other is OH. Further, in each of these embodiments, both R2 and R2' are H, or one is H and the other is OH, preferably one is H and the other is -OH.

[0431] In the compounds of formula (II-a) or (II-b) and their respective embodiments, the two thiophosphate bonds are present in the form of R,R, S,S, S,R or R,S configurations or mixtures thereof; preferably in substantially pure R,R, S,S, S,R or R,S configurations, and particularly preferably in substantially pure R,R configurations.

[0432] The compounds of formula (II-a) / (II-b) disclosed herein also cover any combination between the various embodiments described above and their preferred or exemplary embodiments.

[0433] In a further specific embodiment, D1 in the ADC of this disclosure is a CDN having the following sub-formula.

[0434]

[0435] Wherein B1, B2, R1, R1', R2, R2' have the meanings defined above for compounds of formula (II) or their various specific embodiments; more specifically, they have the meanings defined above for compounds of formula (II-a) / (II-b) or their various specific embodiments.

[0436] In an embodiment of a compound of formula (II-a') or (II-b'), B1 is an adenine optionally substituted with X. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl, or optionally R b Replaced guanine Where R b Selected from -C 1-6 Alkyl; for example In this specific implementation, R1 and R1' are both H, or R1' is H and R1 is F, or R1' is H and R1 is OH.

[0437] In an embodiment of a compound of formula (II-a') or formula (II-b'), when B1 is When both R1 and R1' are H, B1, together with the ribose it is attached to, forms the nucleoside antitumor drug cladribine; or when R1 is F and R1' is H, that is, F and B1 are located on the same side of the ribose, B1, together with the ribose it is attached to, forms the nucleoside antitumor drug clofarabine.

[0438] In an embodiment of a compound of formula (II-a') or formula (II-b'), when B1 is When R1 is OH and R1' is H, B1, together with the ribose it is attached to, forms the nucleoside antitumor drug nerabin.

[0439] In an embodiment of a compound of formula (II-a') or formula (II-b'), when B1 is When R1 is OH and R1' is H, B1, together with the ribose it is attached to, forms the nucleoside antitumor drug fludarabine.

[0440] In an embodiment of a compound of formula (II-a') or (II-b'), B2 is guanine. Both R2 and R2' are H, or R2' is H and R2 is selected from -OH or F, preferably R2' is H and R2 is -OH.

[0441] In an embodiment of a compound of formula (II-a') or (II-b'), B1 is an adenine optionally substituted with X. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl; or optionally Rb Replaced guanine Where R b Selected from -C 1-6 Alkyl group; B2 is guanine. R1 and R1' are both H, or R1' is H and R1 is F, or R1' is H and R1 is OH; R2 and R2' are both H, or R2' is H and R2 is OH, preferably R2' is H and R2 is OH.

[0442] In one embodiment of a compound of formula (II-a'), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H. Further, in each of these specific embodiments, both R2 and R2' are H, or R2' is H and R2 is OH, preferably R2' is H and R2 is OH.

[0443] In one embodiment of a compound of formula (II-a'), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H. Further, in each of these specific embodiments, both R2 and R2' are H, or R2' is H and R2 is OH, preferably R2' is H and R2 is OH.

[0444] In one embodiment of a compound of formula (II-b'), B1 is... And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H; in yet another specific embodiment, R1 is OH and R1' is H. Further, in each of these embodiments, both R2 and R2' are H, or R2 is OH and R2' is H, preferably R2 is OH and R2' is H.

[0445] In one embodiment of a compound of formula (II-b'), B1 is... And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H; in yet another specific embodiment, R1 is OH and R1' is H. Further, in each of these embodiments, both R2 and R2' are H, or R2 is OH and R2' is H, preferably R2 is OH and R2' is H.

[0446] In one embodiment of a compound of formula (II-b'), B1 is... Preferred And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, both R1 and R1' are H; in yet another specific embodiment, R1 is F and R1' is H; in yet another specific embodiment, R1 is OH and R1' is H. Further, in each of these embodiments, both R2 and R2' are H, or R2 is OH and R2' is H, preferably R2 is OH and R2' is H.

[0447] In one embodiment of a compound of formula (II-b'), B1 is Where R b -C 1-6 Alkyl group, such as -CH3, -CH2CH3, -CH2CH2CH3, preferably -CH3; and B2 is... In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H; in yet another specific embodiment, R1 is OH and R1' is H. Further, in each of these embodiments, both R2 and R2' are H, or R2 is OH and R2' is H, preferably R2 is OH and R2' is H.

[0448] In one embodiment of a compound of formula (II-b'), B1 is And B2 is In one specific embodiment, both R1 and R1' are H; in another specific embodiment, R1 is F and R1' is H; in yet another specific embodiment, R1 is OH and R1' is H. Further, in each of these embodiments, both R2 and R2' are H, or R2 is OH and R2' is H, preferably R2 is OH and R2' is H.

[0449] It should be noted that the D1 part of the ADC in this disclosure covers all the above specific embodiments, as well as embodiments consisting of any combination or sub-combination of the above specific embodiments, and embodiments consisting of any combination of any preferred or exemplary embodiments.

[0450] It should be noted that when the CDN(D1) of equations (II), (II-a), (II-b), (II-a'), and (II-b') is covalently bonded to the linker unit (L) in the ADC of equation (X), it is bonded through any of the following sites of the CDN:

[0451] (1) Any -SH; or

[0452] (2) The amino group on the B1 or B2 base; or

[0453] (3) OH on the sugar ring.

[0454] In a preferred embodiment, the CDN (D1) of each of the above formulas is covalently bonded to the linker unit (L) in the ADC of formula (X) via the free thiol group of any of its phosphate esters.

[0455] In some implementations, the CDN (D1) of the above formulas is covalently bonded to the linker unit (L) in the ADC of formula (X) via the thiophosphate ester formed by the OH at position 3 of the nucleotide ring linked by B1 and the hydroxyl group at position 5 of the nucleotide linked by B2.

[0456] The D1 portion of the ADC disclosed herein preferably includes the following compounds, their pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled compounds.

[0457]

[0458]

[0459] It should be noted that this disclosure covers ADC compounds formed by any of the above-described general, specific, or preferred CDN compounds with the linker unit, payload D2, and antibody as defined herein. It should also be noted that the CDN compounds of general or specific examples of this disclosure can be prepared and their structure and activity characterized with reference to the method disclosed in WO2022083584A1.

[0460] Second payload – D2

[0461] The dual-load antibody-drug conjugate disclosed herein, in addition to the first payload as defined above, also covalently binds a second payload through a linker unit. The second payload can be any therapeutic agent compatible with the cGAS-STING pathway, preferably a small molecule compound, and more preferably an antitumor agent.

[0462] In some embodiments, the second payload of the dual-load antibody-drug conjugate of this disclosure is the same as the first payload. For example, the second payload may also be selected from bifunctional CDN STING agonists as defined herein, but may be the same or different specific molecules.

[0463] In other embodiments, the second payload of the dual-load antibody-drug conjugate of this disclosure is different from the first payload, for example, the second payload is selected from other therapeutic agents with different mechanisms of action.

[0464] In some embodiments of this disclosure, D2 in the ADC of this disclosure is selected from the following camptothecin compounds that can be synthesized and characterized based on CN115990269:

[0465]

[0466]

[0467] The following commercially available camptothecin derivatives are preferred:

[0468]

[0469] In some embodiments, the second payload in the ADC of this disclosure is selected from PARP inhibitors, such as the following commercially available PARP inhibitors:

[0470]

[0471] In some embodiments, the second payload in the ADC of this disclosure is selected from nucleoside derivatives, such as the following commercially available nucleoside derivatives:

[0472]

[0473] In some embodiments, the second payload in the ADC of this disclosure is selected from folic acid antagonists, such as the following commercially available folic acid antagonists:

[0474]

[0475] In some embodiments, the second payload in the ADC of this disclosure is selected from microtubule inhibitors, such as the following commercially available microtubule inhibitors:

[0476]

[0477] In some embodiments, the second payload in the ADC of this disclosure is selected from nuclear receptor modulators, such as the following commercially available nuclear receptor modulators:

[0478]

[0479] In some embodiments, the second payload in the ADC of this disclosure is selected from immunomodulators, such as the following commercially available immunomodulators:

[0480]

[0481] In some embodiments, the second payload in the ADC of this disclosure is selected from epigenetic regulators, such as the following commercially available epigenetic regulators:

[0482]

[0483] In some embodiments, the second payload in the ADC of this disclosure is selected from topoisomerase II inhibitors, such as the following commercially available topoisomerase II inhibitors:

[0484]

[0485] In some embodiments, the second payload in the ADC of this disclosure is selected from phosphatase inhibitors, such as the following commercially available phosphatase inhibitors:

[0486]

[0487] In some embodiments, the second payload in the ADC of this disclosure is selected from kinase inhibitors, such as the following commercially available kinase inhibitors:

[0488]

[0489]

[0490]

[0491] In some embodiments, the second payload in the ADC of this disclosure is another antitumor agent, such as the following commercially available antitumor agents:

[0492]

[0493]

[0494] In some embodiments, the second payload in the ADC of this disclosure may be selected from topoisomerase I inhibitors such as camptothecin or derivatives thereof, PARP inhibitors, kinase inhibitors, topoisomerase II inhibitors, nucleoside derivatives, microtubule inhibitors, nuclear receptor modulators, as specifically shown above.

[0495] In a specific implementation, the second payload in the ADC of this disclosure can be selected from...

[0496]

[0497]

[0498] It should be noted that the payloads listed in this article all cover their respective pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels, and each is connected to the linker unit through, for example, hydroxyl, amino, or heterocyclic nitrogen atoms in its structure.

[0499] Connector unit L

[0500] In the ADC described herein, D1 and D2 are linked to an antibody or antigen-binding fragment via a linker unit. The linker unit conjugates D1 and D2 to the antibody by forming a covalent bond with D1 at at least one location, a covalent bond with D2 at at least another location, and a covalent bond with the antibody or antigen-binding fragment at yet another location. The linker unit can be monovalent with respect to D1 or D2, such that a single D1 or D2 is covalently conjugated to the antibody or antigen-binding fragment, or it can be polyvalent with respect to D1 or D2, such that more than one D1 or D2 is covalently conjugated to the antibody or antigen-binding fragment. As used herein, the term "linker unit" is intended to include unconjugated, partially conjugated (i.e., conjugated only to CDN, only to D2, or only to Ab), and fully conjugated forms (i.e., conjugated to both CDN and D2 and Ab).

[0501] The linker unit L suitable for the ADC of this disclosure can be any linker capable of conjugating the drug of this disclosure to an antibody. Suitably, the inclusion of the linker should ensure adequate stability of the ADC of this disclosure in the circulatory system, prevent premature lysis in circulation that could trigger off-target toxicity, and provide rapid and efficient payload release at the target site (e.g., tumor cells or the tumor environment). For example, the linker unit can be stable to the extracellular environment and serum chemistry, or may include intentionally unstable linker units that can release the payload in the extracellular environment or tumor microenvironment.

[0502] In some embodiments, the linker unit includes bonds designed to release the payload after the ADC is internalized within the cell. In some specific embodiments, the linker unit includes bonds designed to be cleaved and / or dissolved or otherwise specifically or nonspecifically degraded within the cell.

[0503] In some embodiments, the linker unit in the ADC of this disclosure is a non-degradable linker. Examples of non-degradable linker units include, but are not limited to, thioether linkers, N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), and maleimide hexanoyl (MC). Typically, such linkers are more stable, and ADCs containing such linkers must be internalized by cells, where the antibody portion of the ADC is degraded by intracellular lysosomal proteases to release the active pharmaceutical ingredient.

[0504] In some further embodiments, the linker unit in the ADC of this disclosure is a degradable linker unit containing one or more chemically or enzymatically degradable chemical bonds. 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).

[0505] In some embodiments, the degradable linker unit is an enzyme-degradable linker unit and is more stable than chemically unstable linkers in plasma and extracellular environments. Such linkers can be peptide-based or include peptide regions, or are non-peptide linkers such as peptide mimics, or sugars, esters, and amides. Such linkers can be cleaved by tumor-specific enzymes, such as tumor-specific proteases that have increased abundance in tumors and / or tumor environments, including but not limited to lysosomal proteases such as cathepsins (e.g., cathepsin B), aspartate endonucleases, plasminogen esterases, or amidases.

[0506] Typically, an enzyme-degradable linker unit can consist of a self-degrading linker, a cleavable linker, an optional property regulation unit, and an antibody linker portion. Self-disintegrating linkers connect the payload to cleavable linkers, facilitating the release of the payload from the rest of the conjugate. Examples include p-aminobenzyl, p-hydroxybenzyl, p-aminobenzyloxyacyl, p-hydroxybenzyloxyacyl, 2-aminomethyl-1-carbonylpiperidine, and 2-aminomethyl-1-carbonylpiperazine. Cleavable linkers, under enzyme-based release mechanisms, will contain enzyme-recognizable peptides or peptide analogs, esters (e.g., carbamates, sulfates), amides, disulfide-containing moieties, sugars, etc. The addition of property-modifying units may be beneficial in improving the properties of the ADC, such as its stability in blood circulation, its efficacy at the target site, and optimizing its hydrophilicity. For example, when the drug is highly hydrophobic, the addition of units such as PEG units or sugar units such as monosaccharides or disaccharides or their derivatives can be considered (but is not necessary) to optimize the hydrophilicity of the ADC, such as reducing precipitation and aggregation. Antibody linkers are used to link the antibody or antigen-binding fragment targeting the antigen to the rest of the conjugate, and they have functional groups that can form bonds with functional groups on the antibody.

[0507] In some embodiments, the connector unit L in the ADC of this disclosure has the following general formula structure:

[0508]

[0509] Among them, G, E, W, P1, P2, A1, A2, S1, and S2 have the meanings defined in Equation (I) above, where the wavy line represents the connection point with the antibody and the asterisk represents the connection point with the payload.

[0510] In some embodiments, depending on the presence or absence of E, S1, and / or S2, the connector unit L in the ADC of this disclosure has the following sub-formula structure:

[0511]

[0512] The following provides general, specific, or preferred embodiments for each possible component of the linker unit L. It should be noted that this disclosure covers linker units L obtained by any combination of the general, specific, or preferred embodiments of each component with the general, specific, or preferred embodiments of any other component; correspondingly, this disclosure covers ADC compounds obtained by any combination of the linker units obtained from the arbitrary combinations with the general, specific, or preferred embodiments of the antibody and drug portions defined in this disclosure.

[0513] G-antibody linker

[0514] The role of the antibody linker is to link the antibody or antigen-binding fragment targeting the antigen to the rest of the ADC. It has functional groups that can form bonds with functional groups on the antibody.

[0515] In some embodiments, the antibody linker in the ADC of this disclosure is linked to the sulfur atom of the antibody; in other embodiments, the antibody linker in the ADC of this disclosure is linked to the nitrogen atom of the antibody; and in still other embodiments, the antibody linker in the ADC of this disclosure is linked to the carbon atom of the antibody. Preferably, in the ADC of this disclosure, the antibody linker is linked to the sulfur atom of the antibody.

[0516] In a specific implementation, G in the ADC of this disclosure has the following structure: -G1-G2-,

[0517] in:

[0518] G1 is selected from:

[0519] • 5-10 membered heterocyclic group, containing one or two heteroatoms selected from N, S and O, wherein the ring carbon atom is optionally oxidized;

[0520]

[0521] The asterisk (*) on the left indicates the binding point with the antibody, and the asterisk (*) on the right... Indicates the connection point with G2;

[0522] G2 is selected from -C 1-10 Alkylene -C(=O)-, -C 1-10 Alkylene-C(=O)-NH-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, wherein G2 is connected to the property regulating unit E (when present) or the branching unit W (when E is absent) via its -C(=O-), and the other end group is connected to G1, or

[0523] G2 is selected from -C 1-10Alkylene-NH-, wherein G2 is connected to property regulating unit E (when present) or branching unit W (when E is absent) via its -NH-, and the other end group is connected to G1;

[0524] Among them, -C in G2 1-10 Alkylene groups are optionally substituted with Bu groups.

[0525] In some implementations, -C in G2 1-10 Alkylene groups are optionally substituted with a Bu group selected from the following: H, deuterium, halogen, NO2, CN, -OR. h -OR h -N(R) h )2、-COR h -CO2R h -C-(O)C(O)R h -C(O)CH2C(O)R h -S(O)R h -SO2R h C(O)N(R) h )2、-SO2N(R h )2、-OC(O)R h -N(R) h SO2R h and -C optionally substituted with prostate groups 1-6 Alkyl, wherein R h H or -C optionally substituted with halogen 1-6 Alkyl groups, or two R atoms attached to the same N atom. h The groups and the nitrogen atoms they are attached to form 4-7 membered heterocyclic groups; preferably, the substituents of G2 are aminoalkyl moieties, such as -(CH2). 1-6 NH2、-(CH2) 1-6 NHR h Or -(CH2) 1-6 N(R h )2, or two R atoms connected to the same N atom h The groups and the nitrogen atoms they are attached to form nitrogen heterocyclic butyl, pyrrolidinyl, or piperidinyl groups.

[0526] In other implementations, -C in G2 1-10 The alkylene group is optionally substituted with a Bu group of the following formula (G):

[0527]

[0528] in,

[0529] T0 is -C 1-6 alkylene-;

[0530] T is selected from -C(R) a )2-、-O-、-NR a -or does not exist;

[0531] M is selected from -CO-, -O-, -NR a -or does not exist;

[0532] Z is selected from glycosyl groups or their derivatives. and contain Hydrophilic peptides, preferably glycosyl groups or their derivatives, and containing Hydrophilic peptides;

[0533] R a Selected from H or C 1-6 alkyl;

[0534] Subunit Selected from natural and non-natural amino acid residues;

[0535] n is an integer from 0 to 4, for example 0-2, 1-2, 1-4, 2-4;

[0536] m is an integer from 0 to 20, for example 0-8, 0-6, 0-4, 0-2, 1-20, 1-10, 2-10, 2-8, 2-6, 2-4, 4-14, 4-12, 4-10, 5-10, 6-10, 6-12, 8-10, 8-14.

[0537] In some Bu implementations, m is 1-20, 1-10, 2-10, 2-8, 2-6, 2-4, 4-14, 5-10, 6-10, 6-12, 8-10, 8-14, such as 4-14, 6-12, or 5-10.

[0538] In some Bu implementations, T0 is -C 1-3 Alkylene, preferably methylene.

[0539] In some Bu implementations, T is -C(R) a )2-, for example, methylene.

[0540] In some Bu implementations, T stands for -O-.

[0541] In some Bu implementations, T is -NR a -, for example -NH-.

[0542] In some Bu implementations, T is absent.

[0543] In some Bu implementations, M stands for -CO-.

[0544] In some Bu implementations, M is -O-.

[0545] In some Bu implementations, M is -NR a -, for example -NH-.

[0546] In some Bu implementations, M is absent.

[0547] In some Bu implementations, T0 is -C 1-6 Alkylene; T is -O- or -NR a - M does not exist; n is 0.

[0548] In some Bu implementations, T0 is -C 1-6 Alkylene; T is absent; M is -O- or -NR a -; n is 0.

[0549] In some Bu implementations, T0 is -C 1-6 Alkylene; T is -O- or -NR a -; M is -CO-; n is 0.

[0550] In some Bu implementations, T0 is -C 1-6 Alkylene; T is absent; M is -CO-; n is 0.

[0551] In some Bu implementation schemes, R a For H, in some other Bu implementations, R a C 1-6 Alkyl, such as C 1-3 Alkyl group, preferably methyl group.

[0552] In any of the above-described Bu embodiments, Z may be a glycosyl group or its derivative, as generally, specifically, or preferably defined as a carbonyl group or its derivative in the “S1 or S2—Solubilizing Unit” section below; preferably selected from monosaccharides, disaccharides, or their derivatives as defined therein, more preferably pentose sugars, hexose sugars or their uronic acids, amino sugars, acylated amino sugars, aminouronic acids or their derivatives, and even more preferably glucose, mannose, galactose or their respective derivatives.

[0553] In any of the above Bu implementation schemes, Z can be a subunit containing repeating subunits. The hydrophilic peptide, wherein the amino acid residues serving as repeating subunits can be any of the 20 conventional natural amino acids, and correspondingly, the combinations of R, R', and R” values ​​in the repeating subunit -CO-CR'R”-NR- correspond to the values ​​of alanine (Ala), arginine (Arg), aspartic acid (Asp), asparagine (Asn), histidine (His), glycine (Gly), glutamic acid (Glu), glutamine (Gln), phenylalanine (Phe), lysine (Lys), leucine (Leu), serine (Ser), tyrosine (Tyr), threonine (Thr), isoleucine (Ile), proline (Pro), tryptophan (Trp), valine (Val), cysteine ​​(Cys), and methionine (Met), preferably corresponding to the values ​​selected from arginine, serine, threonine, tyrosine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, histidine, glycine, and tryptophan. The amino acid residues that serve as subunits can also be amino acids other than the conventional 20 natural amino acids, such as ornithine (Orn), β-alanine (β-Ala), citrulline (Cit), sarcosine (Sar), and those amino acids in the repeating subunit -CO-CR'R”-NR- whose R, R', and R” are different from the corresponding groups or segments in the 20 amino acids.

[0554] In some embodiments, the hydrophilic peptide may be a repeat of a single amino acid unit, such as polysarcosine, polyarginine, and polyglycine; in other embodiments, the hydrophilic peptide may also be a mixed repeat of different subunits, for example, the hydrophilic peptide may be a mixed hydrophilic peptide containing the natural amino acid and the unconventional amino acid.

[0555] In a preferred embodiment, the amino acid units of the hydrophilic peptide are selected from sarcosine, arginine, and glycine.

[0556] In some specific embodiments of the hydrophilic peptide, the hydrophilic peptide is, for example but not limited to, linked via a carboxyl terminus. Or linked via an amino terminus Preferably, it is linked through a carboxyl terminus. Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably H or -CH3; preferably polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0557] In some specific hydrophilic peptide implementation schemes, it includes The hydrophilic peptide is Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably H or -CH3, more preferably -CH3; preferably, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0558] In some specific hydrophilic peptide implementation schemes, it includes The hydrophilic peptide is Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably H or -CH3, more preferably -CH3; preferably, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0559] In some specific hydrophilic peptide implementation schemes, it includes The hydrophilic peptide is Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably H or -CH3, more preferably -CH3; preferably, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0560] In some Bu implementations, Z is selected from glycosyl groups or their derivatives and contains... Hydrophilic peptides; preferably selected from glucose, mannose, galactose, lactose, maltose, sucrose or their respective uronic acids, amino sugars, acylated amino sugars, aminouronic acids or acylated aminouronic acid derivatives, and hydrophilic peptides as generally or specifically defined herein, specifically hydrophilic peptides comprising 4-14 units or 4-10, 5-10 units of polysarcosine, polyarginine, polyglycine, preferably 4-12, 6-12 units or 4-10, 5-10 units of polysarcosine.

[0561] In some specific Bu implementation schemes, equation (G) is Preferred Where T0 is -C 1-3 Alkylene, preferably methylene.

[0562] In some specific Bu implementation schemes, equation (G) is Preferred Where T0 is -C 1-3 alkylene, preferably methylene, Z is Wherein R and m are defined as generally or specifically as above; specifically, the segment indicated by square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0563] In some specific Bu implementation schemes, equation (G) is Preferred Where T0 is -C 1-3 alkylene, preferably methylene, Z is Among them, R, R a , m are as generally or specifically defined above; more specifically, the segment indicated in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10, 5-10 units, more preferably polysarcosine containing 4-12, 6-12 units or 4-10, 5-10 units.

[0564] Alternatively, in some specific Bu implementation schemes, equation (G) is: Preferred, Where T0 is -C 1-3 alkylene, preferably methylene, Z is Among them, R, R a, m are as generally or specifically defined above; more specifically, the segment indicated in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10, 5-10 units, more preferably polysarcosine containing 4-12, 6-12 units or 4-10, 5-10 units.

[0565] In any of the above Bu implementation schemes, Z can also be

[0566] It should be noted that the context of this disclosure involves hydrophilic peptides in the definition of multiple structural fragments, and their definitions are all applicable to the definition given herein for the fragment of formula (G).

[0567] In some embodiments, G1 is a 5-10 membered heterocyclic group, preferably a 5-6 membered heterocyclic group containing one or two heteroatoms selected from N, O, and S, such as a 5-6 membered heterocyclic alkyl or 5-6 membered heteroaryl group, wherein the ring carbon atom is optionally oxidized, for example, but not limited to The asterisk (*) on the left indicates the connection point with the antibody, and the wavy line on the right indicates the connection point with G2.

[0568] In some implementations, G1 is selected from It is the acid-amide moiety formed after partial hydrolysis of succinimide.

[0569] In a preferred embodiment, G1 is

[0570] In some implementations, G2 is -C 1-10 Alkylene-C(=O)-, preferably -C 1-5 Alkylene-C(=O)-, more preferably-(CH2)5-C(=O)-.

[0571] In other implementations, G2 is -C 1-10 Alkylene-C(=O)-, preferably -C 1-5 Alkylene -C(=O)-, more preferably -(CH2)5-C(=O)-, wherein the alkylene group is substituted with a Bu group of formula (G) as defined above, preferably formula (G) is Where T0 is -C 1-3 alkylene, preferably methylene, Z is Among them, R, R a , m, each as generally or specifically defined above; more specifically, R, R a Each independently is C 1-6Alkyl group, preferably -CH3, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0572] In specific implementation schemes, -G1-G2- can be the following groups:

[0573]

[0574] And the corresponding fragment in which the methylene group is replaced by the Bu group of formula (G) as generally or preferably defined above, wherein the left * indicates the linking point with the rest of the antibody, and the right... This indicates the connection point with property adjustment unit E (when it exists) or branch unit W (when E does not exist).

[0575] In some specific implementations, -G1-G2- has the following structure: Preferably More Where T0 is -C 1-3 alkylene, preferably methylene, Z is Preferred Among them, R, R a , m, each as generally or specifically defined above; more specifically, R, R a Each independently is C 1-6 Alkyl group, preferably -CH3, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0576] In an exemplary embodiment, G(-G1-G2-) in the ADC of this disclosure has the following structure:

[0577]

[0578]

[0579]

[0580] In some preferred embodiments, G in the ADC of this disclosure has the following structure:

[0581] E—Property Adjustment Unit The addition of the property regulation unit E may be beneficial in improving the properties of the ADC, such as stability in blood circulation, improved hydrophilicity, leading to a decrease in clearance and an increase in exposure.

[0582] The property-regulating unit E exists in series within the linker unit. It can be directly connected between G and W, or connected via another linking group, such as amino, carbonyl, alkyl carbonyl, amide, ester, urea, disulfide bridge, carbamate, hydrazone, imine, oxime, triazolyl, maleimide, alkenyl, alkynyl, or alkylene. For example, E can be linked via -NH-, -C(=O)-, or -C-. 1-4 Alkylene, -C 1-4 Alkylene -C(=O)-, -NH-C(=O)-(CH2OCH2)-C(=O)-, -C 1-4 Alkylene -NH-C(=O)-(CH2) 1-4 -O-(CH2) 1-4 -C(=O)-、-C 1-4 Alkylene-NH-(CH2) 1-4 -O-(CH2) 1-4 -C(=O)- connects between G and W.

[0583] In some implementations, E is absent.

[0584] In some embodiments, E is selected from polyamines, such as, but not limited to, polyethyleneimine, polylysine, spermine, dimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary ammonium salts of polyamines, and α-helical peptides.

[0585] In some implementations, E is selected from peptides, preferably hydrophilic peptides.

[0586] In some implementations, E stands for cyclodextrin unit.

[0587] In some implementations, E is polyamide.

[0588] In some implementations, E is a polysaccharide, a dendritic polymer, or a bifunctional hydrocarbon chain.

[0589] In some implementations, E is polyethylene glycol (PEG).

[0590] In some implementations, E is -NH-SO2-NH-.

[0591] In some embodiments, the ADC of this disclosure may comprise at least two ethylene oxide (CH2CH2O) subunits forming a PEG chain as a property-modifying unit. The PEG chain may be, for example, linear, branched, or star-shaped. Typically, the PEG chain is covalently linked in tandem between the antibody linker G and the branching unit W of the linker unit. The connections between the PEG chain and the components within the linker unit are cleavable bonds, which may be bonds that are substantially insensitive to cleavage during circulation in plasma but sensitive to cleavage in the intracellular or tumor intracellular environment. Exemplary connections include, but are not limited to, amide bonds, ether bonds, ester bonds, hydrazone bonds, oxime bonds, disulfide bonds, peptide bonds, or triazole bonds.

[0592] The method of attaching a PEG unit to the connector unit of an ADC is well known to those skilled in the art.

[0593] In some embodiments, the PEG unit has 2-12 ethylene oxide (CH2CH2O) subunits, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 ethylene oxide (CH2CH2O) subunits, for example, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 12, 6 to 10, 6 to 8, 8 to 12, or 8 to 10 ethylene oxide (CH2CH2O) subunits. In a preferred embodiment, the PEG unit comprises at least 2, at least 4, at least 6, at least 8, at least 10, or at least 12 ethylene oxide (CH2CH2O) subunits.

[0594] Exemplary implementations of PEG fragments linked to antibody linker G and branching unit W include, but are not limited to, the following:

[0595]

[0596]

[0597] The wavy line on the left indicates the connection point with G2, and the wavy line on the right indicates the connection point with W. Each subscript c is independently selected from an integer between 2 and 12. In some embodiments, c is 2, 4, 8, or 12. In some embodiments, c is 2. In some embodiments, c is 4. In some embodiments, c is 8. In some embodiments, c is 12.

[0598] Branch unit W

[0599] A branching unit is any small molecule with three or more identical or different functional groups that allows for the binding of antibodies and at least two payloads. It can be selected from amino acids (including natural and non-natural amino acids) and their derivatives, sugar units and their derivatives, aromatic-substituted compounds and their derivatives, substituted hydrocarbon groups and their derivatives, amino-containing derivatives, triazole-containing derivatives, and disulfide-substituted compounds.

[0600] In some embodiments, the branch unit W in the ADC of this disclosure is an amino acid residue, for example...

[0601] In some implementations, the branch unit W in the ADC of this disclosure is a structural fragment of the following general formula:

[0602]

[0603] W is selected from O, S, and NR. c CH 2- Or it may not exist;

[0604] R a and R c Each is independently selected from H, or optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 alkenyl;

[0605] y 1 y 2 Each can be independently 0, 1, 2, 3, 4, 5, or 6;

[0606] For example, the central N-terminus is connected to the property regulation unit E or the antibody linker G (when it is absent), and the two carboxyl ends are connected to the cleavable linker P1 or P2, respectively.

[0607] In some other embodiments, the branch unit W in the ADC of this disclosure may include or be selected from the following structures:

[0608]

[0609] In some implementations, the branching unit is connected to either the property regulation unit E (when present) or the antibody linker G (when E is absent) via one of its N-termini.

[0610] Preferably, the branch unit W is an amino acid residue, more preferably containing a structural fragment. It is connected to either the property regulation unit E (when present) or the antibody linker G (when E is absent) through one of its N-termini.

[0611] P1 or P2 — Cuttable connector

[0612] On the one hand, the cleavable linker P1 or P2 in the linker unit of the ADC disclosed herein is an enzyme-cleavable unit, that is, a substrate that can be cleaved or broken by an enzyme. In order to limit or minimize the off-target toxicity of the ADC while ensuring the release of the payload at the target tumor site, the cleavable linker is designed to be a substrate that is specifically cleaved by an enzyme present near or inside the target cell. Preferably, the level or activity of the enzyme near or inside the target cell is higher than the level or activity in other parts of the body, thereby ensuring that the ADC is specifically recognized, cleaved and released by the enzyme in the target cell / tissue to exert its biological function.

[0613] Therefore, the ADCs of this disclosure are stable in biological fluids until they reach their target, such as tumor cells / tissue. Due to the specific release of the ADCs, administration of the ADCs of this disclosure results in less toxicity and maximized activity compared to administration of a single payload.

[0614] In some embodiments, P1 or P2 in the linker unit of the ADC of this disclosure is a dipeptide, tripeptide, or oligopeptide composed of an amino acid sequence that is specifically recognized and cleaved by a protease, preferably specifically recognized and cleaved by a non-mammalian protease or endogenous mammalian protease present in or near the target cell, such enzymes as, but not limited to: β-APP-cleaving enzyme 1 (BACE1), cathepsin D (CTSD), calpain-1 (CAPN1), aspartate endonuclease (Lugemain), caspase 1 (Casp1), caspase 2 (Casp2), caspase 3 (Casp3), etc. Caspase 5 (CASPS), Caspase 6 (Casp6), Caspase 7 (Casp7), Caspase 8 (Casp8), Caspase 9 (Casp9), Cathepsin B (CTSB), Cathepsin K (CTSK), Cathepsin L (CTSL), Cathepsin S (CTSS), Esterases (e.g., cholinesterase, alkaline phosphatase, phosphodiesterase, sulfatase), Amideases, Angiotensin I Converting Enzyme (ACE), Angiotensin I Converting Enzyme 2 (ACE2), ADAM Metallopeptidase Domain 10 (ADAM10), Dipeptidyl Peptidase 3 (DPP3), Pancreatic Insulin-degrading enzyme (IDE), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 12 (MMP12), matrix metalloproteinase 13 (MMP13), matrix metalloproteinase 14 (membrane insertion) (MMP14), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 7 (MMP7), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), membrane metalloendopeptidase (NEPRILYSIN) (MME), ADAM metallopeptidase domain 17 (TACE) (ADAM17), dipeptidyl peptidase 4 (DPP4) Dipeptidyl peptidase 8 (DPP8), dipeptidyl peptidase 9 (DPP9), coagulation factor Xa (factor Xa), coagulation factor VILA (factor VII), fibroblast activation protein α (FAP), furin (paired basic amino acid cleaving enzyme), granzyme A (granzyme 1, cytotoxic T lymphocyte-associated (GZMA), serine esterase 3), granzyme B (granzyme 2, cytotoxic T lymphocyte-associated (GZMB), serine esterase 1), granzyme K (granzyme 3, trypsin II) (GZMK), kallikrein-1 (KLK1), kallikrein-2 (KLK2), plasma kallikrein (PSA),KLK3), kallikrein-11 (KLK11), kallikrein-13 (KLK13), kallikrein-15 (KLK15), matrix enzyme (ST14), spinesin (TMPRSSS), plasmin (PLG), prolyl oligopeptidase (PREP), thrombin (F2), tPA, plasminogen activator tissue type (PLAT), UPA, plasminogen activator urokinase (PLAU), HtrA serine peptidase 2 (hTRA2), casein hydrolysate mitochondrial matrix peptidase proteolytic subunit (CIPP / X), constitutive proteasome chymotrypsin-like form (PSMBS, β5), constitutive proteasome trypsin-like form (PSMB7, β7), constitutive proteasome caspase-like form (PSMB6, β6), immunoproteasome chymotrypsin-like form (PSMB8, LMP7), immunoproteasome trypsin-like form (PSMB, β5). 10 MECL1), immunoproteasome caspase-like forms (PSMB9, LMP2).

[0615] In some embodiments, P1 or P2 in the linker unit of the ADC of this disclosure is specifically recognized and cleaved by cathepsin B, such as Val-Cit, Val-Ala, Gly-Gly-Phe-Gly, and Gly-Phe-Leu-Gly.

[0616] In some embodiments, P1 or P2 in the linker unit of the ADC disclosed herein is specifically recognized and cleaved by an aspartate endonuclease (Legumain), such as Ala-Ala or Ala-Ala-Asn.

[0617] In some embodiments, P1 or P2 in the linker unit of the ADC of this disclosure is specifically recognized and cleaved by, for example, high concentrations of matrix metalloproteinase 2 / 9 (MMP-2 / 9) in tumors, such as Pro-Leu-Gly-Leu-Ala-Gly (PLGLAG), Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln (GPLGIAGQ), Gly-Pro-Val-Gly-Leu-Ile-Gly-Lys (GPVGLIGK).

[0618] In some embodiments, P1 or P2, which is part of the linker unit of the ADC of this disclosure, is a cleavable peptide containing two or more (e.g., 2-12) consecutive or discontinuous amino acids, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptide units, or composed thereof. Each amino acid of the peptide unit may be independently selected from natural or non-natural amino acids and / or D- or L-isomers, provided that P1 or P2 contains a cleavable bond that, upon cleavage, triggers the release of the payload. In some embodiments, P1 or P2 consists only of natural amino acids. In other embodiments, P1 or P2 consists only of non-natural amino acids. In some embodiments, P1 or P2 consists of natural amino acids attached to non-natural amino acids. In some embodiments, P1 or P2 consists of natural amino acids attached to D-isomers of natural amino acids.

[0619] In some embodiments, each amino acid is independently an L-(natural) amino acid. In another embodiment, each amino acid is independently selected from the D-isomer of the said natural amino acid.

[0620] In some embodiments, P1 or P2, or the cleavable peptide contained therein, has the structure of formula (P) from the N-terminus to the C-segment: -(AA) d -(P) Where AA is an amino acid selected from alanine (Ala), arginine (Arg), aspartic acid (Asp), asparagine (Asn), histidine (His), glycine (Gly), glutamic acid (Glu), glutamine (Gln), phenylalanine (Phe), lysine (Lys), substituted lysine, leucine (Leu), serine (Ser), tyrosine (Tyr), threonine (Thr), isoleucine (Ile), proline (Pro), tryptophan (Trp), valine (Val), cysteine ​​(Cys), methionine (Met), selenocysteine, ornithine (Asn), β-alanine (β-Ala), citrulline (Cit) and their derivatives;

[0621] Preferably, AA is selected from alanine, glycine, asparagine, isoleucine, leucine, valine, phenylalanine, citrulline, and glutamic acid; more preferably, AA is selected from alanine, glycine, asparagine, valine, phenylalanine, citrulline, and glutamic acid.

[0622] d is an integer from 2 to 12; preferably an integer from 2 to 8, more preferably an integer from 2 to 4;

[0623] The individual amino acids (AAs) are linked together by peptide bonds.

[0624] In some implementations, the N-terminus of the cleavable peptide contained in P1 or P2 is connected to the branching unit W, and the C-terminus is connected to the self-decomposing linker A1 or A2 portion of the linker unit.

[0625] In some implementations, d is an integer from 2 to 8, such as 2 to 6, 2 to 4, or 2 to 3, such as 2, 3, 4, 6, 8.

[0626] In some implementations, -(AA) d - In this context, d is 2, and P1 or P2 is a dipeptide, for example, selected from the following dipeptides: Ala-Ala, Ala-(D)Asp, Ala-Cit, Ala-Lys, Val-Ala, Asn-Cit, Asp-Cit, Asn-Lys, Asn-(D)Lys, Asp-Val, Cit-Ala, Cit-Asn, Cit-Asp, Cit-Cit, Cit-Lys, Cit-Ser, Cit-Val, Glu-Val, Glu-Gly, Phenylene Gly-(D) Lys, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Lys-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, Phe-Arg, Phe-Cit, Phe-Lys, Pro-(D)Lys, Ser-Cit, Trp-Cit, Val-Ala, Val-(D)Asp, NorVal-(D)Asp, Val-Cit, Val-Glu, Val-Lys, and their salts. In one specific embodiment, the dipeptide is Ala-Ala. In one specific embodiment, the dipeptide is Val-Cit. In another specific embodiment, the dipeptide is Val-Ala. In yet another specific embodiment, the dipeptide is Glu-Gly.

[0627] In some implementations, -(AA) dIn this context, d is 3, and P1 or P2 is a tripeptide, such as Gly-Gly-Gly, Gly-Gly-Arg, Phe-Lys-Gly, Leu-Lys-Gly, Leu-Leu-Gly, Glu-Val-Cit, Cit-Ala-Glu, Val-Lys-Gly, Val-Lys-Ala, Val-Gly-Gly, Val-Cit-Gly, Val-Gln-Gly, Val-Glu-Gly, Val-Lys-Gly, Val-Lys-Leu, Ala-Ala-Ala, Ala-Ala-Asn. In one specific embodiment, the tripeptide is Ala-Ala-Asn. In one specific embodiment, the tripeptide is Glu-Val-Cit. In one specific embodiment, the tripeptide is Cit-Ala-Glu.

[0628] In some implementations, -(AA) d In this context, d is 4, and P1 or P2 is a tetrapeptide, such as Gly-Gly-Gly-Gly, Gly-Gly-Leu-Gly, Gly-Phe-Leu-Gly, Gly-Val-Lys-Gly, A1a-Leu-A1a-Leu, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly, and Val-Lys-Gly-Gly. In one specific embodiment, the tetrapeptide is Gly-Phe-Leu-Gly. In another specific embodiment, the tetrapeptide is Gly-Gly-Phe-Gly.

[0629] In some implementations, -(AA) d - In this context, d is 6 or 8, and P1 or P2 is a hexapeptide or octapeptide, for example, Pro-Leu-Gly-Leu-Ala-Gly (PLGLAG), Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln (GPLGIAGQ), and Gly-Pro-Val-Gly-Leu-Ile-Gly-Lys (GPVGLIGK).

[0630] Preferably, the peptides in the specific examples above are connected to the branching unit W at the N-terminus and to the self-decomposing linker A1 or A2 of the linker unit at the C-terminus.

[0631] In some embodiments, P1 or P2 is a peptide containing a substituted lysine, such as a dipeptide, tripeptide, tetrapeptide, hexapeptide, or octapeptide. In one embodiment, the substituted lysine is:

[0632]

[0633] Among them, R 15 and R 15 'Selected independently from: H, C 1-6 Alkyl, -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 Halogenated alkyl groups and C 3-6 Cycloalkyl.

[0634] In a preferred embodiment, P1 or P2 is selected from the following peptides:

[0635]

[0636] The carbonyl end is connected to the self-decomposing linker A1 or A2, and the N end is connected to the branching unit W.

[0637] A1 or A2 — Self-decomposing connector

[0638] The self-decomposable connectors A1 or A2 in the connector unit L of the ADC disclosed herein may be absent, i.e., the cuttable connectors are directly covalently connected to the payload, or they may be functional groups that promote the connection between the cuttable connectors and the payload, or functional groups that provide additional structural components to further promote the release of the payload from the rest of the ADC.

[0639] In some embodiments, the self-decomposing linkers A1 and / or A2 of the ADC of this disclosure are absent, allowing the cleavage linker to be directly covalently connected to the payload. For example, the carboxyl terminus of the cleavage linker can be linked to an amino or OH group present in the payload via an amide bond or an ester bond.

[0640] In some embodiments, the self-decomposable connectors A1 and / or A2 of the ADC disclosed herein are functional groups that facilitate the connection of the cleavable connectors to the payload, or may provide additional structural components to further facilitate the release of the payload from the ADC. Specifically, such A1 or A2 may be selected from:

[0641]

[0642] in,

[0643] The sites marked with * are the connection points to the payload, and the bonds indicated by the wavy lines are connected to the cuttable connectors;

[0644] Each occurrence of Y1 is independently selected from NH or O;

[0645] Each occurrence of R 16 Each is independently selected from H and -C 1-6Alkyl groups, preferably H or C 1-3 Alkyl, more preferably H or methyl;

[0646] Each occurrence of R 17 Each is independently selected from H, -NO2, -NH2, and -CF3.

[0647] In some implementation schemes, R 16 For H. In other implementations, R 16 -C 1-6 Alkyl, preferably C 1-3 Alkyl, more preferably methyl. R is preferred. 16 For H.

[0648] In some implementation schemes, R 17 For H.

[0649] In some implementations, A1 or A2 is selected from...

[0650] In some implementations, A1 is selected from And A2 is selected from

[0651] In some implementations, as A1 or A2 Examples include

[0652] In some implementations, as A1 or A2 Examples include

[0653]

[0654] In some implementations, as A1 or A2 for

[0655] In a preferred embodiment, A1 or A2 is selected from...

[0656] S1 or S2 — Solubilizing unit

[0657] In the ADC of this disclosure, the solubilizing unit S1 or S2 is connected to the self-decomposing linker A1 or A2 when present. In the ADC of this disclosure, the introduction of solubilizing groups into the linker unit helps to improve the hydrophilicity of the conjugated chain, increase the loading rate to the theoretical maximum value, improve the solubility and purity of the conjugate, reduce the aggregation degree, improve the drug-likeness of the conjugate, and ultimately enhance the inhibitory effect on tumor cell proliferation.

[0658] In some embodiments, the ADC of this disclosure does not include S1 and S2. In some embodiments, the ADC of this disclosure includes S1 but does not include S2. In some embodiments, the ADC of this disclosure includes only S2.

[0659] When the ADC of this disclosure includes S1 and / or S2, it can be connected to A1 and / or A2 in the ADC of this disclosure, and both A1 and A2 are selected from... S1 or S2 can be attached to the benzene ring, i.e.

[0660] In a specific implementation, S1 or S2 has the structure of the following formula (S): Where T' is selected from -C(R) a )2-、-O-、-NR a -or does not exist;

[0661] M' is selected from -CO-, -O-, -NR a -or does not exist;

[0662] Z' is selected from glycosyl groups or their derivatives. and contain Hydrophilic peptides;

[0663] R a Selected from H or C 1-6 alkyl;

[0664] Subunit Selected from natural and non-natural amino acid residues;

[0665] n' is an integer from 0 to 4, for example 0-2, 1-4, 1-2, 2-4;

[0666] m is an integer from 0 to 20, for example 0-8, 0-6, 0-4, 0-2, 1-20, 1-10, 2-10, 2-8, 2-6, 2-4, 4-14, 4-12, 4-10, 5-10, 6-10, 6-12, 8-10, 8-14.

[0667] In some embodiments of formula (S), m is an integer from 0 to 10, such as 0-8, 0-6, 0-4, 0-2, 2-10, 2-8, 2-6, 2-4. In other embodiments of formula (S), m is 1-20, 1-10, 2-10, 2-8, 2-6, 2-4, 4-14, 4-12, 4-10, 5-10, 6-10, 6-12, 8-10, 8-14, such as 4-14, 4-12, 6-12, 4-10, or 5-10.

[0668] In some implementations of formula (S), T is -C(R) a)2-, for example, methylene.

[0669] In some implementations of formula (S), T is -O-.

[0670] In some implementations of formula (S), T is -NR. a -, for example -NH-.

[0671] In some implementations of formula (S), T does not exist.

[0672] In some implementations of formula (S), M is -CO-.

[0673] In some implementations of formula (S), M is -O-.

[0674] In some implementations of formula (S), M is -NR. a -, for example -NH-.

[0675] In some implementations of formula (S), M is absent.

[0676] In some implementations of formula (S), R a For H, in some other implementations of formula (S), R a C 1-6 Alkyl, such as C 1-3 Alkyl group, preferably methyl group.

[0677] In some embodiments of formula (S), when S1 and / or S2 are present in the ADC of this disclosure, Z' is directly connected to the benzene ring of A1 or A2, i.e., neither T' nor M' exists, and n' is 0, such as... As shown. In specific embodiments of this type, Z' is, for example, a glycosyl group or its derivative as generally or specifically defined herein, linked to a benzene ring via a glycosidic bond, a monosaccharide, a disaccharide, or its derivative, more preferably a pentose or hexose or its uronic acid, amino sugar, acylated amino sugar, aminouronic acid or its acylated aminouronic acid derivative, and even more preferably glucose, mannose, galactose or each of the aforementioned derivatives.

[0678] In some implementations of formula (S), when S1 and / or S2 in the ADC of this disclosure are present, Z' is obtained through a segment in formula (S). A fragment attached to a benzene ring of A1 or A2, wherein both T' and M' are present, can be selected from... Alternatively, if T' exists and M' does not exist, the fragment can be selected from... Alternatively, if T' does not exist and M' exists, the fragment can be selected from... Where n' is defined above, preferably 1-2, R aH is preferred.

[0679] In some embodiments of formula (S), Z' is a glycosyl group or a derivative thereof.

[0680] In some embodiments of formula (S), when Z' is a glycosyl group or its derivative and is linked by a glycosidic bond, or when Z is an amino sugar and is linked by an amino group, the above-described... M' in the equation may not exist, for example, M' is not present. For example For example Or M is -CO-, for example, For example For example In this case, Z' is an -O-sugar residue, an -NH-sugar residue, or a derivative thereof.

[0681] In some embodiments of formula (S), when Z' is a uronic acid or a derivative thereof and is linked to M by a carboxyl group, the above-described... M' can be -O or -NR-, in which case Z' is a -CO-uronic acid residue or its derivative.

[0682] In the ADC of this disclosure, the glycosyl group or its derivative in the antibody linker or solubilizing unit is selected from monosaccharide, disaccharide, oligosaccharide or polysaccharide or its derivative, preferably monosaccharide or disaccharide or its derivative, more preferably pentose monosaccharide, hexose monosaccharide or disaccharide formed by their linkage, or their derivatives.

[0683] In some embodiments, the glycosyl group in the ADC of this disclosure can be linked to the solubilizing unit via any of its free hydroxyl groups, or derived amino or carboxyl groups, for example via the OH group at position 1 of the glycosyl group, the carboxyl group of a uronic acid, or the amino group of an amino sugar.

[0684] The aforementioned monosaccharides and their derivatives include their D-configuration, L-configuration, racemic (DL), and meso-configuration, preferably the D-configuration, and also include forms with arbitrary optical activity ((+), (-), (±)). The monosaccharides and their derivatives described in this disclosure include their chain isomers, cyclic isomers, or mixtures thereof, wherein the cyclic forms include α-terminal isomers and β-terminal isomers, or mixtures thereof, and include, for example, pyranose or furanose forms.

[0685] In some embodiments, the glycosyl group in the ADC of this disclosure is linked to the solubilizing unit via a glycosidic bond, for example via a glycosidic bond at position 1 of the glycosyl group, the configuration of which may be α-type, β-type, or a mixture thereof.

[0686] In a preferred embodiment, the monosaccharide in the ADC of this disclosure is selected from glucose, mannose, galactose, or their respective uronic acid, amino sugar, acylated amino sugar, aminouronic acid, or acylated aminouronic acid forms, such as glucose, galactose, mannose, glucosamine, galactosamine, mannosamine, galacturonic acid, glucuronic acid, mannosamine, glucosamine uronic acid, galactosamine uronic acid, mannosamine uronic acid, acetaminophen, acetaminoglucosamine, acetaminophen, acetaminophen, acetaminophen, acetaminophen, acetaminophen, acetaminophen, acetaminophen, acetaminophen, acetaminophen, acetaminophen, acetaminophen; D-glucose and D-galactose are further preferred. D-mannose, D-glucosamine, D-galactosamine, D-mannosamine, D-galacturonic acid, D-glucuronic acid, D-mannuronic acid, D-glucosamineuronic acid, D-galactosamineuronic acid, D-mannosamineuronic acid, 2-acetamino-2-deoxy-D-galactose, 2-acetamino-2-deoxy-D-glucose, 2-acetamino-2-deoxy-D-mannose, 2-acetamino-2-deoxy-D-galacturonic acid, 2-acetamino-2-deoxy-D-glucuronic acid, 2-acetamino-2-deoxy-D-mannuronic acid, more preferably the monosaccharides shown in Table 1 below or their derivatives:

[0687]

[0688]

[0689] Preferred disaccharide groups suitable for the ADCs of this disclosure include lactose or derivatives thereof, maltose or derivatives thereof, sucrose or derivatives thereof, including their α and β terminal isomers, wherein the derived forms are, for example, in which any monosaccharide group constituting the disaccharide is a uronic acid, amino sugar, acylated amino sugar, aminouronic acid, acylated aminouronic acid, or combinations thereof, such as D-lactose, D-maltose, D-sucrose, or their respective acetamino groups (e.g., 2-acetamino groups) and / or 6-carboxyl derivatives; specific examples include, but are not limited to, those shown in Table 2 below:

[0690]

[0691]

[0692] The dashed line at position 1 of the sugar unit indicates that the configuration of the glycosidic bond can be α-type, β-type, or a mixture thereof.

[0693] In some embodiments, the solubilizing glycosides and their derivatives in the ADC of this disclosure are selected from glucose, mannose, galactose, lactose, maltose, sucrose or derivatives thereof, with the derivatives preferably in the form of their respective uronic acid, amino sugar, acylated amino sugar, aminouronic acid and / or acylated aminouronic acid forms; as specifically shown in the table above.

[0694] In some implementations of formula (S), Z' is Where R a And m have the values ​​defined above as generally or preferably.

[0695] In some implementations of the above formula (S), Z' is a subset of... The hydrophilic peptide, as generally, specifically, or preferably defined in the "G-antibody linker" section above, is an example of a peptide of formula (S) linked via a carboxyl terminus. Or linked via an amino terminus (preferably) ), where R is as defined above, including but not limited to the amino group residues of the various amino acids listed in this article for hydrophilic peptides, for example, R can be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably -CH3; for example, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0696] In some other embodiments of the above formula (S), Z' is a subset of... Hydrophilic peptides, selected from Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably H or -CH3, more preferably -CH3; preferably, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0697] In some specific formulation (S) embodiments, Z' is selected from glycosyl groups or their derivatives and contains... The hydrophilic peptide; wherein the glycosyl group or its derivative is preferably glucose, mannose, galactose, lactose, maltose, sucrose or their respective uronic acid, amino sugar, acylated amino sugar, aminouronic acid or acylated aminouronic acid derivative, more preferably glucose, mannose, galactose or their respective derivatives, wherein the hydrophilic peptide is preferably... More Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably H or -CH3, more preferably -CH3; preferably, the segment indicated in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0698] In some specific implementations of formula (S), formula (S) In this context, T' is selected from -C(R) a )2-、-O-、-NR a -, preferably -O-, M is -CO-, for example Where R a H or -C 1-3 Alkyl group, and Z' is Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably -CH3, for example, Z is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units; or T' is selected from -C(R a )2-、-O-、-NR a -, preferably -O-, M does not exist, for example Z' is selected from glucose, mannose, galactose, lactose, maltose, sucrose, or their respective uronic acids, amino sugars, acylated amino sugars, aminouronic acids, or derivatives of acylated aminouronic acids, preferably glucose, mannose, galactose, or their respective derivatives; wherein R a H or -C 1-3 Alkyl group, n' is 1 to 2.

[0699] Self-decomposing linker fragments carrying solubilizing units

[0700] The connecting subunits of the ADC disclosed herein include structural segments that are connected as appropriate to any combination of the self-decomposing connecting units A1 or A2 as defined above in general or specific terms and the solubilizing units S1 or S2 as defined above in general or specific terms.

[0701] In some specific implementation plans,

[0702] A1 and A2 are both selected from Preferred

[0703] S1 or S2 is attached to the benzene ring of A1 or A2, and is Where T' is selected from -C(R) a )2-、-O-、-NR a -or does not exist, M' is selected from -CO-, -O-, -NR a - or does not exist, n' is an integer from 0 to 4, R a For H or C 1-6 alkyl;

[0704] Z' is a monosaccharide or disaccharide or a derivative thereof, for example selected from glucose, mannose, galactose, lactose, maltose, sucrose or their uronic acid, amino sugar, acylated amino sugar, aminouronic acid or acylated aminouronic acid derivatives thereof, preferably the monosaccharide glucose, mannose, galactose or their respective derivatives as shown above; or

[0705] Z' is inclusive Hydrophilic peptides, as generally or specifically defined above for formula (S), are preferred.

[0706] In a further specific implementation plan, S1 or S2 is For example Furthermore, Z' is a monosaccharide or disaccharide, or a derivative thereof, as generally or specifically defined herein, linked by an α- or β-glycosidic bond. Examples of self-decomposing linkers A1 or A2 carrying a glycosyl group or a derivative thereof include, but are not limited to:

[0707]

[0708] In the specific examples above, the α / β indicated by the glycosidic bond at position 1 means that the configuration of the glycosidic bond can be α-type, β-type, or a mixture thereof.

[0709] In a further specific implementation plan, S1 or S2 is Where T' is selected from -C(R) a )2-, -O- or -NR a -; M' is selected from -CO-; n is 1 to 4, for example 1-2; R a For H or C 1-6 Alkyl; Z indicates inclusion Hydrophilic peptides, preferably Where R is defined above, it includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a R a Selected from H or -C 1-6Alkyl group, preferably -CH3, for example, the fragment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10, 5-10 units, more preferably polysarcosine containing 4-12, 6-12 units or 4-10, 5-10 units. Examples of self-decomposing linkers A1 or A2 carrying hydrophilic peptides include, but are not limited to:

[0710]

[0711] Implementation scheme of connector unit L

[0712] It should be noted that the connector unit L of the ADC of this disclosure includes any combination of the general or specific or preferred definition of any one of the components of the connector unit as described above and the general or specific or preferred definition of the remaining one or more components.

[0713] The connector unit L in the ADC of this disclosure has the structure of formula (III) and its various sub-formulas described above, and preferably has the following structure.

[0714]

[0715] G, W, P1, P2, A1, A2, and S2 are each defined in general, specific, or preferred terms as described above for each part.

[0716] In some specific implementations, the connector units of the ADC disclosed herein are in formulas (III-2) and (III-3).

[0717] G is Preferred The asterisk-marked end connects to the antibody, and the wavy line end connects to the property regulation unit E or the branching unit W; the alkylene group is optionally replaced by the Bu substituent of formula (G) as generally, specifically, or preferably defined in the "G-antibody linker" section above, and preferably by... Replacement, where T0 is -C 1-3 alkylene, preferably methylene, Z is Where R, as defined above, includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably -CH3, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0718] W is Connected to the antibody linker G via the N-terminus;

[0719] P1 or P2 is a cleavable peptide with a structure of formula (P) from the N-terminus to the C-segment: -(AA) d - , wherein AA is selected from alanine, glycine, asparagine, isoleucine, leucine, valine, phenylalanine, citrulline, and glutamic acid; preferably, AA is selected from alanine, glycine, asparagine, valine, phenylalanine, citrulline, and glutamic acid; d is an integer from 2 to 8, more preferably an integer from 2 to 4; wherein each AA is linked by a peptide bond; more preferably selected from...

[0720] More

[0721] A1 or A2 is selected from Where R 16 Let H be the number of 'R', and R be the number of 'R'. 17 H is selected from NH or O; preferably. One end marked with an asterisk (*) is connected to the payload, and the other end marked with a wavy line is connected to P1 or P2.

[0722] S2 is the expression (S) It is attached to the benzene ring of A2, where T' is selected from -C(R a )2-、-O-、-NR a - or not present, preferably -O-, M' is selected from -CO-, -O-, -NR a - or does not exist, preferably -CO- or does not exist, n is an integer from 0 to 4, preferably 1-2, R a For H or C 1-6 alkyl;

[0723] Z' is a monosaccharide or a disaccharide or a derivative thereof; or

[0724] Z' is inclusive Hydrophilic peptides, preferably Where R, as defined above, includes, but is not limited to, the amino group residues of the various amino acids listed in this article for hydrophilic peptides. For example, R could be R a ;R a Selected from H or -C 1-6 Alkyl group, preferably -CH3; for example, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

[0725] In some embodiments of formula (III-2) or (III-3), A1 is For example A2 is selected from For example

[0726] In some implementations of formula (III-2), A2 is For example S2, which carries the formula (S) as generally, specifically or preferably defined herein, preferably includes Z' as a monosaccharide or disaccharide or a derivative thereof, or includes Z' as a hydrophilic peptide as generally, specifically or preferably defined above.

[0727] In specific embodiments of formula (III-2) or (III-3), the monosaccharide or disaccharide or its derivative as Z' is as generally, specifically or preferably shown above, preferably glucose, mannose, galactose, lactose, maltose, sucrose, more preferably glucose, mannose, galactose, or their uronic acid, amino sugar, acylated amino sugar, aminouronic acid and / or acylated aminouronic acid derivatives.

[0728] Therefore, the connecting body unit of equation (III-2) or (III-3) can have the general formula shown in Table 3 below:

[0729]

[0730]

[0731]

[0732]

[0733]

[0734] Each variable or segment has the general, specific, or preferred definition given above for each component. That is, this disclosure also covers linker units in the above formulas where each variable or segment has the general, specific, or preferred definition given above for each component. In particular, the segments in the general formulas above corresponding to formulas (G) and (S) have the general, specific, or preferred definitions given above, wherein Z or Z' is preferably a glycosyl group or its derivative (preferably a monosaccharide or its derivative as defined herein) or a hydrophilic peptide (preferably as defined above). ).

[0735] In a specific implementation, the connector unit L segment of the ADC of this disclosure is selected from the structures shown in Table 4 below.

[0736]

[0737]

[0738]

[0739] It should be noted that this disclosure covers not only the connector units as defined above in the general, specific or preferred sense that are part of the ADC of this disclosure, but also the connector units themselves as defined above in the general, specific or preferred sense, and all conjugates containing the connector unit structure.

[0740] That is, in another respect, this disclosure relates to linker units as defined above in general, specific or preferred terms, preferably those linker units containing hydrophilic peptides and / or glycosyl groups or derivatives thereof.

[0741] In another respect, this disclosure relates to conjugates comprising linker units as defined above in general, specific or preferred terms, preferably those conjugates containing linker units of hydrophilic peptides and / or glycosyl groups or derivatives thereof.

[0742] Payload-connector unit segment

[0743] The connector element L of the ADC disclosed herein comprises a payload-connector element segment consisting of any combination of the general or specific or preferred definition of the connector element in this disclosure and the general or specific or preferred definition of D1 or D2.

[0744] Based on the above, in this disclosure (X) or (X-1)... It has the following formula:

[0745] For example

[0746]

[0747] in:

[0748] The wavy line represents the linkage with the targeting ligand, such as an antibody or its antigen-binding fragment;

[0749] D1 is the compound of formula (II) as defined herein and its various sub-formulas, connected to the linker unit via an amino group on any of the -SH, B1, or B2 bases or a -OH group on the sugar ring, preferably as follows: More specifically, the subform of formula (II) connected by the following sites,

[0750] Further for

[0751] The asterisk indicates the connection point with the linker unit. B1, B2, R1, R1', R2, and R2' have the general, specific, or preferred meanings defined above for part D1, and molecule D1 has the general, preferred, or specific meanings defined above for part D1.

[0752] D2 is a small molecule compound, as defined above as generally, preferably or specifically;

[0753] The connecting body unit portions respectively have the structures defined in this paper as (III), (III-1), (III-2), (III-3), (III-4), (III-5), (III-6), and (III-7), and preferably have the structures defined in this paper as " Implementation scheme of connector unit L The structure of formula (III-2) or (III-3) defined in the “Part” section is preferred, and the structure of formula (III-2-1), (III-2-2), (III-3-1), (III-3-2), (III-3-3) or (III-3-4) defined above is even more preferred, and the example structure of formula (III-2-1), (III-2-2), (III-3-3) or (III-3-4) defined above is even more preferred.

[0754] In some implementations of formula (IV) and its various subforms,

[0755] B1 is adenine that can be substituted by X. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl; or optionally R b Replaced guanine Where R b Selected from -C 1-6 alkyl;

[0756] B2 is guanine.

[0757] R1 and R1' are both H, or one of them is H and the other is F, or one of them is H and the other is OH;

[0758] R2 and R2' are both H, or one of them is H and the other is F, or one of them is H and the other is OH;

[0759] In some implementations, in formula (IV), B1 is, for example... Preferred B2 is guanine. In this specific embodiment, R1 and R1' are both H, or one of them is H and the other is F, or one of them is H and the other is OH. In a further specific embodiment, one of R2 and R2' is H and the other is OH.

[0760] The exemplary payload-connector unit fragments of this disclosure include, but are not limited to, PL-3 to PL-59 prepared and characterized in the Embodiments section, or stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts or solvates thereof.

[0761] It should be noted that this disclosure covers not only the payload-connector unit segments defined above in the general, specific or preferred sense that are part of the ADC of this disclosure, but also the payload-connector unit segments themselves as defined above in the general, specific or preferred sense, and all conjugates containing the payload-connector unit segments.

[0762] That is, in another aspect, this disclosure relates to the payload-connector unit fragment compound as generally, specifically, or preferably defined above, denoted as: Specifically

[0763] For example

[0764]

[0765] Each component and each variable within each component has the corresponding general, specific or preferred definition as described above. This disclosure covers any combination of the general, specific or preferred definition of any component and its variables with the general, specific or preferred definition of one or more other components and their variables to form a payload-connector unit fragment compound.

[0766] Preferred compounds are those containing payload-linker unit fragments of hydrophilic peptides and / or glycosyl groups or their derivatives as generally, specifically or preferably defined herein.

[0767] In another respect, this disclosure relates to conjugates comprising payload-linker unit fragments as defined above in general, specific or preferred terms, preferably those conjugates containing payload-linker unit fragments of hydrophilic peptides and / or glycosyl groups or derivatives thereof.

[0768] This disclosed ADC

[0769] The ADC disclosed herein includes a technical solution formed by combining any general or specific or preferred definition of any of its components with the general or specific or preferred definitions of one or more other components.

[0770] Based on the above, the ADC of this disclosure (X-1) can be expressed as:

[0771] Specifically For example

[0772]

[0773]

[0774] Or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotopic labels;

[0775] The components of the linker unit, the linker unit itself, D1, and D2 are defined as generally, specifically, or preferably as described above; q has the value defined above for formula (X) or (X-1), specifically representing 1 to 10 or the average DAR value of any two values ​​between 1 and 10, preferably 1 to 8 or the average DAR value of any two values ​​between 1 and 8, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, 4 to 6, 4 to 8, 4 to 10, 6 to 8, or 6 to 10; Ab is an antibody or antigen-binding fragment thereof that binds to a tumor-specific antigen or a tumor-associated antigen, as generally, specifically, or preferably defined above for the Ab part.

[0776] In some preferred embodiments, q represents an average DAR of about 3. In some preferred embodiments, q represents an average DAR of about 6. In some preferred embodiments, q represents an average DAR of about 8.

[0777] In some preferred embodiments, the antigen bound to the Ab in the ADC of this disclosure is selected from HER2, KIT, FORR1, CD276, PD-L1, NECTIN4, Mesothelin, MUC1, GCPII, BCMA, cMet, RPR1, CD22, CD19, TOP1, Claudin 18.2, EGFR, and Trop-2; preferably selected from HER2, EGFR, Claudin 18.2, and Trop-2.

[0778] In a more preferred embodiment, the Ab in the ADC of this disclosure is an antibody that specifically binds to HER2 or an antigen-binding fragment thereof.

[0779] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure includes three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs), wherein: according to the Kabat definition,

[0780] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:6.

[0781] HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:7.

[0782] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:8.

[0783] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:3.

[0784] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:4, and

[0785] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:5.

[0786] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure 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:10, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9.

[0787] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure comprises:

[0788] (a) A heavy chain containing the amino acid sequence of SEQ ID NO:2, and

[0789] (b) A light chain containing the amino acid sequence of SEQ ID NO:1.

[0790] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure is trastuzumab.

[0791] In another preferred embodiment, the Ab in the ADC of this disclosure is an antibody that specifically binds to Trop-2 or an antigen-binding fragment thereof.

[0792] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure includes three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs), wherein: according to the Kabat definition,

[0793] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:18.

[0794] HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:19.

[0795] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:20.

[0796] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:15.

[0797] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:16, and

[0798] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:17.

[0799] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure 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:22, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:21.

[0800] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure comprises:

[0801] (a) A heavy chain containing the amino acid sequence of SEQ ID NO:14, and

[0802] (b) A light chain containing the amino acid sequence of SEQ ID NO:13.

[0803] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure is sacituzumab.

[0804] In another preferred embodiment, the Ab in the ADC of this disclosure is an antibody that specifically binds to Claudin 18.2 or an antigen-binding fragment thereof.

[0805] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure includes three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs), wherein: according to the Kabat definition,

[0806] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:29.

[0807] HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:30.

[0808] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:31.

[0809] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:26.

[0810] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:27, and

[0811] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:28.

[0812] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure 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:33, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32.

[0813] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure comprises:

[0814] (a) A heavy chain containing the amino acid sequence of SEQ ID NO:25, and

[0815] (b) A light chain containing the amino acid sequence of SEQ ID NO:24.

[0816] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure is zotuximab.

[0817] In another preferred embodiment, the Ab in the ADC of this disclosure is an antibody that specifically binds to EGFR or an antigen-binding fragment thereof.

[0818] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure includes three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs), wherein: according to the Kabat definition,

[0819] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:40.

[0820] HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:41.

[0821] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:42.

[0822] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:37.

[0823] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:38, and

[0824] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:39.

[0825] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure 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:44, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:43.

[0826] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure comprises:

[0827] (a) A heavy chain containing the amino acid sequence of SEQ ID NO:36, and

[0828] (b) A light chain containing the amino acid sequence of SEQ ID NO:35.

[0829] In a more preferred embodiment of this group, the Ab in the ADC of this disclosure is cetuximab.

[0830] It should be noted that the ADC disclosed herein covers any combination of any embodiment of the D1 or D2 unit as generally or specifically defined in this disclosure, any embodiment of the linker unit L as generally or specifically defined in this disclosure, and any embodiment of the antibody Ab as generally or specifically defined in this disclosure.

[0831] In a particularly preferred embodiment, specific examples of the ADC disclosed herein include:

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842] It can be in the form of pharmaceutical salts, esters, isomers, prodrugs, solvates, or isotopic variants.

[0843] Where q represents the average DAR value from 1 to 20, preferably the average DAR value from 1 to 10 or 1 to 8, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, 4 to 6, 4 to 8, 4 to 10, 6 to 8 or 6 to 10.

[0844] In some preferred embodiments, q represents an average DAR of about 3. In some preferred embodiments, q represents an average DAR of about 6. In some preferred embodiments, q represents an average DAR of about 8.

[0845] II: Beneficial Effects of the ADC Disclosed in this Paper

[0846] The ADC according to this disclosure has at least one or more of the following advantages:

[0847] It targets tumor cells, activates the STING innate immune pathway within tumor cells, and kills tumor cells. At the same time, after CDN decomposition, it releases pre-designed cytotoxic substructural units to further kill tumor cells, giving it a highly efficient tumor-killing effect in animals and providing tumor marker fragments for the acquired immune system, giving the body immune memory and consolidating long-term immune effects.

[0848] Compared with the blood circulation distribution of CDN, it significantly promotes the distribution of ADC into the tumor environment, effectively limits the premature release of CDN load in plasma, and reduces off-target toxicity;

[0849] There is no obvious aggregation phenomenon, and a high drug loading capacity can be achieved, with DAR up to 8;

[0850] ADC-CDN conjugates can effectively protect CDN from hydrolysis by phosphatases in the blood, thereby greatly improving the blood circulation stability of drug CDN;

[0851] ADC-CDN is internalized by cancer cells that highly express the corresponding antigen under antibody guidance, and can provide rapid and efficient CDN release in tumor cells. After CDN exerts its STING agonist function, it decomposes and releases cytotoxic molecules.

[0852] CDN is broken down in cells to produce phosphorylated fragment molecules, bypassing the key activation steps of traditional cytotoxic molecules and improving drug efficacy.

[0853] After CDN is broken down inside the cell, it produces phosphorylated fragment molecules that are not easily pumped out of cancer cells, thus reducing drug resistance.

[0854] By using connectivity technology to introduce a second payload, it is possible to fuse payloads with different mechanisms of action into a single ADC. This not only enables the delivery of antitumor drugs to the target tissue, but also ensures that different drugs can reach the same cell, thereby achieving efficient synergistic effects of drugs, reducing the effective dose of drugs, and reducing toxicity.

[0855] Adding a solubilizing unit to the linker unit can promote the coupling reaction, increase the DAR value, and reduce ADC aggregation, thereby increasing the activity of the ADC.

[0856] In animal models, the dual-load ADC showed significantly improved antitumor activity compared to the single-load ADC1 and the single-load ADC2, and exhibited an effect beyond the sum of the two single-load ADCs, i.e., synergistic enhancement was achieved.

[0857] It exhibits a wide safety window among all (numerous) in vivo pharmacodynamic models;

[0858] Acceptable PK characteristics;

[0859] More traditional, mature and user-friendly administration methods (such as intravenous, subcutaneous, intramuscular, and topical administration) can be used instead of intratumoral administration to prolong efficacy and reduce toxic side effects.

[0860] Specifically, the targeted inhibitory effect of the ADC compound described in this application can be that, when the compound is added to the culture medium of tumor cells highly expressing a specific target, the proliferation ability of the tumor cells highly expressing the specific target decreases by more than 1%, more than 5%, more than 20%, more than 50%, or even more than 90% or 95% compared to the addition of a negative control or control drug. For example, the targeted inhibitory effect can be that the IC50 value (nM) of the tumor cells highly expressing the specific target is below 1000, below 500, or below 100, such as 1-500 or 1-100; for example, the tumor cells can include, but are not limited to, solid tumor cells, such as breast adenocarcinoma cells, gastric cancer cells, lymphoma cells, ovarian adenocarcinoma cells, etc.; the specific target includes, but is not limited to, HER2, TROP2, EGFR, and Claudin 18.2.

[0861] Specifically, the in vivo tumor-suppressing effect of the ADC compound described in this application can be defined as a reduction in tumor volume of more than 1%, 5%, 20%, 50%, or even 90% or 95% at 1, 3, 5, 7, 14, 21, or 30 days after administration to animals compared to the addition of a control or control drug. For example, compared to the administration of a negative control and control drug, the tumor volume of the animals reduced by more than 1.1 times, 1.5 times, 2 times, 5 times, 10 times, or even 100 times or 500 times at 1, 3, 5, 7, 14, 21, or 30 days. The animals include, but are not limited to, mammals. The administration includes, but is not limited to, oral, intravenous injection or infusion, intraperitoneal injection, or local administration.

[0862] Specifically, the distribution of the ADC compound described in this application in animal tumor tissues after administration, compared to other tissues and organs, or compared to the distribution of CDN, can be increased by more than 1%, 5%, 20%, 50%, or even 90% or 95%. For example, the distribution can be increased by more than 1.1 times, 1.5 times, 2 times, 5 times, 10 times, or even 100 times or 500 times. The animals include, but are not limited to, mammals, and the tissues or organs include, but are not limited to, the heart, liver, spleen, lungs, kidneys, and brain. The administration includes, but is not limited to, oral administration, intravenous injection or infusion, intraperitoneal injection, or local administration.

[0863] Specifically, the ADC compound described in this application has good in vivo safety, with the release rate of free toxins in animals not exceeding 50%, 20%, 10%, or even 1% or 0.1% after administration. For example, the in vivo safety refers to the concentration of the compound administered without producing toxic effects in animals, which can be 0.5 mg / kg or higher, 5 mg / kg or higher, 10 mg / kg or higher, 50 mg / kg or higher, 100 mg / kg or higher, or even 500 mg / kg or higher. The animals include, but are not limited to, mammals. The administration includes, but is not limited to, oral, intravenous injection or infusion, intraperitoneal injection or local administration.

[0864] Specifically, the ADC compound described in this application has plasma stability, and the release rate of the cytotoxic drug after being added to plasma does not exceed 50%, 20%, 10%, or even 1% or 0.1% at 1, 3, 5, 7, 14, 21, or 30 days.

[0865] III. Pharmaceutical Composition

[0866] Another aspect of this disclosure provides pharmaceutical compositions comprising the ADC of this disclosure and one or more pharmaceutically acceptable excipients, the compositions of which may comprise components conventional to pharmaceutical formulations, such as diluents, carriers, pH adjusters, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for altering osmotic pressure, buffers, masking agents, antioxidants, and other active agents. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, in Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000), Lippincott, Williams & Wilkins, Philadelphia.

[0867] This disclosure also provides methods for preparing the compositions using the ADCs of this disclosure. Typical pharmaceutical compositions or dosage forms are prepared by mixing the compounds of this disclosure with a carrier or excipient, and can be formulated in a manner well known to those skilled in the art and consistent with good medical practice, for example, see Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams & Wilkins, Philadelphia or national pharmacopoeias.

[0868] The pharmaceutical compositions disclosed herein can be in any suitable form, such as tablets, powders, capsules, sterile injectable formulations, solutions, dispersants, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc., and can be administered to patients via a variety of routes, such as intravenous, intratumoral, subcutaneous, intramuscular, oral, intranasal, intrathecal, transdermal, or topical. The route of administration in any given situation may depend on the specific antibody and / or ADC, the subject, the nature and severity of the disease, and the subject's physical condition. In some embodiments, the pharmaceutical composition will be administered intravenously, intratumorally, subcutaneously, or intramuscularly as a liquid formulation.

[0869] In preferred embodiments, the ADCs described herein and pharmaceutical compositions comprising them are administered systemically, such as subcutaneously, intraperitoneally, intramuscularly, or intravenously, particularly intravenously, especially in sterile injectable formulations, such as sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents, or prepared as lyophilized powders; acceptable media or solvents may include, for example, water, 1,3-butanediol, Ringer's solution, or isotonic sodium chloride solution; furthermore, sterile non-volatile oils may be routinely used as solvents or suspension media, for which any mild non-volatile oil may be used, including, for example, synthetic monoglycerides or diglycerides, fatty acids, etc. In other embodiments, the ADCs described herein and pharmaceutical compositions comprising them are administered locally at the tumor site, such as within the tumor or in the tumor microenvironment.

[0870] The dosage of the disclosed compounds can vary over a wide range, and of course, can be adjusted by a clinician according to individual needs in each specific case. For preventative or therapeutic purposes, the appropriate dosage of the disclosed ADC (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease, whether the antibody is administered for preventative or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0871] The pharmaceutical compositions or formulations disclosed herein may also comprise more than one other active ingredient required for the specific indication being treated, preferably those 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.

[0872] IV: Drug Combinations and Pillboxes

[0873] In some embodiments, this disclosure also provides pharmaceutical combinations or pharmaceutical combination products comprising the ADC of this disclosure or a pharmaceutically acceptable salt, ester, isomer, prodrug, solvate, or isotopic variant thereof, and one or more other therapeutic agents for the prevention or treatment of diseases, such as those related to or mediated by STING, more specifically for the treatment or prevention of inflammation, allergic or autoimmune diseases, infectious or proliferative diseases, especially tumors or viral infections; thereby, this disclosure also provides methods for treating or preventing diseases, such as those related to or mediated by STING, more specifically for inflammation, allergic or autoimmune diseases, infectious or proliferative diseases, especially tumors or viral infections, in subjects, the method comprising administering the pharmaceutical combination of this disclosure to a human or animal.

[0874] Another object of this disclosure is to provide a kit containing the drug combination of this disclosure, preferably in the form of drug dosage units. This allows the dosage units to be provided according to a dosing regimen or drug administration interval.

[0875] In one embodiment, the kit of this disclosure comprises, within the same package:

[0876] - A first container containing a pharmaceutical composition comprising the ADC of the present disclosure or a pharmaceutically acceptable salt, ester, isomer, prodrug, solvate or isotopic variant thereof;

[0877] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0878] Based on factors such as 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 combined product. The combined product of this disclosure can be used in the treatment methods of this disclosure. In some embodiments, this disclosure provides a combined product in which the other therapeutic agents are, for example, antibodies that effectively stimulate an immune response to further enhance, stimulate, or upregulate the immune response of the subject. In some embodiments, the combined product is used to prevent or treat diseases, such as those related to or mediated by STING, particularly tumors.

[0879] V. Uses and Methods

[0880] Given that the first payload CDN compound of the ADC disclosed herein can activate STING, induce the expression of type I interferon and pro-inflammatory cytokines such as IL-6, TNF-α and IFN-γ, and has cytotoxic activity, and that the second payload can be selected from a variety of therapeutic agents with mechanisms compatible with STING agonists, this disclosure also provides therapeutic uses and methods of the ADC disclosed herein.

[0881] In some implementations, this disclosure provides the ADC disclosed herein for therapeutic purposes.

[0882] The ADCs provided in this disclosure can be used alone or in combination with each other and / or in combination with other therapeutic agents. As shown in the examples below, ADCs can promote an immune response when administered to a subject. For example, the ADCs provided in this disclosure, alone or in combination, can induce interferon B (IFNB) in human subjects, partly due to their ability to activate STING. The binding of CDN in the ADC to antibodies or antigen-binding fragments that bind to tumor-associated antigens or immune cell antigens targets and delivers it to tumor-associated immune cells or the tumor microenvironment to trigger STING activation and generate an immune response, killing tumor cells and producing anti-inflammatory and antiviral effects, while prolonging or enhancing the immune response; subsequently, the CDN released from the ADC further decomposes to release the cytotoxic molecular portion of the structure, further inhibiting the survival of tumor cells.

[0883] Therefore, the ADC of this disclosure can promote immune responses and tumor cell killing, with a stronger effect than that of antibodies contained in uncoupled CDNs or ADCs. In other words, this disclosure achieves a synergistic effect by combining the prepared CDN with specific immunotherapeutic antibodies, and this synergistic effect is further enhanced by the cytotoxic portion released from the further breakdown of the CDN, thereby achieving a triple synergistic effect. Furthermore, the ADC of this disclosure, by introducing a second payload, can further achieve a synergistic effect of biological activity with the first payload.

[0884] Therefore, in one aspect, this disclosure consequently provides a method for inducing, stimulating, or assisting an immune response in an individual, including administering the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates, or isotopic variants, or pharmaceutical compositions comprising thereof, to the individual. In one embodiment, the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates, or isotopic variants, or pharmaceutical compositions comprising thereof, are administered to an individual as immunotherapy to induce the production in vivo of a variety of cytokines that are therapeutically useful in humans or animals, including type I interferon and pro-inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, to modulate the immune system of the human or animal to achieve certain therapeutic benefits.

[0885] On the other hand, this disclosure thereby provides methods for treating or preventing diseases related to or mediated by immune responses, specifically diseases related to or mediated by STING, including inflammation, allergic or autoimmune diseases, infectious diseases or cancer, including administering a therapeutically effective amount of the disclosed ADC or its pharmaceutically acceptable salt, ester, isomer, prodrug, solvate or isotopic variant or pharmaceutical composition containing the thereof to a subject in need.

[0886] On the other hand, this disclosure provides for the use of the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates or isotopic variants or pharmaceutical compositions comprising the thereof in the prevention or treatment of diseases, such as those related to or mediated by STING, and more specifically in the treatment or prevention of inflammatory, allergic or autoimmune diseases, infectious or hyperproliferative diseases, especially tumors or viral infections.

[0887] On the other hand, this disclosure provides the ADC of the present disclosure or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates or isotopic variants or pharmaceutical compositions containing the thereof for use as therapeutic agents for treating or preventing diseases, such as diseases related to or mediated by STING, more specifically as therapeutic agents for treating or preventing inflammatory, allergic or autoimmune diseases, infectious diseases or hyperproliferative diseases, especially as antitumor or antiviral agents, or as vaccine adjuvants; specifically, as cytotoxic agents for treating or preventing hyperproliferative diseases, especially tumors, or as cytotoxic agents for treating or preventing viral infections.

[0888] In a preferred embodiment, the ADC of this disclosure, or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates, or isotopic variants, or pharmaceutical compositions comprising thereof, are used as cytotoxic agents for the treatment or prevention of hyperproliferative diseases, particularly tumors. In another preferred embodiment, the ADC of this disclosure, or its pharmaceutically acceptable salts or solvates, or pharmaceutical compositions comprising thereof, are used for the treatment of recurrent tumors or for the prevention of tumor recurrence.

[0889] On the other hand, this disclosure provides the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates, or isotopic variants, or pharmaceutical compositions comprising thereof, as multifunctional active agents, possessing both immunotherapeutic and cytotoxic therapeutic activities. These activities include the ability to activate the immune system by stimulating the STING signaling pathway to exert antitumor and antiviral replication functions, to induce tumor cell death or inhibit viral replication by releasing cytotoxic agents, to continuously activate STING to kill tumor cells by releasing tumor DNA, and to provide "immune memory" or durable immunity against tumors by releasing tumor neoantigens to generate antibody-antigen responses. In this regard, this disclosure also provides the disclosed ADC or its pharmaceutically acceptable salts or solvates, or pharmaceutical compositions comprising thereof, for achieving the above-mentioned multiple functions, and its use in achieving the above-mentioned multiple functions.

[0890] On the other hand, this disclosure provides methods for treating or preventing diseases in subjects, such as diseases related to or mediated by STING, more specifically inflammatory, allergic or autoimmune diseases, infectious diseases or hyperproliferative diseases, especially tumors or viral infections, methods comprising administering to a human or animal an ADC of the present disclosure or a pharmaceutically acceptable salt, ester, isomer, prodrug, solvate or isotopic variant thereof or a pharmaceutical composition comprising thereof; specifically, this disclosure provides methods for treating or preventing hyperproliferative diseases, especially tumors, in subjects, or methods for treating or preventing viral infections in subjects, said methods comprising administering to a human or animal an ADC of the present disclosure or a pharmaceutically acceptable salt, ester, isomer, prodrug, solvate or isotopic variant thereof or a pharmaceutical composition comprising thereof.

[0891] On the other hand, this disclosure provides the use of the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates, or isotopic variants, or pharmaceutical compositions comprising thereof, in the preparation of medicaments for the prevention or treatment of diseases, such as those related to or mediated by STING, more specifically inflammatory, allergic, or autoimmune diseases, infectious or hyperproliferative diseases, especially tumors or viral infections, or in the preparation of vaccine adjuvants; specifically, this disclosure provides the use of the disclosed ADC or its pharmaceutically acceptable salts or solvates, or pharmaceutical compositions comprising thereof, in the preparation of medicaments for the treatment or prevention of hyperproliferative diseases, especially tumors, and the use of the disclosed ADC or its pharmaceutically acceptable salts, esters, isomers, prodrugs, solvates, or isotopic variants, or pharmaceutical compositions comprising thereof, in the preparation of medicaments for the treatment or prevention of viral infections.

[0892] The hyperproliferative diseases for the purposes and methods described above refer to physiological conditions in subjects characterized by uncontrolled or disordered cell growth or death, particularly tumors or cancers, including solid tumors and hematogenous tumors, including but not limited to brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, leukemia, lung cancer (small cell lung cancer, non-small cell lung cancer), and bone cancer. Examples of the aforementioned cancer types include neuroblastoma, colorectal cancer such as rectal cancer, colon cancer, colorectal cancer, familial adenomatous polyposis carcinoma and hereditary non-lymphatic colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal carcinoma, pharyngeal squamous cell carcinoma, oral cancer, head and neck cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urothelial carcinoma, sweat gland cancer, gastric cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, pancreatic cancer, prostate cancer, testicular cancer, breast cancer (including HER2-negative breast cancer), urinary tract cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, and lymphoma. Examples of tumor types include Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL), lymphocytic carcinoma, acute myeloid leukemia (AML), myeloid leukemia (chronic myeloid leukemia (CML), adult T-cell lymphoma, diffuse lymphoma (DLBCL), liver cancer, hepatocellular carcinoma, multiple myeloma, seminoma, osteosarcoma, chondrosarcoma, anal canal cancer, renal cell carcinoma, adrenocortical carcinoma, chordoma, fallopian tube cancer, gastrointestinal stromal tumor, myeloproliferative disorders, mesothelioma, biliary tract cancer, Ewing sarcoma, and other rare tumor types, as well as recurrent forms of the above tumors.

[0893] In a preferred embodiment, the proliferative diseases for which the above-described uses and methods are: small cell lung cancer, non-small cell lung cancer, colorectal cancer, liver cancer, breast cancer, ovarian cancer, gastric cancer, prostate cancer, melanoma, renal cell carcinoma, head and neck cancer, pancreatic cancer, soft tissue sarcoma, Hodgkin's lymphoma, leukemia, or bladder cancer.

[0894] Viral infection as described in the above-mentioned uses and methods refers to the process by which viruses invade the body through various pathways and multiply in susceptible host cells. The viruses involved include, but are not limited to, double-stranded DNA viruses and single-stranded DNA viruses, single-stranded positive-sense RNA viruses, single-stranded negative-sense RNA viruses and double-stranded RNA viruses, and retroviruses. Examples include hepatitis B virus, TTV virus, adenovirus, papillomavirus, herpes zoster virus, smallpox virus and vaccinia virus, influenza virus, classical swine fever virus, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, rabies virus, Ebola virus, enterovirus, and human immunodeficiency virus. The therapeutic uses and methods provided in this disclosure can be used for the above-mentioned viral infections and the diseases they cause.

[0895] The use of the ADC of this disclosure or its pharmaceutically acceptable salts or solvates or pharmaceutical compositions comprising the thereof as vaccine adjuvants and in the preparation of vaccine adjuvants refers to the use of the ADC of this disclosure or its pharmaceutically acceptable salts or solvates or pharmaceutical compositions comprising the thereof as adjuvants in a vaccine-based treatment or prevention strategy, i.e., the ADC of this disclosure or its pharmaceutically acceptable salts or solvates or pharmaceutical compositions comprising the thereof are used with one or more vaccines selected to stimulate an immune response to one or more predetermined antigens, said vaccines comprising inactivated or attenuated bacteria or viruses, such as inactivated tumor cells expressing and secreting one or more of GM-CSF, CCL-20, CCL3, IL-12p70, FLT-3 ligands, and cytokines.

[0896] VI: Fabrication of the ADC disclosed herein

[0897] 1. General Synthesis Method

[0898] The compounds disclosed herein, their stereoisomers, tautomers, stable isotopic variants, pharmaceutically acceptable salts or solvates, can be prepared by a variety of methods well known in the field of organic synthesis, including the methods given below, the methods given in the examples, or similar methods as understood by those skilled in the art.

[0899] The following examples illustrate general synthetic schemes for synthesizing the compounds disclosed herein. For each reaction step, appropriate reaction conditions are known to those skilled in the art or can be conventionally determined. Specifically, the method steps for synthesizing the compounds disclosed herein can be carried out under reaction conditions known per se (including those specifically mentioned), in the absence or generally in the presence of solvents or diluents (including, for example, solvents or diluents that are inert to the reagents used and soluble in the reagents used), in the absence or in the presence of catalysts, condensing agents, or neutralizing agents (e.g., ion exchangers, such as cation exchangers, e.g., in the H+ form), depending on the nature of the reaction and / or the reactants, at reduced, normal, or elevated temperatures (e.g., from about -100°C to about 190°C, including, for example, from about -78°C to about 150°C, e.g., from about 0°C to about 125°C, room temperature, -20°C to 40°C, or reflux temperature), at atmospheric pressure or in a closed container, under pressure when appropriate, and / or in an inert atmosphere such as oxygen or nitrogen.

[0900] The starting materials and reagents used in the preparation of these compounds are generally commercially available or can be prepared by the methods described below, similar methods, or methods known in the art. If desired, the starting materials and intermediates in the synthetic reaction process can be separated and purified using conventional techniques, including but not limited to filtration, distillation, crystallization, and chromatography. The materials can be characterized using conventional methods, including physical constants and spectroscopic data.

[0901] Unless otherwise specified in the description of the method, solvents suitable for any particular reaction include: those solvents specifically mentioned, or, for example, water; esters, such as lower fatty acid alkyl esters, such as ethyl acetate; ethers, such as aliphatic ethers, such as diethyl ether, or cyclic ethers, such as tetrahydrofuran or dioxane; liquid aromatic hydrocarbons, such as benzene or toluene; alcohols, such as methanol, ethanol, or 1- or 2-propanols, such as acetonitrile; halogenated hydrocarbons, such as dichloromethane or chloroform; amides, such as N,N-dimethylformamide or N,N-dimethylacetamide; bases, such as heterocyclic nitrogen bases, such as pyridine; carboxylic anhydrides, such as lower aliphatic chain carboxylic anhydrides, such as acetic anhydride; cyclic, straight-chain, or branched hydrocarbons, such as cyclohexane, hexane, or isopentane; or mixtures of these solvents, such as aqueous solutions. Such solvent mixtures may also be used for post-processing, such as post-processing by chromatography or partitioning.

[0902] Those skilled in the art will recognize the presence of a stereocenter in the Formula I compound. At all stages of the reaction, the mixture of isomers formed can be separated into individual isomers, such as diastereomers or enantiomers, or into any desired mixture of isomers, such as racemates or mixtures of diastereomers, see, for example, "Stereochemistry of Organic Compounds" by E.L. Leel, S.H. Wilen, and L.N. Mander (Wiley-Interscience, 1994).

[0903] 2. First payload – Bifunctional cyclic dinucleotide CDN STING agonist fragment

[0904] The first payload of the dual-loaded antibody-drug conjugate disclosed herein is a bifunctional CDN STING agonist disclosed in patents WO2024153127 and WO2022083584, the synthesis and characterization of which are described in detail in those documents, which are incorporated herein by reference in their entirety.

[0905] Specifically, the first effective load in the dual-load antibody-drug conjugate of this disclosure is selected from the following compounds or their pharmaceutically acceptable salts or solvates, and is prepared and characterized according to the corresponding examples of patents WO2024153127 and WO2022083584:

[0906]

[0907]

[0908] 3. Second payload

[0909] The second payload in the dual-loaded antibody-drug conjugate disclosed herein is a pharmaceutical compound or a pharmaceutically acceptable salt or solvation thereof as generally or specifically described in the detailed description of the invention herein. These compounds may be prepared based on the relevant patent document (CN115990269) or are commercially available.

[0910] In specific implementations, the second payload in the dual-load antibody-drug conjugate of this disclosure may be selected from the following commercially available drugs or their pharmaceutically acceptable salts or solvates.

[0911]

[0912] 4. Synthesis of the key intermediate PL

[0913] As described above, the dual payloads (bifunctional cyclic dinucleotide payload 1 and payload 2) of the ADC disclosed herein are linked to the antibody via a key intermediate PL, such as Figure 6 As shown.

[0914] The following process 1 illustrates the general formula of a key intermediate PL that can be used to prepare the dual-load conjugated drug of this disclosure. The variables have the same meaning as those defined herein for the compound or its various specific embodiments, unless otherwise stated.

[0915] Process 1

[0916]

[0917] The amino group in intermediate B reacts with an active ester containing a succinamide group to generate intermediate C.

[0918]

[0919] The free carboxyl group of C reacts with the free amino group of intermediate A under the action of a condensing agent (such as DMTMM) to generate the key intermediate PL.

[0920]

[0921] Process 2 illustrates the general formula for the preparation of intermediate A.

[0922] Process 2

[0923]

[0924] Commercially available intermediate D, wherein AA1, AA2, AA3 and AA4 represent amino acids, as defined in the “cleavable linker” section of this invention, is used to convert the benzyl alcohol of intermediate D into benzyl halide using a halogenating agent (such as thionyl chloride, etc.), with benzyl iodide E being the most preferred.

[0925]

[0926] In a suitable solvent (such as DMF), the iodine in intermediate E reacts directly with a -SH group in the CDN molecule to generate intermediate F.

[0927]

[0928] Intermediate F undergoes deprotection of its protecting group Fmoc under the action of an organic base (such as diethylamine) to yield intermediate A. Alternatively, other amino protecting groups (such as Cbz) can be used, and the protecting group can be removed in this step using the corresponding deprotection method to obtain the free amine intermediate A.

[0929]

[0930] Process 3 illustrates a general formula for the preparation of intermediate B.

[0931] Process 3

[0932]

[0933] Commercially available intermediate D, wherein AA5, AA6, AA7, and AA8 represent amino acids as defined in the "Cleavable Linker" section detailed herein, is obtained by treating with di-p-nitrocarbonate to yield activated carbonate G.

[0934]

[0935] The free amine or hydroxyl group in load 2 reacts with G to generate intermediate H.

[0936]

[0937] The allyl ester in intermediate H can be hydrolyzed in the presence of triphenylphosphine palladium to obtain free acid intermediate I; if the raw material D is a methyl ester, I can be prepared by hydrolysis with an inorganic base (such as LiOH).

[0938]

[0939] Intermediate I is deprotected by an organic base (such as diethylamine) to obtain intermediate B. Alternatively, other amino protecting groups (such as Cbz) can be used, and the protecting group can be removed in this step by a corresponding deprotection method to obtain the free amine intermediate B.

[0940]

[0941] Intermediate H can also be prepared using the method in process 4. For details, please refer to process 2, which will not be repeated here.

[0942] Process 4

[0943]

[0944] When the connection point of load 2 is O, intermediate B can be prepared using a process similar to process 2 via intermediates J and K, process 5. For specific details, please refer to processes 2 and 3, which will not be repeated here.

[0945] Process 5

[0946]

[0947] The leaving groups of the active esters in the process can also be pentafluorophenol, etc.; some loaded fragments require functional group protection-deprotection operations that match the reaction conditions in the process; as shown in the examples, a variety of solubilizing groups can be introduced into the molecular structure of each flowchart by commonly used organic synthesis methods and the process operation can be followed.

[0948] 5. Antibody selection and synthesis

[0949] The antibodies used in this disclosed ADC are derived from commercial sources, including but not limited to antibodies targeting the following targets: KIT, FOLR1, CD276, PD-L1, NECTIN4, Mesothelin, MUC1, GCPII, BCMA, cMet, RPR1, CD22, CD19, TOP1, Claudin 18.2, EGFR, Trop-2, HER2, etc., and their bispecific antibodies.

[0950] 6. Synthesis of dual-load CDN-linker-antibody conjugate ADC

[0951] 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 linker L with a leaving group is used to conjugate drug D to a cysteine ​​residue of the antibody to prepare a conjugate of formula A of this disclosure. In some embodiments, the interchain disulfide bonds of the antibody can be disrupted and free thiol groups exposed for conjugation with the 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 linker disulfide bonds can be reduced, thereby generating up to eight reactive thiol groups for conjugation. Conjugates prepared by this method can conjugate zero, one, two, three, four, five, six, seven, or eight drug molecules in each antibody molecule.

[0952] When the prepared conjugates are compositions of conjugates with different drug conjugation sites and / or numbers, the drug loading of the conjugates is expressed as the average drug-adjustable range (DAR). In this case, the average DAR of the prepared antibody-drug conjugate composition can be characterized by conventional methods such as mass spectrometry, ELISA, and HPLC. The quantitative distribution of the antibody-drug conjugates, expressed as q, can also be determined. The separation, purification, and characterization of homogeneous antibody-drug conjugates with a certain q value from antibody-drug conjugates with other drug loadings can be achieved using methods such as reversed-phase HPLC or electrophoresis.

[0953] The following procedure 6 illustrates a general synthetic route that can be used to prepare the dual-loaded CDN compounds described herein and their various specific embodiments. The variables in the general formulas of the following procedure have the same meaning as those defined herein for the compounds or their various specific embodiments, unless otherwise stated.

[0954] Process 6

[0955]

[0956] The antibody molecule is first reduced, preferably by reducing the disulfide bonds. The heavy and light chains of the antibody molecule are linked by four pairs of disulfide bonds. These disulfide bonds can be reduced to a maximum of 0-8 thiol groups using reducing agents (such as tris(2-carboxyethyl)phosphine (TCEP), mercaptoethanol, dithiothreitol, cysteine, reduced glutathione, etc.). Each thiol group may react with succinamide to generate antibody-drug conjugates (ADCs) derived from 0-8 bifunctional CDN-linkers. The average drug loading (DAR) of this compound can be experimentally determined.

[0957] Unless otherwise specified, the experimental materials and reagents used in the above synthesis methods and procedures can be obtained from commercially available sources, prepared according to existing techniques, or prepared according to methods similar to those disclosed in this application. Unless otherwise specified, the synthesis conditions used in the above synthesis methods and procedures can be routinely determined by those skilled in the art.

[0958] This disclosure also relates to preparation methods in which a compound that can be obtained as an intermediate in any step of the various preparation methods and processes described herein is used as a starting material and the remaining method steps are carried out, or in which the starting material is formed in situ under reaction conditions or used in the form of a derivative, for example, in a protected form or in salt form, or a compound that can be obtained according to the methods of this disclosure is generated under said method conditions and further treated in situ. Attached Figure Description

[0959] Figure 1A This demonstrates the in vivo tumor-suppressive effect of bifunctional CDN intratumoral administration in a mouse CT26 cell transplantation model (administered tumor).

[0960] Figure 1B This demonstrates the in vivo tumor-suppressive effect of bifunctional CDN intratumoral administration in a mouse CT26 cell transplantation model (non-administered tumor).

[0961] Figure 2 This demonstrates the in vivo tumor-suppressing effect of the dual-load CDN-ADC disclosed herein in a mouse EMT6-hHer2 cell transplantation model.

[0962] Figure 3This demonstrates the in vivo tumor-suppressing effect of the dual-load CDN-ADC of this disclosure in a mouse GP2D cell transplantation model.

[0963] Figure 4 This demonstrates the in vivo tumor-suppressing effect of the dual-load CDN-ADC disclosed herein in a mouse AsPC1 cell transplantation model.

[0964] Figure 5 This demonstrates the in vivo tumor-suppressing effect of the dual-load CDN-ADC disclosed herein in a mouse SK-OV-3 cell transplantation model.

[0965] Figure 6 This diagram shows a bifunctional cyclic dinucleotide payload 1 and payload 2 in the dual-load ADC of this disclosure, which are linked to an antibody via a key intermediate PL. Detailed Implementation

[0966] The present disclosure will be further described below with reference to embodiments. It should be noted that the following embodiments should not be construed as limiting the scope of protection of the present disclosure.

[0967] Unless there is an obvious error in the structural formula, the structural formula shall prevail when the chemical name of any compound disclosed herein is inconsistent with the given structural formula.

[0968] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard conditions for such reactions or as recommended by the manufacturer. Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available, prepared using existing methods, or prepared using methods similar to those disclosed in this application.

[0969] Unless otherwise stated, percentages and parts are weight percentages and weight parts; ratios of liquids are volume ratios; unless otherwise stated, all temperatures are given in degrees Celsius.

[0970] In the following embodiments, 1 H NMR spectrum 31P NMR spectra are typically recorded using a Bruker 400MHz and 500MHz NMR spectrometer, with chemical shifts expressed as δ (ppm). Mass spectrometry is recorded using an Agilent 1290 HPLC system with a 6120B LCMS. Silica gel column purification is performed using a Biotage Selekt SEL-2SV or ISO-1SV. Preparative HPLC purification is performed using a Gilson 281 (column: Waters Xbridge 19mm × 250mm × 5μm or Welch C18, 21.2mm × 250mm × 10μm. Mobile phase: A: water (10mM NH4HCO3 or 0.05% formic acid), B: acetonitrile (or containing 0.05% formic acid). Flow rate: 20-30 mL / min. Detection wavelength: 214nm / 254nm), or as otherwise specified.

[0971] The following abbreviations are used in the synthetic embodiments below. The abbreviations not listed have the meanings commonly understood by those skilled in the art.

[0972] A (absorbance); AA (amino acid); ACN or CH3CN or MeCN (acetonitrile); ADC (antibody-drug conjugate); Boc (tert-butoxycarbonyl); (Boc)2O (di-tert-butyl dicarbonate); BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate); t-Bu (tert-butyl); t-BuOK (sodium tert-butoxide); CBr4 (carbon tetrabromide); Cbz (benzyloxycarbonyl); CDCl3 (deuterated chloroform); CDN (cyclic dinucleotide); CD3OD-d4 (tetradeuterated methanol); cm (cm); cm 2 (square centimeters); CsCO3 (cesium carbonate); DAR (drug-antibody ratio); DCM (dichloromethane); DIEA or DIPEA (N,N-diisopropylethylamine); DMF (N,N-dimethylformamide); DMSO (dimethyl sulfoxide); DMSO-d6 (hexadeuterated dimethyl sulfoxide); DMTMM (4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride); D2O (deuterated water); DTT (dithiothreitol); E (trans-olefin); EA (ethyl acetate); ED50 (50% effective dose); ELISA (enzyme-linked immunosorbent assay); eq (equivalent); Et2NH (diethylamine); 19 F (fluorine nucleus); FCC (fast column chromatography); Fmoc (9-fluorenylmethoxycarbonyl); g (gram); h (hour); 1H (proton); H2 (hydrogen); HATU (tetramethylurea hexafluorophosphate); HCl (hydrogen chloride); HF (hydrogen fluoride); HFIP (hexafluoroisopropanol); HMDSLi (lithium bis(trimethylsilylamine)); H2O (water); HOBt (1-hydroxybenzotriazole); HPLC (high performance liquid chromatography); Hz (Hertz); I (optical path length); I2 (iodine); IC50 (50% inhibition concentration); IFNβ (interferon beta); in vitro (in vitro experiment); in vivo (in vivo); IV or iv (intravenous administration); J (nuclear magnetic resonance coupling constant); KI (potassium iodide); L (liter); L (relative stereoconfiguration); LCMS (liquid chromatography-mass spectrometry); LC-MS / MS (liquid chromatography-mass spectrometry-mass spectrometry); LiOH (lithium hydroxide); M or MW (molecular weight); M (molar concentration); NaI (sodium iodide); MeOH (methanol); MeONa (sodium methoxide); mg (milligram); MHz (megahertz); min (minute); mL (milliliter); mm (millimeter); mm 3 (millimeters); mM (millimolecular concentration); mmol (millimole); MS (mass spectrometry); MS-ESI (electron spray ionization mass spectrometry); MW (microwave); m / z (mass-charge ratio); N2 (nitrogen); NaCl (sodium chloride, table salt); Na2CO3 (sodium carbonate); NaOAc (sodium acetate); Na2SO4 (sodium sulfate); NCS (chlorosuccinimide); NH3 (ammonia); NH4Cl (ammonium chloride); NH4HCO3 (ammonium bicarbonate); nm (nanometer); NMR (nuclear magnetic resonance); 31 P (phosphorus nucleus); PABC (p-aminobenzyl alcohol); Pb(OAc)4 (lead tetraacetate); PBS (phosphate-buffered saline); Pd / C (palladium / carbon); Pd(OAc) (palladium acetate); Pd(OH)2 (palladium hydroxide); Pd(PPh3)4 (tetra(triphenylphosphine palladium)); PE (petroleum ether); PPh3 (triphenylphosphine); PO or PO or po (oral administration); psi (pounds per square inch); Py (pyridine); R (R configuration of chiral molecules); RP-HPLC (reversed-phase high-performance liquid chromatography); rt (room temperature); R t(Chromatographic retention time); S (S configuration of chiral molecules); s / d / t / q / m (NMR single / double / triple / tetraple / multiple splitting peaks); SFC (supercritical fluid chromatography); SiO2 (silica gel); SO2Cl (sulfonyl chloride); TCEP (tris(2-carboxyethyl)phosphine); TEA or Et3N (triethylamine); TBSCl (dimethyl-tert-butylchlorosilane); TFA (trifluoroacetic acid); TGI (tumor growth inhibition rate); THF (tetrahydrofuran); TLC (thin-layer chromatography); TMSI (trimethyliodosilane); TsOH (p-toluenesulfonic acid); TsOH·H2O (p-toluenesulfonic acid monohydrate); v / v (volume ratio); w / w (weight ratio); Z (cis-olefin); δ (chemical shift); μL (microliter); μm (micrometer); μM (micromolar concentration); μmol (micromolar).

[0973] Preparation of load-connector intermediate PL

[0974] 1) Preparation of linker intermediate Pep

[0975] Preparation of Pep-1

[0976]

[0977] Step A: At 0°C, Fmoc-L-citrulline (5.0 g, 12.58 mmol) was added to anhydrous DMF (200 mL), followed by the sequential addition of HATU (5.7 g, 15.09 mmol) and 4-(hydroxymethyl)aniline (1.85 g, 15.09 mmol), and then dropwise DIEA (2.4 g, 18.87 mmol). The resulting mixture was stirred at 0°C to room temperature for 2 hours. After the reaction was complete, the mixture was poured into ice water (1000 mL), stirred at room temperature for 10 minutes, filtered, and the filter cake was collected and vacuum dried to give a yellow solid compound, Pep-1-2 (5.8 g, yield 92.1%). MS-ESI[M+H] + :503.2.

[0978] Step B: At 0°C, diethylamine (20 mL) was added to a solution of compound Pep-1-2 (2.5 g, 4.97 mmol) in tetrahydrofuran (20 mL), and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated to remove the solvent, and a mixture of methyl tert-butyl ether and tetrahydrofuran (300 mL: 10 mL) was added and stirred for 10 minutes. The mixture was filtered, the filter cake was collected and dried under vacuum to give a yellow solid compound Pep-1-3 (1.0 g, yield 71.9%). MS-ESI [M+H] + :281.2.

[0979] Step C: At 0°C, 10 g (24.42 mmol) of N-fluorenylmethoxycarbonyl-L-glutamic acid 5-allyl ester (compound Pep-1-4, CAS No. 133464-46-7, purchased from Shanghai Bid Pharmaceutical) was added to anhydrous DMF (200 mL), followed by the addition of HATU (11.1 g, 29.3 mmol) and L-valine tert-butyl hydrochloride (5.1 g, 24.42 mmol), and then DIEA (6.3 g, 48.84 mmol) was slowly added dropwise. The resulting mixture was stirred at 0°C to room temperature for 1 hour. After the reaction was complete, the reaction solution was poured into ice water (1000 mL), stirr...

Claims

1. A dual-load antibody-drug conjugate of formula (X-1), in, Ab represents an antibody or antigen-binding fragment; L represents the connecting body unit that connects D1 and D2 to Ab; D1 represents the first effective load, which is a compound of formula (II): in B1 is adenine that can be substituted by X. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl; or optionally R b Replaced guanine Where R b Selected from H or -C 1-6 alkyl, R1 and R1' are each independently selected from H, F, or -OH. B2 is guanine. R b Selected from H or -C 1-6 alkyl, This indicates that the phosphate bond can be attached to either the 2' or 3' position of the pentose sugar, where the site not cyclized with the phosphate is replaced by R2 and R2'. R2 and R2' are each independently selected from H, -OH, or F. D2 represents the second payload; q indicates connection to Ab. The quantity is approximately 1 to 20, either an integer or a non-integer. D1 is connected to L through an amino group on any one of the -SH, B1 or B2 bases or a -OH group on a sugar ring, preferably through any one of the -SH bases; Or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled forms.

2. The dual-load antibody-drug conjugate according to claim 1, wherein B1 in formula (II) is adenine. Where X is selected from H, Cl, F or -NHC 1-6 Alkyl groups, or guanine Where R b -C 1-6 Alkyl, for example 3. The dual-loaded antibody-drug conjugate of claim 1 or 2, wherein R1 and R1' in formula (II) are both H, or one of them is H and the other is F, or one of them is H and the other is OH.

4. The dual-load antibody-drug conjugate of any one of claims 1 to 3, wherein B2 in formula (II) is guanine.

5. The dual-loaded antibody-drug conjugate of any one of claims 1 to 4, wherein one of R2 and R2' in formula (II) is H and the other is OH.

6. The dual-load antibody-drug conjugate of any one of claims 1 to 5, wherein formula (II) has the following formula: Specifically, the segment of equation (II) of L is connected in the following way:

7. The dual-loaded antibody-drug conjugate of claim 6, wherein R1 and R1' are both H, or wherein R1' is H and R1 is F, or wherein R1' is H and R1 is OH.

8. The dual-load antibody-drug conjugate of claim 6 or 7, wherein B1 is And R1 is F and R1' is H; or B1 is And R1 is OH and R1' is H; or B1 is And R1 is F and R1' is H.

9. The dual-load antibody-drug conjugate of any one of claims 6 to 8, wherein B2 is guanine.

10. The dual-loaded antibody-drug conjugate of any one of claims 6 to 9, wherein R2' is H and R2 is -OH.

11. The dual-loaded antibody-drug conjugate of claim 1, wherein the compound of formula II is selected from: Or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled forms.

12. The dual-load antibody-drug conjugate according to any one of claims 1 to 11, wherein the second payload D2 is any therapeutic agent compatible with the cGAS-STING pathway, preferably an antitumor agent, such as selected from topoisomerase I inhibitors like camptothecin or derivatives thereof, further preferably cytotoxic agents, PARP inhibitors, nucleoside derivatives, folic acid antagonists, microtubule inhibitors, nuclear receptor modulators, immunomodulators, epigenetic modulators, topoisomerase II inhibitors, phosphatase inhibitors, kinase inhibitors; or For example, the second payload D2 is selected from topoisomerase I inhibitors such as camptothecin or its derivatives, PARP inhibitors, kinase inhibitors, topoisomerase II inhibitors, nucleoside derivatives, microtubule inhibitors, and nuclear receptor modulators; or For example, the second effective load D2 is selected from Or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled forms.

13. The dual-load antibody-drug conjugate according to any one of claims 1 to 12, wherein formula (X-1) has the following formula: in, Ab represents an antibody or antigen-binding fragment; G indicates the antibody linker that is connected to Ab; E represents an optional property adjustment unit; W indicates a branch connector; P1 and P2 are each independently cleavable linkers, selected from peptide residues of 2-8 amino acids, preferably dipeptides, tripeptides or tetrapeptides; A1 and A2 are each independently optional self-decomposing connectors; S1 and S2 are each an independently arbitrarily existing solubilizing unit; D1 is defined as in any one of claims 1 to 11; D2 as defined in claim 12; q represents an integer or non-integer from 1 to 20; For example, the equation (I-1) when E does not exist: For example, equation (I-2) when E and S1 do not exist: For example, equation (I-3) when E, S1, and S2 all do not exist:

14. The dual-load antibody-drug conjugate of claim 13, wherein G has the following structure: -G1-G2-, in: G1 is selected from: • 5-10 membered heterocyclic groups, containing one or two heteroatoms selected from N, S, and O, wherein the ring carbon atom is optionally oxidized; and • The asterisk (*) on the left indicates the binding point with the antibody, and the asterisk (*) on the right... Indicates the connection point with G2; G2 is selected from -C 1-10 Alkylene -C(=O)-, -C 1-10 Alkylene-C(=O)-NH-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, wherein G2 is connected to the property regulating unit E (when present) or the branching unit W (when E is absent) via its -C(=O-), and the other end group is connected to G1, or G2 is selected from -C 1-10 Alkylene-NH-, wherein G2 is connected to property regulating unit E (when present) or branching unit W (when E is absent) via its -NH-, and the other end group is connected to G1; Among them, -C in G2 1-10 The alkylene group is optionally substituted with a Bu group having the formula (G): in, T0 is -C 1-6 alkylene-; T is selected from -C(R) a )2-、-O-、-NR a -or does not exist; M is selected from -CO-, -O-, -NR a -or does not exist; Z is selected from glycosyl groups or their derivatives and containing... The hydrophilic peptide is preferably selected from glucose, mannose, galactose or their respective uronic acids, amino sugars, acylated amino sugars, aminouronic acids or acylated aminouronic acid derivatives, and hydrophilic peptides. R, R a Each independently is C 1-6 Alkyl group, preferably -CH3, the segment shown in square brackets is polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, more preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units; R a Selected from H or C 1-6 alkyl; Subunit Selected from natural and non-natural amino acid residues; n is an integer from 0 to 4, for example 0-2, 1-2, 1-4, 2-4; m is an integer from 0 to 20, such as 1-20, 1-10, 2-10, 2-8, 2-6, 2-4, 4-14, 4-12, 4-10, 5-10, 6-10, 6-12, 8-10, 8-14, for example 4-14, 4-12, 6-12 or 4-10, 5-10.

15. The dual-load antibody-drug conjugate according to claim 13 or 14, wherein G is selected from: For example, selected from 16. The dual-loaded antibody-drug conjugate according to any one of claims 13 to 15, wherein E, when present, is a polyethylene glycol chain composed of 2-12 ethylene oxide (CH2CH2O) subunits, via -NH-, -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)-(CH2) 1-4 -O-(CH2) 1-4 -C(=O)- or -C 1-4 Alkylene-NH-(CH2) 1-4 -O-(CH2) 1-4 -C(=O)- connects between G and W.

17. The dual-loaded antibody-drug conjugate according to any one of claims 13 to 16, wherein W is an amino acid residue, preferably The -NH terminal is connected to E or G, and the -CO- terminal is connected to P1 and P2.

18. The dual-load antibody-drug conjugate according to any one of claims 13 to 17, wherein P1 or P2 is a cleavable peptide having the following structure from the N-terminus to the C-segment: -(AA) d - where each of AA is independently selected from alanine, glycine, asparagine, isoleucine, leucine, valine, phenylalanine, citrulline, and glutamic acid; preferably, AA is selected from alanine, glycine, asparagine, valine, phenylalanine, citrulline, and glutamic acid; d is an integer from 2 to 12, preferably an integer from 2 to 8, and more preferably an integer from 2 to 4. For example, P1 or P2 is selected from: The carbonyl end is connected to the self-decomposing linker A1 or A2, and the N end is connected to the branching unit W.

19. The dual-load antibody-drug conjugate according to any one of claims 13 to 18, wherein A1 or A2 is selected from... in, The site marked with * is the connection point with D1 or D2, and the bond indicated by the wavy line is connected to the cuttable connector P1 or P2; Each occurrence of Y1 is independently selected from NH or O; Each occurrence of R 16 Each is independently selected from H and -C 1-6 Alkyl groups, preferably H or C 1-3 Alkyl, more preferably H or methyl; Each occurrence of R 17 Each is independently selected from H, -NO2, -NH2, and -CF3; For example, A1 or A2 is selected from For example, A1 is selected from And A2 is selected from For example, A1 is selected from And A2 is selected from 20. The dual-load antibody-drug conjugate according to any one of claims 13 to 19, wherein when A1 and / or A2 comprises a benzene ring structure, it is optionally substituted with a solubilizing unit S1 or S2 of the following formula (S): in: T' is selected from -C(R) a )2-、-O-、-NR a -or does not exist; M' is selected from -CO-, -O-, -NR a -or does not exist; Z' is selected from glycosyl groups or their derivatives and contains The hydrophilic peptide, wherein the glycosyl group or its derivative is preferably selected from glucose, mannose, galactose, lactose, maltose, sucrose or their respective uronic acid, amino sugar, acylated amino sugar, aminouronic acid or acylated aminouronic acid derivative, more preferably glucose, mannose, galactose or their respective derivatives, wherein the hydrophilic peptide is preferably... More Where R can be R a Preferably, the fragments shown in square brackets are polysarcosine, polyarginine, and polyglycine, with polysarcosine being the most preferred. R a Selected from H or C 1-6 Alkyl group, preferably H or -CH3, more preferably -CH3; Subunit Selected from natural and non-natural amino acid residues; n' is an integer from 0 to 4, for example, 0-2, 1-4, 1-2, 2~4; m is an integer from 0 to 20, such as 1-20, 1-10, 2-10, 2-8, 2-6, 2-4, 4-14, 4-12, 4-10, 5-10, 6-10, 6-12, 8-10, 8-14, for example 4-14, 4-12, 6-12 or 4-10, 5-10.

21. The dual-load antibody-drug conjugate according to any one of claims 13 to 20, wherein... A1 and A2 are each selected independently. Preferred S1 or S2 is attached to the benzene ring of A1 or A2, and is Where T' is selected from -C(R) a )2-、-O-、-NR a -or does not exist, M' is selected from -CO-, -O-, -NR a - or does not exist, n is an integer from 0 to 4, R a For H or C 1-6 alkyl; Z' is a monosaccharide or disaccharide or a derivative thereof, for example selected from glucose, mannose, galactose, lactose, maltose, sucrose or their uronic acid, amino sugar, acylated amino sugar, aminouronic acid or acylated aminouronic acid derivatives thereof, preferably the monosaccharide glucose, mannose, galactose or their respective derivatives as shown; or Z' is Where R is -C 1-6 Alkyl group, preferably -CH3, the segment shown in square brackets specifically refers to polysarcosine, polyarginine, or polyglycine containing 4-14 units or 4-10 or 5-10 units, preferably polysarcosine containing 4-12 or 6-12 units or 4-10 or 5-10 units.

22. The dual-load antibody-drug conjugate according to any one of claims 1 to 21, wherein the linker unit L is: The wavy line indicates the key connected to Ab, and the asterisk indicates the connection point with D1 or D2.

23. The dual-load antibody-drug conjugate according to any one of claims 1 to 22, wherein Ab is an antibody or antigen-binding fragment thereof that binds to a tumor-specific antigen or a tumor-associated antigen, wherein the tumor-specific antigen or tumor-associated antigen is selected from: HER2, Her3, HER1 (ErbB1), HER4 (ErbB4), TROP2, Nectin4, tissue factor, PD-L1, PD-1, PD-L1 / PD-L2, MET, CLDN18.2, KIT, CTL A-4, RPR1, EphA2 receptor, folate receptor (FRa), mesothelin, endothelin receptor, GCPII, IL-13Ra, BCMA, GD2, CLL-1, CA-IX, MUC1, 5T4, AOC3, ALK, AXL, C242, CA-125, CCL11, CCR5, CD2, CD3, CD4, CDS, CD15, CA15-3, CD16, CD18, CD19, CD20, CD21 CD22, CD25, CD30, CD32, CD33, CD37, CD38, CD44, CD52, CD56, CD64, CD66e, CD70, CD72, CD74, CD79a, CD79b, CD123, CD138, CD142, CD174, CD276, CDH3, CDH6, CDH17, CCD79b, CLDN9 / CLDN6, CA19-9, DPEP3, AGS-16, IGF1 R, IGF2R, VEGFR1, VEGFR2, VEGFR3, PDGFR-α, PDGFR-β, EGFR, EGFRvIII, ENPP3, FcRH5, FRα, KAAG1, LIV-1, Me sothelin, cMet, ROR1, SLTRK6, TF, BMPR1B, E16, TOP1, STEAP1, Steap2, 0772P, MUC16, Napi2b, Napi3b, Sema 5b. PSCAhIg, ETBR, RNF124, prostate cancer-associated gene 1, TrpM4, teratoma-derived growth factor 1, C3DR, FcRH2, NCA, MDP, IL20R-α, Brevican, EphB2R, ASLG659, prostate stem cell antigen precursor, GEDA, BAFF-R, CXCR5, CCR2, CCR8, HLA-DOB, P2X5, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, integrin α4β7, FGF2, FGFR1, FGFR2, FGFR3, FGFR4, PSMA, somatostatin receptor, RANK, SLAMF7, ITGB6, CEACAM5, CA9, EGFRv111, IL2RA, AXL receptor tyrosine kinase, TGF-βR, TNFRSF8, cancer / testis-associated antigen, CLEC14A, GRP78, stem cell-specific antigen, ASG -5, PRR4, GUCY2C, SLC39A6, TPBG, tumor-associated antigen CA242, FOLR1, GPNMB, HAVCR1, prostate tumor target Mindin, VTCN1, PTK7 protein tyrosine kinase 7, macrophage stimulation receptor 1, TACSTD2, CA6, DLL3, DLL4, EpCAM, FAP, Fibronectin-EDB, DKK-1, Endoglin, VCAM1, GPC3, DR5, ASCT2, B7H1, B7H3, B7H4; preferably, the tumor-specific antigens or tumor-associated antigens are selected from AXL, B7H1, B7H3, B7H4, BCMA, CD16, CD19, CD22, CD25, CD30, CD32, CD33, CD44, CD64, CD70, CD74, CD79, CD138, CD142, CD276, CDH3, CEACAM5, Claudin 18.2, CLDN9 / CLDN6, DPEP3, EGFR, ENPP3, EphA, FcRH5, FOLR1, FRα, GCPII, HER2, HER3, KAAG1, KIT, LIV-1, Mesothelin, cMet, MUC1, NECTIN4, PD-L1, PD-L1 / PD-L2, PSMA, ROR1, RPR1, TF, TOP1, TROP2; More preferably, the tumor-specific antigen or tumor-associated antigen is selected from antibodies or antigen-binding fragments that specifically bind to HER2, Claudin 18.2, EGFR and / or TROP2.

24. The dual-load antibody-drug conjugate of any one of claims 1 to 23, wherein the Ab comprises three heavy chain complementarity-determining regions (HCDRs) and three light chain complementarity-determining regions (LCDRs), wherein: According to Kabat's definition, 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:

3. LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:4, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:5; or HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:

18. HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:

19. HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:

20. 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 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:

26. LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:27, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:28; or HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:

40. HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:

41. HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:

42. LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:

37. LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:38, and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:

39.

25. The dual-load antibody-drug conjugate of any one of claims 1 to 23, wherein the Ab 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:10, and wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; or The heavy chain variable region comprises the amino acid sequence described in SEQ ID NO:22, and the light chain variable region comprises the amino acid sequence described in SEQ ID NO:21; or The heavy chain variable region comprises the amino acid sequence described in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32; or The heavy chain variable region comprises the amino acid sequence described in SEQ ID NO:44, and the light chain variable region comprises the amino acid sequence described in SEQ ID NO:

43.

26. The dual-load antibody-drug conjugate of any one of claims 1 to 23, wherein the Ab comprises: (a) A heavy chain containing the amino acid sequence of SEQ ID NO:2, and (b) A light chain containing the amino acid sequence of SEQ ID NO:1; or (a) A heavy chain containing the amino acid sequence of SEQ ID NO:14, and (b) A light chain containing the amino acid sequence of SEQ ID NO:13; or (a) A heavy chain containing the amino acid sequence of SEQ ID NO:25, and (b) A light chain containing the amino acid sequence of SEQ ID NO:24; or (a) A heavy chain containing the amino acid sequence of SEQ ID NO:36, and (b) A light chain containing the amino acid sequence of SEQ ID NO:

35.

27. The dual-loaded antibody-drug conjugate of any one of claims 1 to 23, wherein the Ab is selected from trastuzumab, pertuzumab, margetuximab, or HT-19, or an antibody fragment thereof, or other anti-human HER2 antibodies that recognize the same epitope or competitively bind to human HER2, preferably trastuzumab; or The Ab is selected from cetozumab, datopotamab, or its antibody fragments, or other anti-human TROP2 antibodies that recognize the same epitope or competitively bind to human TROP2, preferably cetozumab; or The Ab is selected from zotuximab, osemitamab (TST001), CMG901, ASKB589, ZL-1211, or antibody fragments thereof, or other anti-human Claudin18.2 antibodies that recognize the same epitope or competitively bind to human Claudin18.2, preferably zotuximab; or The Ab is selected from cetuximab, panitumumab, nimotuzumab, necitumuma, amivantamab, depatuxizumab, NECITUMUMAB, IZALONTAMAB, BAFISONTAMAB, Petosemtamab, PIMURUTAMAB, FUTUXIMAB, and MODOTUXIMAB, or antibody fragments thereof, or other anti-human EGFR antibodies that recognize the same epitope or competitively bind to human EGFR, with cetuximab being preferred.

28. The dual-load antibody-drug conjugate according to any one of claims 1 to 27, wherein q is an integer or non-integer from 1 to 10 or a range consisting of any two values ​​between 1 and 10, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, 4 to 6, 4 to 8, 4 to 10, 6 to 8 or 6 to 10; preferably q is about 1 to 8 or the average DAR value of a range of any two values ​​between 1 and 8, for example about 1, 2, 3, 4, 5, 6, 7, 8, about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, 4 to 6, 4 to 8, 4 to 10, 6 to 8 or 6 to 10.

29. The dual-load antibody-drug conjugate according to claim 1, wherein the drug is selected from... Or its pharmaceutically acceptable salts, esters, isomers, solvates, prodrugs, or isotope-labeled forms.

30. Connector unit of formula (IV) - payload fragment compound, For example in: The wavy line represents the connection bond with the targeted ligand; D1 is as defined in any one of claims 1 to 11, preferably, Further for Wherein the asterisk indicates the connection point with the connecting body unit, B1, B2, R1, R1', R2, R2' are defined as in any one of claims 1 to 10; preferably D1 is defined as in claim 11; D2 as defined in claim 12; G, E, W, P1, P2, A1, A2, S1, and S2 are respectively defined as in any one of claims 13 to 22; Preferably, the fragment compounds of formula (IV) are the fragment compounds PL-3 to PL-41 and PL-45 to PL-59 described in the specification.

31. A pharmaceutical composition comprising the dual-loaded antibody-drug conjugate of any one of claims 1 to 29 and one or more pharmaceutically acceptable excipients.

32. The pharmaceutical composition of claim 31, which is administered intravenously, intratumorally, subcutaneously, intramuscularly, orally, intranasally, intrathecally, transdermally, or locally, preferably intravenously, intraperitoneally, subcutaneously, or intramuscularly.

33. A method for treating or preventing diseases associated with or mediated by STING, including inflammatory, allergic or autoimmune diseases, infectious diseases such as viral infections or hyperproliferative diseases, comprising administering to a subject in need a therapeutically effective amount of the dual-loaded antibody-drug conjugate of any one of claims 1-29 or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotopic label thereof or a pharmaceutical composition of claim 31 or 32.

34. Use of the dual-loaded antibody-drug conjugate of any one of claims 1-29, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotopic label thereof, or the pharmaceutical composition of claim 31 or 32, in the prevention or treatment of diseases associated with or mediated by STING, more specifically in the treatment or prevention of inflammatory, allergic, or autoimmune diseases, infectious diseases such as viral infections, or hyperproliferative diseases.

35. The use of the dual-loaded antibody-drug conjugate of any one of claims 1-29, or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug, or isotopic label thereof, or the pharmaceutical composition of claim 31 or 32, in the preparation of a drug for the prevention or treatment of diseases associated with or mediated by STING, more specifically inflammatory, allergic, or autoimmune diseases, infectious diseases such as viral infections, or hyperproliferative diseases.

36. The use of claim 34 or 35, wherein the hyperproliferative disease refers to a tumor or cancer selected from brain cancer, skin cancer, bladder cancer, ovarian cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, leukemia, lung cancer, bone cancer, neuroblastoma, intestinal cancer such as rectal cancer, colon cancer, colorectal cancer, familial adenomatous polyposis carcinoma and hereditary non-lymphatic colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal carcinoma, pharyngeal squamous cell carcinoma, oral cancer, head and neck cancer, salivary gland cancer, peritoneal cancer, soft tissue sarcoma, urethral epithelial carcinoma, sweat gland cancer, gastric cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, cervical cancer, uterine cancer, endometrial cancer, testicular cancer, breast cancer (including HER2-negative breast cancer), urinary cancer, melanoma, and brain tumors such as glioblastoma. Astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL) and lymphocytic carcinoma, acute myeloid leukemia (AML), myeloid leukemia (chronic myeloid leukemia (CML), adult T-cell lymphoma, diffuse lymphoma (DLBCL), liver cancer, hepatocellular carcinoma, multiple myeloma, seminoma, osteosarcoma, chondrosarcoma, anal canal cancer, renal cell carcinoma, adrenocortical carcinoma, chordoma, fallopian tube cancer, gastrointestinal stromal tumor, myeloproliferative disorders, mesothelioma, biliary tract cancer, Ewing sarcoma and other rare tumor types, as well as recurrent forms of the above tumors; Preferred cancer types include small cell lung cancer, non-small cell lung cancer, colorectal cancer, liver cancer, breast cancer, ovarian cancer, stomach cancer, prostate cancer, melanoma, renal cell carcinoma, head and neck cancer, soft tissue sarcoma, pancreatic cancer, Hodgkin's lymphoma, leukemia, or bladder cancer.

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