Improved linker-payloads for antibody conjugation, pharmaceutical compositions and applications thereof

Abstract

The present disclosure provides linker-payload conjugates including a cysteine-reactive group and a hydrophilic moiety. The improved linker-payloads for biomolecules conjugation and improved drug conjugates exhibit greater stability in blood circulation and enhanced drug delivery and drug release efficiencies in target cells. The present disclosure also relates to antibodies and antigen-binding fragments thereof for several antigen targets (e.g. cMET, HER3, EGFR, TROP2, HER2, Nectin-4, etc.), as well as pharmaceutical compositions including ADCs. Also described herein are methods of using ADCs for treatment of subjects associated with pathological conditions.

Description

IMPROVED LINKER-PAYLOADS FOR ANTIBODY CONJUGATION, PHARMACEUTICAL COMPOSITIONS AND APPLICATIONS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Patent Applications No. 63 / 640,190 (filed on April 29, 2024). The entirety of the aforementioned application is incorporated herein by reference. FIELD

[0002] The present disclosure relates to linker-payloads, antibody conjugates using drug unit or probe unit, and methods for treatment of cancers using the antibody conjugates. BACKGROUND OF THE INVENTION

[0003] A great deal of interest in cancer therapies is focused on the use of monoclonal antibodies (mAbs) for the targeted delivery of cytotoxic agents to cancer cells. The design of antibody-drug conjugates (ADCs), by attaching a drug to an antibody, typically via a linker, involves consideration of a variety of factors. These factors include the types of the antibody and the linker for conjugation of the drug, how the antibody, the linker, and the drug are linked. Further, if the drug is to be released after antibody internalization, the mechanism of drug release, and the associated structural elements, and the structural modification of the drug after release, if any, also need to be taken into account. For example, the structural elements and mechanisms of drug release must be consistent with the intracellular trafficking of the conjugate.

[0004] The antibodies for ADC construction can be small protein formats (e.g., scFv’s, Fab fragments, designed ankyrin repeat proteins, Affibodies, Nanobody, etc.) and monoclonal antibodies (mAbs) which have been selected based on their high selectivity and affinity for a given antigen, their long circulating half-lives, and little to no immunogenicity. Thus, mAbs as protein ligands for a carefully selected biological receptor provide an ideal platform for the delivery of selective drugs to target cells. For example, a monoclonal antibody known to bind selectively with a tumor-associated antigen can be used for delivery of a conjugated cytotoxic agent to the tumor through site-specific binding, internalization, intracellular processing, and final release of the cytotoxic agent. The cytotoxic agent may be a small molecule toxin, a protein toxin, or in other formats, like oligonucleotides. Hence, the concept of targeted drug delivery to a specific cellular location of choice is a powerful approach for the treatment of a wide range of diseases, with many beneficial aspects versus systemic delivery of the same drug.

[0005] In the field of ADCs, a chemical linker is typically employed to attach a drug to an antibody. This linker needs to possess several key attributes, including the requirement to be stable in blood during circulation after ADC administration for an extended period of time. This is intended to reduce the risks of inadvertently releasing the cytotoxic agent prematurely and potentially improve the safety of ADCs. A stable linker enables the localization of the ADC to the projected site or cells in the body and prevents the premature release of thedrug in circulation, which would indiscriminately induce undesired biological response of all kinds, thereby lowering the therapeutic index of the ADC. Upon internalization, the ADC is processed such that the drug is effectively released to kill the targeted cells.

[0006] Although recent clinical trials have demonstrated the efficacy of ADCs in cancer therapy, several challenges remain. One of these is the off-target side effects caused by premature drug release into the bloodstream, which also affects the therapeutic index of ADCs. Therefore, there is an urgent need to develop new linker-payloads to generate ADCs with improved stability and well-controlled drug release efficiency. SUMMARY OF THE INVENTION

[0007] The inventors have found that linker-payloads with cleavable linkers are highly suitable for cysteine or thiol conjugation of drugs to antibodies. The resulting ADCs were found to display significant in vitro and in vivo therapeutic index.

[0008] In one aspect, the present disclosure provides a linker-payload for conjugation with antibodies. The linker-payload includes a branched hydrophilic moiety and a payload, such as a drug unit or a probe unit, that is conjugated with a linker unit that includes a cleavable moiety, for example, a protease-cleavable peptide. The hydrophilic moiety may be a hydrophilic polymer, such as polyethylene glycol (PEG), and may be extended to a suitable length.

[0009] In another aspect, the present disclosure provides an antibody conjugate, such as an antibody-drug conjugate (ADC) or an antibody-probe conjugate prepared by conjugating the linker-payload with an antibody or an antigen-binding fragment thereof. The incorporation of the linker-payload disclosed herein in ADCs may enhance drug delivery and the subsequent drug release efficiency, improving therapeutic index of the ADCs and lowering the administration of the effective dose.

[0010] In certain embodiments, the drug unit may be an immune stimulator, a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), a ligand, or a radioactive isotope; and the probe unit may be an imaging agent (e.g., a fluorophore). In certain embodiments, the antibody or antigen-binding fragment thereof is an monospecific antibody, such as an antibody specific to DLL3, CEACAM-5, CEACAM-6, ROR1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfr1, TNF alpha, tissue factor, folate receptor alpha, c-MET, HER3, EGFR, HER2, TROP2, or Nectin-4, or a multispecific antibody, such as a anti-HER2 and anti-TROP2 bispecific antibody.

[0011] In certain embodiments, the antibody is R4702, an anti-TROP2 monoclonal antibody. R4702 is as described in PCT Publication No. WO2022222992A1), the content of which is incorporated herein by reference in its entirety.

[0012] In certain embodiments, the antibody is an anti-TROP2 antibody selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamb, or sacituzumab. In certain embodiments, the antibody is an anti-Nectin 4 antibody, for examples, 08B04, 10K06, 14I08, 05O04, 12E03, 02P14, 11O23, 14B21, 08C24, 13C24 or enfortumab.

[0013] In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor, including a topoisomerase I inhibitor and a topoisomerase II inhibitor.

[0014] In certain embodiments, the chemotherapeutic agent is a topoisomerase I inhibitor, including but not limited to, camptothecin (CPT) compounds and non-camptothecin compounds. Examples of topoisomerase I inhibitor include Irinotecan, Topotecan, Camptothecin, Rubitecan, MLN576, Exatecan, Belotecan, Seconeolitsine, SN-38, Genz-644282, Betulinic acid, β-Lapachone, Karenitecin, Gimatecan, Namitecan, Edotecarin, SW044248, LMP744, T-2513, Podocarpusflavone A, Indimitecan, Lurtotecan, TP3011, or 10-hydroxycamptothecin.

[0015] In another aspect, the present disclosure concerns a process for preparing the linker-payload described herein and a process for preparing an antibody conjugate using the linker-payload.

[0016] In another aspect, the present disclosure provides a method for inhibiting proliferation of cancer cells, including administration of an effective amount of the ADC described herein to the cancer cells.

[0017] In another aspect, the present disclosure provides a method for treating cancer, including administering to a subject in need thereof an effective amount of the ADC described herein.

[0018] In another aspect, the present disclosure provides a pharmaceutical composition, including the ADC described herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition includes a therapeutically effective amount of the ADC. For each specific ADC, the therapeutically effective amount used depends on various factors, including but not limited to the physiological conditions (e.g., general health or age) of a subject, the type and severity of the cancer, the type of treatments, or the presence of other diseases. Generally, this amount is in the range of 0.01 μg-250 mg per kilogram body weight of a human subject. In some embodiments, the therapeutically effective amount (i.e., an effective dosage) may range from about 0.001 ^g / kg to 250 mg / kg, about 0.01 μg / kg to 100 mg / kg, about 0.1 μg / kg to 50 mg / kg, or may be at least about: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009; 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09;0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 100, 125, 150, 175, 200, 225, or 250 grams or micrograms per kilogram body weight of a human subject, or may range between any two of the amounts listed above.

[0019] In certain embodiments, the disease is characterized by expressing DLL3, CEACAM-5, CEACAM- 6, ROR1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfr1, TNF alpha, tissue factor, folate receptor alpha, c-MET, HER3, EGFR,HER2, TROP2, or Nectin-4. In certain embodiments, the cancer is selected from the group including but not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, or oral cancer.

[0020] The details of one or more embodiments of the invention are set forth in the description below. The features or advantages of the present invention will be apparent from the detailed description of preferred embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1. The non-reducing and reducing PAGEs and hydrophobic interaction chromatography (HIC) results of R4702 (anti-TROP2) ADC. FIG.1A shows SDS-PAGE and FIG.1B shows DAR distribution. LN0628-146: MCCa-R4702 ADC / LN0760-010: 2SP-R4702 ADC.

[0022] Figure 2. The non-reducing and reducing PAGEs and hydrophobic interaction chromatography (HIC) results of 10K06 (anti-Nectin-4) ADC. FIG. 2A shows SDS-PAGE and FIG. 2B shows DAR distribution. LN0819-022: 2SP-10K06 ADC.

[0023] Figure 3. Cytotoxicity assay of R4702 (anti-TROP2) ADC in human pancreatic carcinoma cell line (BxPC-3). FIG. 3A shows the results of cell viability assay and FIG. 3B shows cytotoxicity IC50 value.

[0024] Figure 4. Bio-activity comparison assay of 10K06 and commercial anti-Nectin-4 ADCs. FIG. 4A shows the results of binding activity EC50values and FIG. 4B shows cytotoxicity IC50values in human lung cancer cell line (NCI-H460).

[0025] Figure 5. In-vivo efficacy comparison of R4702 (anti-TROP2) ADCs. FIG. 5A shows the changes in tumor volume of NCI-H1975 human non-small cell lung cancer xenograft mice. FIG.5B shows the changes in tumor volume of DLD-1 human colorectal cancer xenograft mice.

[0026] Figure 6. In-vivo efficacy assay of 10K06 (anti-Nectin-4) ADCs in FaDu human hypopharyngeal squamous cell cancer xenograft mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Abbreviations

[0027] 2SP: 2-sulfonylpyrimidine group; ACN: acetonitrile; ADC: antibody-drug conjugate; ADCC: antibody-dependent cellular cytotoxicity; CDR: complementarity-determining region; DAR: drug-to-antibody ratio; DL: drug-linker compound; DLD: drug load distribution; DMSO: dimethyl sulfoxide; FA: formic acid; HATU: Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium; HC: heavy chain; HFIP: 1,1,1,3,3,3- hexafluoro-2-propanol; HIC: hydrophobic interaction chromatography; HRMS: high resolution mass spectrometer; LC: light chain; mAb: monoclonal antibody; NaOAc: sodium acetate; NMM: N- methylmorpholine; NaOH: sodium hydroxide; PBS: phosphate buffered saline; TFA: trifluoroacetic acid; ADC-1: R4702-MCCa-PEG24-VA-PAB-Exatecan; ADC-2: R4702-2SP-PEG24-VA-PAB-Exatecan; ADC-3:R4702-2SP-GGVA-PAB-PEG24-Exatecan; ADC-4: 10K06-MCCa-GGVA-PAB-PEG24-Exatecan; ADC- 5:10K06-2SP-GGVA-PAB-PEG24-Exatecan; DL-1: 2SP-GGVA-PAB-PEG24-Exatecan; DL-2: 2SP- Glu(PEG24)-VA-PAB-PEG24-Exatecan; DL-3: 2SP-PEG24-VA-PAB-Exatecan; DL-4: 2SP-VC-PAB-PEG24- Exatecan; DL-5: 2SP-GGVC-PAB-PEG24-Exatecan; DL-6: 2SP-GGFG-PAB-PEG24-Exatecan; DL-7: 2SP- GGFG-PAB-PEG24-MMAE; DL-8: Preparation of 2SP-GGFG-PAB-PEG24-T785; DL-9: 2SP-GGFG-PAB- PEG24-Prexasertib; DL-10: Benzoylacryl-GGVA-PAB-PEG24-Exatecan; DL-11: Maleimide-phenyl-GGVA- PAB-PEG24-Exatecan. Definitions

[0028] As used herein, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

[0029] As used herein, the term “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted immunoglobulin (Ig) bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

[0030] As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.

[0031] As used herein, the term “immunogenicity” refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0032] As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

[0033] As used herein, the term “vaccine” refers to a preparation that contains an antigen, for example, whole disease-causing organisms (killed or weakened) or components of such organisms, such as proteins, peptides, or saccharides, that is used to confer immunity against the disease that the organisms cause. Vaccine preparations can be natural, synthetic or derived by recombinant DNA technology.

[0034] As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

[0035] As used herein, the term “specifically binding” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10-6moles / liter, about 10-7moles / liter, or about 10-8moles / liter, or less.

[0036] The phrase “substantially similar,” “substantially the same”, “equivalent”, or “substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10% as a function of the value for the reference / comparator molecule.

[0037] The phrase “substantially reduced” or “substantially different”, as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value for the reference / comparator molecule.

[0038] The phrase “binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

[0039] The term “antibody” as used herein can be a full-length or a fragment (or a combination of fragments) of an antibody having an antigen-binding portion according to the context. The fragment includes, but are not limited to, Fab, F(ab')2, Fab', F(ab)', Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri- scFv), Fd, dAb fragment, an CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies formed from antibody fragments. Single-chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present disclosure.

[0040] The antibody disclosed herein or antigen-binding fragment thereof may be monospecific, bi-specific or multispecific.

[0041] The antibody disclosed herein may include a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof. The antibody or antigen- binding fragment thereof may be mammalian-derived, including murine and human antibodies.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof can bind to cMET, HER3, EGFR, TROP2, HER2, Nectin-4, or any combination thereof. In certain embodiments, the antibody or antigen-binding fragment thereof may have one or more amino acids being substituted, deleted, or added while these alternations do not have a substantial effect on the antibody’s biological properties such as binding affinity. In certain embodiments, the antibody or antigen-binding fragment thereof may have amino acid substitutions in the framework region to improve the binding affinity of the antibody to an antigen. In certain embodiments, a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids. The donor framework can be a mature or germline human antibody framework sequence or a consensus sequence.

[0043] The antibody or antigen-binding fragment thereof, can be derivatized or linked to other functional molecules. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag), amino acid linkers, signal sequences, immunogenic carriers, or ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and staphylococcal protein A. One type of derivatized protein is produced by crosslinking two or more proteins (of the same type or of different types). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, 111. Useful detectable agents with which a protein can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting, exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase and the like. A protein can also be derivatized with a prosthetic group (e.g., streptavidin / biotin and avidin / biotin).

[0044] The term “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity. Insome instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0045] The term “Framework” or “FW” residues refer to those variable domain residues other than the hypervariable region residues as herein defined.

[0046] The term “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0047] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein.

[0048] A “disorder” is any condition that would benefit from treatment with an antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer.

[0049] The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0050] “Tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0051] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, or oral cancer.

[0052] As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and / or tissue / organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.

[0053] An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice, or rats. In certain embodiments, the vertebrate is a human.

[0054] “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is human.

[0055] An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0056] A “therapeutically effective amount” of a substance / molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

[0057] A “combination therapy” refers to a combination of an amount of an ADC and an amount of other biological or chemical drugs that when administered together (either as co-administration and / or co- formulation), either sequentially or simultaneously, on the same or different days during a treatment cycle, have a synergistic effect that is therapeutically effective and more than therapeutically additive.

[0058] The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. Cytotoxic agents include radioactive isotopes (e.g.,211At,131I,125I,90Y,186Re,188Re,153Sm,212Bi,32P,60C, and radioactive isotopes of lutetium-177, strontium-89 and samarium (153Sm) ), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0059] The term “photodynamic therapy (PDT)’, sometimes called photochemotherapy, is a form of phototherapy involving light and a photosensitizing chemical substance, used in conjunction with molecular oxygen to elicit cell death (phototoxicity). It is used clinically to treat a wide range of medical conditions, including wet age-related macular degeneration, psoriasis, atherosclerosis and has shown some efficacy inanti-viral treatments, including herpes. It also treats malignant cancers including head and neck, lung, bladder, skin and prostate cancer (Wang, SS et al. Cancer Journal. 8 (2): 154–63. 2002). The “photodynamic therapeutic agent” is selected from Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m-tetrahydroxyphenylchlorin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, Verteporfin, Purlytin, ATMPn, Zinc phthalocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa), or Pheophorbide a (PhA).

[0060] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (also called DM1), anthracycline, pyrrolobenzodiazepine, ^-amanitin, tubulysin, benzodiazepine, erlotinib (TARCEVA®), Genentech / OSI Pharm.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®), Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARASAR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone), camptothecin (including the synthetic analogue topotecan), bryostatin, callystatin, CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues), cryptophycins (particularly cryptophycin 1 and cryptophycin 8), dolastatin, duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1), dynemicin, including dynemicin A; aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), cyclophosphamide, thiotepa, taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE®doxetaxel (Rhône- Poulenc Rorer, Antony, France); chloranbucil, GEMZAR®gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin; vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE®vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine (XELODA®, Roche), and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0061] The term “prodrug” as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to its parent form and is capable of being enzymatically activated or converted into the more active parent form. Examples of prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified pro drugs, glycosylated prodrugs, β-lactam- containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, or other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in ADCs include, but are not limited to, those chemotherapeutic agents described above.

[0062] The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an ADC. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3- naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion.

[0063] The term “pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and an ADC. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine. Antibody conjugates

[0064] The present disclosure provides an antibody conjugate prepared by conjugating a linker-payload as described below with an antibody or an antigen-binding fragment thereof. Depending on the nature of the linker-payload, which may be a drug-linker compound (DL) that includes a drug unit as the payload or a probe-linker compound that includes a probe unit, the antibody conjugate may be an antibody-drug conjugate or an antibody-probe conjugate.

[0065] In certain embodiments, the antibody-drug conjugate (ADC) is represented by Formula (I): Ab-(DL)n (I); wherein Ab is an antibody or an antigen-binding fragment thereof capable of binding to one or more of tumor- associated antigens or cell-surface receptors; DL is a linker-payload as described below where the payload is a drug unit; and n is a drug-to-antibody ratio (DAR) ranging from 1 to 20.

[0066] In contrast, in certain embodiments of the present disclosure, the linker-payload has the structure of Formula :C - - ; wherein E is a hydrophilic moiety including: polyethylene Glycol (PEG), polysarcosine (pSar), poly lactic- co-glycolic acid (PLGA), poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N- acryloylmorpholine) (PAcM), saccharides, or any combination thereof; C is a cysteine-reactive group for cysteine conjugation to an antibody or an antigen-binding fragment thereof;L is a linker unit comprising a cleavable moiety or a spacer; and P is a payload selected from a drug unit or a probe unit.

[0067] The term “cysteine-reactive group (C)” refers to a non-native chemical group that can be used to conjugate the linker-payload with an antibody or an antigen-binding fragment thereof under the conditions of living systems without affecting the activity of the antibody or antigen-binding fragment thereof. In certain embodiments, cysteine-reactive group is selected from a 2-sulfonylpyrimidine group (abbreviated as 2SP) Osuch as or a derivativeOthereof, an iodoacetamide group such asor a derivative thereof, a vinylpyridine group such asa derivative thereof, a cyclopentenone group such asor a derivative thereof, a chlorooxime group such asor a derivative thereof, a cyclopropenyl ketone group such as or a derivative thereof, a heteroaromatic sulfone group such as S NCderivative thereof, a 2-cyanobenzothiazole group such asNor a derivativethereof, a 2-formyl phenylboronic acid group such as or a derivative thereof, a 2,3-dibromomaleimide such as a ketone such as O , a vinyl sulfone groupsuch as O , an alfa-nitrile vinyl ketone group such as group suchas O , a 5-methanesulfonyl-1,2,3,4-tetrazole group such as N , a 2-methanesulfonyl-[1,3,4]oxadiazole group such as , a cyclic alkyne group, a ^,^-unsaturated carbonyl group, or a sulfonyl pyrimidine group.

[0068] The term “linker unit (L)” refers to an element connecting the cysteine-reactive group with the payload and including a cleavable moiety. In certain embodiments, the linker unit includes a protease cleavable peptide, a glycosidase cleavable sugar or a sulfatase cleavable unit.

[0069] The term “payload” as used herein refers to a molecule that is to be carried and delivered by an antibody or an antigen-binding fragment thereof. The term “drug unit” refers to a drug molecule (e.g., a cytotoxic agent or an immune stimulator) or a pharmaceutically acceptable salt or prodrug thereof. The term “probe unit” refers to a probe molecule that assists the visualization of target cells or tissues or a body part of a subject. In certain embodiments, the payload is selected from a toxin, a cytokine, a growth factor, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, or an immunoregulatory agent (e.g., an immunostimulatory agent), a fluorophore, a dye, or a contrast agent.

[0070] In certain embodiments, the toxin is selected from a pyrrolobenzodiazepine or its derivative (e.g., PBD), an auristatin or its derivative (e.g., MMAE, MMAF), a maytansinoid or its derivative (e.g., maytansine, DM1, DM4, DM21), a duocarmycin or its derivative, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor or its derivative, a tubulysin or its derivative, an enediyne compound or its derivative (e.g., calicheamicin), an anthracycline or its derivative (e.g., doxorubicin), a pyrrole-based kinesin spindle protein (KSP) inhibitor or its derivative, a cryptophycin or its derivative, an efflux pump inhibitor or its derivative, a sandramycin compound or its derivative, an amanitin compound or its derivative, a camptothecin compound or its derivative (e.g., exatecan, deruxtecan), an ataxia-telangiectasia mutated (ATM) kinase inhibitor or its derivative, an ataxia telangiectasia and Rad3-related protein (ATR) kinase inhibitor or its derivative, a checkpoint kinase inhibitor or its derivative (e.g. Chk1 or Chk2), or a Wee1 inhibitor or its derivative (e.g. adavosertib). The ataxia-telangiectasia mutated (ATM) kinase inhibitor is selected from caffeine, wortmannin, dactolisib, torin2, ETP-46464, NVP-BEZ235, CGK733, CP466722, GSK635416A, AZ31, AZ32, AZD0156, AZD1390, M4076, KU-55933, KU-60019 or KU-59403. The ataxia telangiectasia and Rad3-related protein (ATR) kinase inhibitor is selected from berzosertib, ceralasertib, camonsertib, dactolisib, elimusertib, gartisertib, VE-821, AZD6738, M6620, BAY1895344 or M4344

[0071] In certain embodiments, the linker unit (L) of Formula (II) has the structure of LP-QCL-QSP, and the linker-payload is represented by Formula (III):C - LP- QCL- QSP- P (III); wherein:(a) C is a cysteine-reactive group for conjugation to an antibody or an antigen-binding fragment thereof; (b) E is a hydrophilic moiety as defined in Formula (II), and preferably includes PEG and has the formula of:wherein the wavy line indicates the site of covalent attachment to LP, R20is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, orR21is select from H, SO3H, PO3H2, a sugar derivative, C1-C10(hetero) alkyl group, C3-C10(hetero) cycloalkyl group, C2-C10 alkyl-NH2, C1-C10 alkyl-COOH, C2-C10 alkyl-NH(C1-C3 alkyl), C2-C10 alkyl-N (C1-C3 alkyl)2, or sarcosine, and the subscript n is an integer ranging from 2 to 72; (c) QSPis a spacer unit including an aromatic group or amino methylene; (d) QCLis a cleavable unit selected from a protease cleavable peptide, a glycosidase cleavable sugar or a sulfatase cleavable unit; (e) LPis a connector unit that connects C and E, wherein the connector unit includes an amino acid or an aromatic tri-functional group and preferably includes(f) P is a payload selected from a drug unit or a probe unit.

[0072] In one embodiment, the linker-payload is MCCa-PEG24-VA-PAB-Exatecan and has the structure of Formula (IV): .

[0073] In one embodiment, the linker-payload is 2SP-PEG24-VA-PAB-Exatecan (also termed DL-3) and has the structure of Formula (V): O N .

[0074] In certain embodiments, the linker unit (L) of Formula (II) has the structure of LB-QCL-BP, and the linker-payload is represented by Formula (VI): Cwherein (a) C is a cysteine-reactive group for conjugation to an antibody or an antigen-binding fragment thereof; (b) E is a hydrophilic moiety as defined in Formula (II), and preferably includes PEG and has the formula of:wherein the wavy line indicates the site of covalent attachment to BP, R20is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or, R21is select from H, SO3H, PO3H2, a sugar derivative, C1-C10 (hetero) alkyl group, C3-C10 (hetero) cycloalkyl group, C2-C10alkyl-NH2, C1-C10alkyl-COOH, C2-C10alkyl-NH(C1-C3alkyl), C2-C10alkyl-N (C1-C3alkyl)2, or sarcosine, and the subscript n is from 2 to 72; (c) BPis a branch unit that connects E and the backbone of the linker-payload, wherein the branch unit includesan aromatic tri-functional group and preferably includes ; (d) QCLis a cleavable unit selected from a protease cleavab e pep de, a g ycos dase cleavable sugar or a sulfatase cleavable unit; (e) LBis a bridge unit; and (f) P is a payload selected from a drug unit or a probe unit.

[0075] In one embodiment, the linker-payload is Maleimide-phenyl-GGVA-PAB-PEG24-Exatecan (also termed DL-11) and has the structure of Formula (VII): O.

[0076] In one embodiment, the linker-payload is 2SP-GGVA-PAB-PEG24-Exatecan (also termed DL-1) and has the structure of Formula (VIII): O.

[0077] In certain embodiments, the linker unit (L) of Formula (II) has the structure of (L1)n1 - QCL- (L2)n2, and the (IX):C – (L1)n1 – Q – (L2)n2 - P (IX), wherein: (a) E is a hydrophilic moiety as defined in Formula (II); (b) C is a cysteine-reactive group for cysteine conjugation to an antibody or an antigen-binding fragment thereof;(c) L1and L2are the same or different connector units, wherein the connector units include an amino acid or an aromatic tri-functional group and preferably includes are independently 0, 1 or 2, with the proviso that n1 and n2 are(d) QCLis a cleavable unit selected from a protease cleavable peptide, a glycosidase cleavable sugar or a sulfatase cleavable unit; and (e) P is a payload selected from a drug unit or a probe unit.

[0078] In one embodiment, the linker-payload is 2SP-Glu(PEG24)-VA-PAB-PEG24-Exatecan (also termed DL-2) and has the structure of Formula (X):.

[0079] In certain embodiments, the antibody or antigen-binding fragment thereof of the ADC is monospecific or multispecific. In certain embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to cMET, HER3, EGFR, TROP2, HER2, or Nectin-4. In one preferred embodiment, the antibody or antigen-binding fragment thereof is bispecific to HER2 and TROP2. The ADC with specificity to cMET, HER3, EGFR, TROP2, HER2, or Nectin-4 may inhibit the growth of tumor cells which expresses cMET, HER3, EGFR, TROP2, HER2, or Nectin-4. Therapeutic Applications

[0080] The present disclosure further provides a method for killing or inhibiting the proliferation of tumor cells or cancer cells, including contacting the cells with an effective amount of the ADC disclosed herein.

[0081] The present disclosure also provides a method for treating a disease, including administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition that includes one or more of the ADCs described herein.

[0082] In some embodiments, the subject (e.g., a human patient) in need of the treatment is diagnosed with, suspected of having, or at risk for cancer. Examples of the cancer include, but are not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer,colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, or oral cancer.

[0083] The treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and / or prolongation of time to tumor cancer progression.

[0084] In some embodiments, the method for treatment further includes administering an additional therapy to said subject prior to, during or subsequent to said administering of the ADCs. In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiation therapy, photodynamic therapy, chemotherapy, immunotherapy, targeted therapy, or hormone therapy.

[0085] The phrase “an effective amount” refers to the amount of each active agent required to confer therapeutic index on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0086] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has cancer, a symptom of cancer, or a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect cancer, the symptom of cancer, or the predisposition toward cancer.

[0087] As used herein, “development” or “progression” of cancer means initial manifestations and / or ensuing progression of cancer. Development of cancer can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein "onset" or “occurrence” of cancer includes initial onset and / or recurrence. Applications in imaging

[0088] The present disclosure further provides a method of selecting a subject for a cancer therapy by imaging, including:(a) administering to the subject an effective amount of the antibody conjugate disclosed herein where the payload is the probe unit, wherein the probe unit is an imaging agent selected from a fluorophore, a dye, a contrast agent, or a radionuclide; (b) detecting visually or instrumentally a reporting signal of the imaging agent in the subject; and (c) identifying the subject as suitable for the cancer therapy when the reporting signal is detected.

[0089] In certain embodiments, the subject is diagnosed with, suspected of having, or at risk for cancer.

[0090] In certain embodiments, the method further comprises detecting metastasis of a cancer. Administration of ADC Pharmaceutical Formulations

[0091] The pharmaceutical compositions of ADC may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

[0092] The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL / hour can occur. Subcutaneous (bolus) administration may be effected with about 1.5 mL or less of total volume and a concentration of about 100 mg ADC per mL. For ADC that require frequent and chronic administration, the subcutaneous route may be employed, such as by pre-filled syringe or autoinjector device technology.

[0093] As a general proposition, the initial pharmaceutically effective amount of ADC administered per dose will be in the range of about 0.01-100 mg / kg, namely about 0.1 to 20 mg / kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg / kg / day. The dose may be escalated to the maximally tolerated dose (MTD). The dosing schedule may be about every 3 weeks, but according to diagnosed condition or response, the schedule may be more or less frequent. The dose may be further adjusted during the course of treatment to be at or below MTD which can be safely administered for multiple cycles, such as about 4 or more. Combination Therapy

[0094] The ADC described herein may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second therapeutic agent having anti-cancer properties. The second therapeutic agent of the pharmaceutical combination formulation or dosing regimen preferably has complementary and / or synergistic activities to the ADC of the combination such that they do not adversely affect each other.

[0095] The second therapeutic agent may be a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an anti-hormonal agent, an aromatase inhibitor, a protein kinase inhibitor, a lipid kinase inhibitor, an anti-androgen, an antisense oligonucleotide, a ribozyme, a gene therapy vaccine, an anti- angiogenic agent, a cardioprotectant, or an immunotherapeutic agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. A pharmaceutical composition containing an ADC may also have a therapeutically effective amount of a chemotherapeutic agent such as a tubulin- forming inhibitor, a topoisomerase inhibitor, or a DNA binder. Articles of Manufacture

[0096] In another embodiment, an article of manufacture, or “kit”, containing ADC and materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, or blister pack. The containers may be formed from a variety of materials such as glass or plastic. The container holds an ADC composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an ADC. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.

[0097] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES Example 1: Preparation of the drug-linker compounds 1-1. Preparation of 2SP-GGVA-PAB-PEG24-Exatecan (also termed DL-1) Step 1:

[0098] (2R)-hydroxy(4-nitrophenyl)acetic acid (150.0 mg, 0.76 mmol), m-PEG24-amine (909.4 mg, 0.84 mmol) and HATU (346.4 mg, 0.91 mmol) were dissolved in anhydrous DMF (7.0 mL) at room temperature. NMM (0.25 mL, 2.30 mmol) was added. The resulting mixture was stirred at room temperature for 6-8 hours.7% NaHCO3(aq) (7.0 mL) was then added, and the mixture solution was extracted with CH2Cl2(three times, each with 10 mL). The combined organic layers were dried with MgSO4 and then concentrated in vacuo at 30-35 ^C. The crude 2-hydroxy-(4-nitrophenyl) PEG24 amide (also termed H-PAB-1) was dried with high vacuum and then used in the next step without further purification. H Pd / C, EA / MeOH(2R)-hydroxy(4-nitrophenyl) m-PEG24-amine H-PAB-1 aceticacidChemical Formula: C57H106N2O28Exact Mass: 1266.69 Molecular Weight: 1267.46 Step 2:

[0099] To a solution of crude H-PAB-1 in EA / MeOH (25 mL, 95 / 5), 10% Pd / C (165.0 mg, 5 wt%) was added. The reaction was stirred at room temperature with H2 balloon for twenty-four hours. After reaction was completed, the reaction mixture was passed through celite (3.3 g) and the celite was washed with MeOH (10 mL x 3 times). The desired fraction was concentrated in vacuo at 30-35 ^C. The crude Preparation of PAB- PEG24(also termed H-PAB-2) was dried with high vacuum and then used in next step without further purification. HRMS (ESI) m / z found [M+H]+, 1237.7278 C57H109N2O26+, required 1237.7269.Chemical Formula: C57H106N2O28Chemical Formula: C57H108N2O26Exact Mass: 1266.69 Exact Mass: 1236.72 Molecular Weight: 1267.46 Molecular Weight: 1237.48 Step 3:

[0100] To a solution of crude H-PAB-2 in anhydrous CH2Cl2 (25 mL), Boc-VA-OH (792.8 mg, 2.75 mmol) and EEDQ (741.8 mg, 3.00 mmol) were added. The mixture was stirred at room temperature for 18-24 hours. After reaction was completed, 1 M HCl (aq) (15 mL) was added and then the mixture solution was extracted with CH2Cl2 (15 mL x 3 times). The combined organic layers were washed with H2O (20 mL) and then concentrated in vacuo at 30-35 ^C. The resulting mixture was purified with preparative HPLC to obtain 440.0mg of Boc-VA-PAB-PEG24-OH (also termed H-PAB-3) with 38.4% yield for three steps. HRMS (ESI) m / z found [(M+2Na) / 2]+, 776.4278 C70H130N4O30 Na22+, required 776.4282. OExact Mass: 1506.88 Exact Mass: 1236.72 Molecular Weight: 1507.81 Molecular Weight: 1237.48

[0101] Table 1. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 6 8 1 1 1 1 1 2Steps 4 and 5:

[0102] H-PAB-3 (440.0 mg, 0.29 mmol) and Bis(4-nitrophenyl) carbonate (177.5 mg, 0.58 mmol) in anhydrous CH2Cl2(5.8 mL) was cooled to 0-4 ^C. 2,6-lutidine (51.0 µL, 0.43 mmol) and DIPEA (50.9 µL, 0.29 mmol) were added sequentially at 0-4 ^C. The resulting mixture was stirred at 0-4 ^C and monitored by HPLC. After H-PAB-3 was consumed, Et2NH (6.0 µL x 5) was added separately at 0-4 ^C to quench excess Bis(4-nitrophenyl) carbonate. Anhydrous DMF (11.6 mL), Exatecan mesylate (278.4 mg, 0.52 mmol), DIPEA (188.0 µL, 1.07 mmol) were added into previous reaction solution at 0-4 ^C. The reaction mixture was stirred at 0-4 ^C until Boc-VA-PAB-PEG24-PNP (also termed H-PAB-4) was consumed by checking with HPLC. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 229.8 mg of Boc-VA-PAB-PEG24-Exatecan (also termed H-PAB-5) with 40.0% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 985.0144 C95H152FN7O352+, required 985.0156.O2N NO2

[0103] Table 2. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 3 4 6 8 1 1 1 1 1 122 min 20 10% 90% Step 6:

[0104] To a solution of H-PAB-5 (45.0 mg, 0.02 mmol) in CH2Cl2(0.9 mL) was cooled to 0 ^C. TFA (225.0 µL) was added by dropwise. The reaction mixture was stirred at 0 ^C for 4-6 hours. After reaction was completed, the reaction mixture was added by dropwise into a stirring ether (11.3 mL) to get precipitate. The solids were collected by filtration and washed with ether (5 mL x 3 times), and followed by dried with highvacuum to obtain crude NH2-VA-PAB-PEG24-Exatecan TFA salt (also termed H-PAB-6). The crude product was used in the next step without further purification. H O NStep 7:

[0105] To a solution of crude H-PAB-6 (150.0 mg, 0.076 mmol) in anhydrous DMF (1.51 mL), HATU (34.5 mg, 0.091 mmol) and Boc-GG-OH (17.6 mg, 0.076 mmol) were added. Et3N (31.6 µL, 0.227 mmol) was added slowly. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 96.3 mg of Boc-GGVA-PAB- PEG24-Exatecan (also termed H-PAB-8) with 61.0% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1078.5447 C109H157FN8O352+, required 1078.5367.Chemical Formula: C92H143F4N7O35Exact Mass: 1981.96 Molecular Weight: 1983.16O H H N OExact Mass: 2082.06 Molecular Weight: 2083.36

[0106] Table 3. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 5 1 3 3 4Step 8:

[0107] To a solution of H-PAB-8 (96.0 mg, 0.046 mmol) in CH2Cl2 (1.92 mL) was cooled to 0 ^C. TFA (0.48 mL) was added by dropwise. The reaction mixture was stirred at 0 ^C for 3-4 hours. After reaction was completed, ether (50.0 mL) was added into reaction solution to get precipitate. The solids were collected by filtration and washed with ether (15 mL x 3 times), and followed by dried with high vacuum to obtain crude NH2-GGVA-PAB-PEG24-Exatecan TFA salt (also termed H-PAB-9). The crude product was used in the next step without further purification.Exact Mass: 2082.06 Molecular Weight: 2083.36HStep 9:

[0108] To a solution of 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (9.7 mg, 0.036 mmol) and HATU (20.5 mg, 0.054 mmol) in anhydrous DMF (0.72 mL). NMM (7.91 µL, 0.072 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. H-PAB-9 TFA salt (62.6 mg, 0.030 mmol) in anhydrous DMF (0.72 mL) was added into previous solution and then NMM (7.91 µL, 0.072 mmol) was added. The reaction was stirred at room temperature for 1 h. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 39.8 mg of DL-1 (2SP-GGVA-PAB-PEG24- Exatecan) with 60.0% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1117.5339 C105H160FN11O38S2+, required 1117.0315. - OMolecular Weight: 2233.5164

[0109] Table 4. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) Gradient: T 0 1 5 2 2 2 2 31-2. Preparation of 2SP-Glu(PEG24)-VA-PAB- PEG24-Exatecan (also termed DL-2) Step 1:

[0110] H-PAB-6 (57.3 mg, 0.029 mmol), Fmoc-Glu(PEG24) (43.7 mg, 0.030 mmol) and HATU (22.7 mg, 0.060 mmol) were dissolved in anhydrous DMF (0.29 mL). NMM (9.5 μL, 0.087 mmol) was added and the reaction mixture was stirred at room temperature for 1-2 hours. After the reaction completed, the resulting mixture was purified with preparative HPLC to obtain 72.6 mg of H-PAB-10 with 76.1% yield. HRMS (ESI) m / z found [(M+4H) / 4]+, 823.1910 C159H262FN9O614+, required 823.1915.F [00 G T 01 min 20 80% 20%5 min 20 70% 30% 8 3 3 3 3Step 2:

[0112] H-PAB-10 (70.4 mg, 0.021 mmol) was dissolved in CH2Cl2 / MeOH (1.69 mL, 1 / 1) and then cooled to 0oC. Et2NH (0.42 mL) was added and the reaction mixture was stirred at 0-4oC for 8-10 hours. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35oC, and azeotroped with Toluene (5 mL x 3) to remove excess Et2NH. This crude product H-PAB-11 was used in next step without further purification.Step 3:

[0113] To a solution of H-PAB-11 (25.7 mg, 0.0084 mmol), 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5- ynoic acid (2.3 mg, 0.0084 mmol) and HATU (4.8 mg, 0.0126 mmol) in anhydrous DMF (0.17 mL). Et3N (3.5 µL, 0.0252 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 h. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 16.5 mg of DL- 2 with 59.4% yield. HRMS (ESI) m / z found [(M+3H) / 3]+, 1106.5778 C155H261FN11O62S3+, required 1106.5771.

[0114] Table 6. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 5 8 3 3 3 35min 20 10% 90% 1-3. Preparation of 2SP-PEG24-VA-PAB- Exatecan (also termed DL-3) Step 1:

[0115] Fmoc-Glu(tBu)-OH (200.0 mg, 0.470 mmol), m-(dPEG)24-amine (511.6 mg, 0.470 mmol) and HATU (268.1 mg, 0.705 mmol) were dissolved in anhydrous DMF / CH2Cl2(4.0 mL, v / v= 1 / 1). NMM (155.0 μL, 1.410 mmol) was added and the reaction mixture was stirred at room temperature for two hours. After the reaction completed, the resulting mixture was concentrated in vacuo at 30-35oC, and azeotroped with toluene (5 mL x 3) to remove excess DMF. The crude product was purified by flash silica gel column (EA / MeOH = 95 / 5 to CH2Cl2 / MeOH = 94 / 6) to obtain 704 mg of Fmoc-Glu(PEG24) with 100.0% yield.Step 2:

[0116] Fmoc-Glu(PEG24) (704.0 mg, 0.470 mmol) was dissolved in CH2Cl2 / MeOH (12.6 mL, 1 / 1). Et2NH (1.4 mL) was added and the reaction mixture was stirred at room temperature overnight. After the reaction completed, the reaction mixture was concentrated in vacuo at 30-35oC, and azeotroped with toluene (5 mL x 3) to remove excess Et2NH. The crude compound was high vacuum drying to obtain 612.0 mg of NH2- Glu(tBu)-PEG24and used in the next step without further purificationStep 3:

[0117] NH2-Glu(tBu)-PEG24(474.7 mg, 0.373 mmol), 6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100.0 mg, 0.373 mmol) and HATU (212.6 mg, 0.559 mmol) in anhydrous DMF (11.4 mL). NMM (163.9 µL, 1.491 mmol) was added. The reaction mixture was concentrated in vacuo at 30-35oC, and azeotroped with toluene (5 mL x 3) to remove excess DMF. The crude product was purified by flash silica gel column (EA / MeOH = 95 / 5, then CH2Cl2 / MeOH = 94 / 6 to 92 / 8) to obtain 447.0 mg of 2SP-Glu(tBu)-PEG24with 93.0% yield.Step 4:

[0118] 2SP-Glu(tBu)-PEG24 (447.0 mg, 0.293 mmol) in CH2Cl2 (8.8 mL) was added Et3SiH (140.6 µL, 0.880 mmol). The reaction mixture was cooled to 0 ⁰C. After 5 minutes, TFA (2.2 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C for two hours and then room temperature for additional one hour. After reaction was completed, the resulting mixture was concentrated in vacuo at 30-35oC. The crude product was purified by flash silica gel column (EA / MeOH = 90 / 10, then CH2Cl2 / MeOH = 90 / 10 to 80 / 20) to obtain 2SP-PEG24-COOH (224.2 mg) with 52.1% yield.Step 5:

[0119] 2SP-PEG24-COOH (224.2 mg, 0.153 mmol) and HATU (87.1 mg, 0.229 mmol) were dissolved in anhydrous DMF (2.2 mL). NMM (33.6 µL, 0.305 mmol) was added, and the reaction mixture was stirred at room temperature for one hour. N-PM-0018 (146.0 mg. 0.168 mmol) in anhydrous DMF (2.2 mL) was added into previous solution. NMM (33.6 µL, 0.305 mmol) was added. The reaction mixture was stirred at room temperature for one hour. After reaction was completed, the resulting mixture was concentrated in vacuo at 30-35oC. The crude compounds was purified by MPLC to obtain DL-3 (251.7 mg) with 74.0% yield. HRMS (ESI) m / z found [(M+2Na) / 2]+, 1124.5159 C107H162F2N10O37Na22+, required 1124.5184.

[0120] Table 7. MPLC condition (YMC-DsipoPack AT, OD3-25, 40 g) E H 9 8 7 6 6 6 101-4. Preparation of 2SP-VC-PAB-PEG24-Exatecan (also termed DL-4) Step 1:

[0121] Fmoc-VC-OH (489.3 mg, 0.985 mmol) and PAB(COOtBu)-OH (200.0 mg, 0.895 mmol) were dissolved in anhydrous CH2Cl2 (2.0 mL) and anhydrous MeOH (2.0 mL). EEDQ (310.1 mg, 1.254 mmol) was added. The resulting mixture was stirred at room temperature overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (50.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude Fmoc-VC-PAB(COOtBu)- OH (445.1 mg, 71.0% yield). This crude product was used in next step without further purification.

[0122] Fmoc-VC-PAB(COOtBu) (445.0 mg, 0.634 mmol) and Bis(4-nitrophenyl) carbonate (578.7 mg, 1.902 mmol) were dissolved in DMF (4.5 mL). DIPEA (0.33 mL, 1.902 mmol) was added into the reactionsolution. The reaction was stirred at room temperature overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (50.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude Fmoc-VC-PAB(COOtBu)-PNP (300.0 mg, 81.0% yield). This crude product was used in next step without further purification.

[0123] To a suspension of Exatecan mesylate (183.9 mg, 0.346 mmol), Fmoc-VC-PAB(COOtBu)-PNP (300.0 mg, 0.346 mmol) in anhydrous DMF (1.73 mL), DIPEA (0.18 mL, 1.038 mmol) was added at room temperature. The suspension became clear brown solution within five minutes. This mixture was stirred at room temperature overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (20.0 mL) to get precipitate. The solids were collected by filtration and followed with vacuum drying to obtain crude product. The crude product was purified by flash silica gel column (CH2Cl2 / MeOH = 12 / 1 to 7 / 1) to obtain 250.0 mg of H-PAB-13 with 62.0% yield.H-PAB-13 Step 2:

[0124] A stirring suspension of H-PAB-13 (100.0 mg, 0.115 mmol) in CH2Cl2(4.0 mL) was cooled to 0 ⁰C. TFA (1.98 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (60 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude Fmoc-VC-PAB(COOH)-Exatecan (64.5 mg, 68% yield). This crude product was used in next step without further purification.

[0125] Fmoc-VC-PAB(COOH)-Exatecan (64.5 mg, 0.058 mmol), NH2-PEG24-OMe (75.7 mg, 0.070 mmol) and HATU (33.1 mg, 0.087 mmol) were dissolved in anhydrous DMF 0.65 mL. NMM (12.8 µL, 0.116 mmol) was added and the reaction mixture was stirred at room temperature for 1-2 hours. After the reaction completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (10 mL) to get precipitate. The solids were collected by filtration and followed with vacuum drying to obtain crude product. The crude product was purified with preparative HPLC to obtain 47.8 mg of Fmoc-VC-PAB-PEG24-Exatecan with 37.0% yield.

[0126] Table 8. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 5 2 2 2

[0127] Fmoc-VC-PAB-PEG24-Exatecan (219.0 mg, 0100 mmol) was dissolved in CH2Cl2 / MeOH (6.57 mL, 1 / 1). Et2NH (0.77 mL) was added and the reaction mixture was stirred at 0-4oC overnight. After the reaction completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (130 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude H- PAB-14 (207 mg). This crude product was used in next step without further purification. Step 3:

[0128] To a solution of H-PAB-14 (20.0 mg, 0.010 mmol), 2SP-OSu (4.1 mg, 0.011 mmol) in anhydrous DMF (0.40 mL). DIPEA (5.4 µL, 0.031 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 hours. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 10.2 mg of DL-4 with 45.0% yield. HRMS (ESI) m / z found [(M+3Na) / 3]+, 757.6732 C104H158FN11O37SNa33+, required 757.6739.O

[0129] Table 9. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 5 2 3 3 351-5. Preparation of 2SP-GGVC-PAB-PEG24-Exatecan (also termed DL-5)

[0130] H-PAB-15 (100.0 mg, 0.090 mmol) and 2SP-OSu (36.2 mg, 0.099 mmol) were dissolved in anhydrous DMF (2.5 mL). DIPEA (48.1 µL, 0.270 mmol) was added. The resulting mixture was stirred at room temperature for 1-2 hours. After reaction was completed, added by dropwise into a stirring Ethyl ether (25 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude 2SP-GGVC-PAB(COOH)-Exatecan 86.3 mg without purification. Crude BCN-GGVC- PAB(COOH)-Exatecan (86.3 mg, 0.069 mmol), NH2-PEG24-OMe (90.2 mg, 0.083 mmol) and HATU (39.4 mg, 0.104 mmol) were dissolved in anhydrous DMF 1.4 mL. NMM (227.6 µL, 2.070 mmol) was added and the reaction mixture was stirred at room temperature overnight. After the reaction completed, the reactionmixture was purified with preparative HPLC to obtain 68.9 mg of DL-5 with 43.9% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1160.0553 C106H162FN13O38S2+, required 1160.0555.

[0131] Table 10. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 5 2 3 3 351-6. Preparation of 2SP-GGFG-PAB-PEG24-Exatecan (also termed DL-6)

[0132] H-PAB-16 (50.0 mg, 0.053 mmol) and 2SP-OSu (21.2 mg, 0.058 mmol) were dissolved in anhydrous DMF (1.0 mL). DIPEA (28.2 µL, 0.158 mmol) was added. The resulting mixture was stirred at room temperature for 0.5-1 hour. After reaction was completed, added by dropwise into a stirring Ethyl ether (10 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude 2SP-GGFG-PAB(COOH)-Exatecan 51.8 mg without purification. Crude BCN-GGFG- PAB(COOH)-Exatecan (51.8 mg, 0.043 mmol), NH2-PEG24-OMe (70.6 mg, 0.065 mmol) and HATU (24.5 mg, 0.065 mmol) were dissolved in anhydrous DMF 0.9 mL. NMM (14.2 µL, 0.129 mmol) was added and the reaction mixture was stirred at room temperature overnight. After the reaction completed, the reaction mixture was purified with preparative HPLC to obtain 50.2 mg of DL-6 with 51.5% yield. HRMS (ESI) m / z found [(M+2H) / 2]+, 1134.0228 C108H158FN11O38S2+, required 1134.0237.

[0133] Table 11. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) T 0 1 5 2 3 3 31-7. Preparation of 2SP-GGFG-PAB-PEG24-MMAE (also termed DL-7) Step 1:

[0134] To a solution of N-DT-0024 (25.0 mg, 0.032 mmol), MMAE (26.9 mg, 0.037 mmol) and HOAt (6.6 mg, 0.049 mmol) in anhydrous DMF (1.0 mL). DIPEA (16.9 μL, 0.097 mmol) and pyridine (7.8 μL, 0.097 mmol) were added sequentially. The resulting mixture was stirred at room temperature until N-DT-0024 was consumed. After reaction was completed, the reaction mixture was concentrated in vacuo at 30-35oC, and azeotroped with Toluene (5 mL x 3) to remove excess DMF. The crude product was purified by flash silica gel column (CH2Cl2, then CH2Cl2 / MeOH = 98 / 2 to 92 / 8) to obtain 33.0 mg of H-PAB-17 with 75.6% yield.Step 2:

[0135] To a solution of H-PAB-17 (80.0 mg, 0.065 mmol) in CH2Cl2(2.40 mL) was cooled to 0 ⁰C. TFA (1.20 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C for 20-24 hours. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (72.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude intermediate (88.0 mg). This crude product was used in next step without further purification.

[0136] The intermediate (88.0 mg, 0.074 mmol) and 2SP-OSu (29.6 mg, 0.081 mmol) were dissolved in anhydrous DMF (1.76 mL). DIPEA (38.4 μL, 0.221 mmol) was added into the reaction solution at room temperature. The reaction mixture was stirred at room temperature for one hour. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (35 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude 2SP-intermediate (90.1 mg). This crude product was used in next step without further purification.

[0137] The 2SP-intermediate (90.1 mg, 0.062 mmol), NH2-PEG24-OMe (81.4 mg, 0.075 mmol) and HATU (35.6 mg, 0.093 mmol) were dissolved in anhydrous DMF (1.8 mL). NMM (20.6 μL, 0.187 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 hours. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 82.6 mg of DL-7 with 53.0% yield. HRMS (ESI) m / zH-PAB-17

[0138] Table 12. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) Gradient: T 0 1 5 2 3 3 31-8. Preparation of 2SP-GGFG-PAB-PEG24-T785 (also termed DL-8) Step 1:

[0139] To a solution of N-DT-0024 (49.4 mg, 0.064 mmol), T785 (21.9 mg, 0.070 mmol) in anhydrous DMF (0.64 mL). DIPEA (33.4 μL, 0.192 mmol) was added slowly. The resulting mixture was stirred at room temperature overnight. After reaction was completed, the reaction mixture added by dropwise into a stirring Ethyl ether (10.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain H-PAB-18 (71.7 mg). This crude product was used in next step without further purification.H-PAB-18Step 2:

[0140] To a solution of H-PAB-18 (71.7 mg, 0.076 mmol) in CH2Cl2 (1.26 mL) was cooled to 0 ⁰C. TFA (0.54 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C for 20-24 hours. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (20.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude intermediate (63.9 mg). This crude product was used in next step without further purification.

[0141] The intermediate (31.9 mg, 0.035 mmol) and 2SP-OSu (15.5 mg, 0.042 mmol) were dissolved in anhydrous DMF (0.71 mL). Et3N (14.8 μL, 0.106 mmol) was added into the reaction solution at room temperature. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (10.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude 2SP-intermediate (33.5 mg). This crude product was used in next step without further purification.

[0142] The 2SP-intermediate (33.5 mg, 0.032 mmol), NH2-PEG24-OMe (52.7 mg, 0.048 mmol) and HATU (14.7 mg, 0.039 mmol) were dissolved in anhydrous DMF (0.32 mL). NMM (10.7 μL, 0.097 mmol) was added. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 26.3 mg of DL-8 with 35.3% yield for four steps. HRMS (ESI) m / z found [(M+H+Na) / 2]+, 1066.0491 C99H162N13NaO34S2+, required 1066.0483.[0014 T l 1 HPL iti YM A t T it 1 2 2 mm, 5 µL, 12 nm) G F 1 2 220 55% 45%20 10% 90% 21-9. Preparation of 2SP-GGFG-PAB-PEG24-Prexasertib (also termed DL-9) Step 1:

[0144] To a solution of N-DT-0024 (50.8 mg, 0.066 mmol), Prexasertib (26.4 mg, 0.072 mmol) in anhydrous DMF (0.66 mL). DIPEA (34.4 μL, 0.197 mmol) was added slowly. The resulting mixture was stirred at room temperature overnight. After reaction was completed, the reaction mixture added by dropwise into a stirring Ethyl ether (10.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain H-PAB-19 (66.3 mg). This crude product was used in next step without further purification.Step 2:

[0145] To a solution of H-PAB-19 (66.3 mg, 0.066 mmol) in CH2Cl2(1.16 mL) was cooled to 0 ⁰C. TFA (0.50 mL) was added at 0oC slowly. The reaction mixture was stirred at 0-4 ⁰C for 20-24 hours. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (15.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude intermediate (56.2 mg). This crude product was used in next step without further purification.

[0146] The intermediate (25.1 mg, 0.026 mmol) and 2SP-OSu (11.5 mg, 0.031 mmol) were dissolved in anhydrous DMF (0.52 mL). Et3N (11.0 μL, 0.079 mmol) was added into the reaction solution at room temperature. The reaction mixture was stirred at room temperature overnight. After reaction was completed,the reaction mixture was added by dropwise into a stirring Ethyl ether (10.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude 2SP-intermediate (30.1 mg). This crude product was used in next step without further purification.

[0147] The 2SP-intermediate (30.1 mg, 0.027 mmol), NH2-PEG24-OMe (44.9 mg, 0.041 mmol) and HATU (12.6 mg, 0.033 mmol) were dissolved in anhydrous DMF (0.28 mL). NMM (9.1 μL, 0.083 mmol) was added. The reaction mixture was stirred at room temperature overnight. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 14.0 mg of DL-9 with 24.7% yield for four steps. HRMS (ESI) m / z found [(M+2H) / 2]+, 1082.0345 C99H157N15O36S2+, required 1082.0318.(1) (2) (3)

[0148] Table 14. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 2 3 335 min 20 10% 90% 1-10. Preparation of Benzoylacryl-GGVA-PAB-PEG24-Exatecan (also termed DL-10)

[0149] Step 1:Benzoylacryl-acid (200.0 mg, 1.135 mmol), EDC-HCl (435.2 mg, 2.270 mmol) and NHS (261.3 mg, 2.270 mmol) were dissolved in anhydrous CH2Cl2(2.0 mL). This mixture was stirred at room temperature overnight. After reaction was completed, the reaction was quenched by adding 0.1N HCl(aq) (2.0 mL) and then extracted with CH2Cl2 (5 mL x 3). The organic layer was dried with MgSO4, filtered, and thenconcentrated in vacuo at 30-35oC. The crude product was purified by flash silica gel column (CH2Cl2, then EA / CH2Cl2 = 1 / 1) to obtain 162.0 mg of benzoylacryl-OSu with 52.0% yield.Step 2:

[0150] Benzoylacryl-OSu (18.0 mg, 0.066 mmol) and N-PM-0024 (50.0 mg, 0.055 mmol) were dissolved in anhydrous DMF (1.0 mL). DIPEA (28.6 μL, 0.164 mmol) was added. The reaction mixture was stirred at room temperature for 0.5-1 hour. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (20.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude H-PAB-20 (61.6 mg). This crude product was used in next stepStep 3:

[0151] H-PAB-20 (61.6 mg, 0.058 mmol), NH2-PEG24-OMe (75.2 mg, 0.069 mmol) and HATU (32.8 mg, 0.086 mmol) were dissolved in anhydrous DMF (1.23 mL). NMM (25.3 μL, 0.230 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 hours. After reaction was completed, the resultingmixture was purified with preparative HPLC to obtain 80.2 mg of DL-10 with 65.1% yield. HRMS (ESI) m / z found [(M+3H) / 3]+, 714.3590 C104H157FN9O373+, required 714.3555. O

[0152] Table 15. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) G T 0 1 2 3 3 31-11. Preparation of Maleimide-phenyl-GGVA-PAB-PEG24-Exatecan (also termed DL-11) Step 1:

[0153] N-PM-0024 (50.0 mg, 0.055 mmol) and N-phenyl-maleimide-OSu (21.6 mg, 0.066 mmol) were dissolved in anhydrous DMF (1.0 mL). DIPEA (28.6 μL, 0.164 mmol) was added. The reaction mixture was stirred at room temperature for 0.5-1 hour. After reaction was completed, the reaction mixture was added by dropwise into a stirring Ethyl ether (20.0 mL) to get precipitate. The solids were collected by filtration and followed with high vacuum drying to obtain crude H-PAB-21 (62.1 mg). This crude product was used in next step without further purification.Step 2:

[0154] H-PAB-21 (62.1 mg, 0.055 mmol), NH2-PEG24-OMe (72.0 mg, 0.066 mmol) and HATU (31.4 mg, 0.083 mmol) were dissolved in anhydrous DMF (1.24 mL). NMM (24.2 μL, 0.221 mmol) was added. The reaction mixture was stirred at room temperature for 1-2 hours. After reaction was completed, the resulting mixture was purified with preparative HPLC to obtain 22.7 mg of DL-11 with 18.7% yield. HRMS (ESI) m / z found [(M+3H) / 3]+, 732.6975 C106H158FN10O383+, required 732.6908.

[0155] Table 16. HPLC condition (YMC-Actus Trait C18250 x 20 mm, 5 µL, 12 nm) Gr di nt T 0 1 5 2 3 3 3Example 2: ADC preparation 2-1. Preparation of average DAR4 ADC

[0156] Antibody (10 mg / mL, 5.0 mL) in reaction buffer (50 mM Histidine, 20 mM EDTA, pH 7.0) was cooled to 12-16 ^C. Antibody was treated with TCEP.HCl (0.23 mg, 0.0008 mmol) in reaction buffer (115 µL) for two hours at 12-16 ^C. To this partial reduced antibody solution, the linker-payload DL-1 (4.46 mg, 0.002 mmol) in DMSO (223 µL) was added and conjugated for twenty-four hours at 12-16 ^C. After conjugation completed, the buffer of ADC was changed to storage buffer (20 mM Sodium acetate, pH 5.0) via ultrafiltration / diafiltration to afford a final ADC (9.37 mg / mL, 4.8 mL). The drug-to-antibody ratio (DAR) value of the final ADC is determined by hydrophobic interaction chromatography (HIC). 2-2. Preparation of DAR8 ADC

[0157] Antibody (10 mg / mL, 3.0 mL) in reaction buffer (50 mM Histidine, 20 mM EDTA, pH 7.0). Antibody was treated with TCEP.HCl (0.57 mg, 0.002 mmol) in reaction buffer (57 µL) for two hours at room temperature. To this fully reduced antibody solution, payload-linker (5.36 mg, 0.0024 mmol) in DMSO (268 µL) was added and conjugated for twenty-four hours at room temperature. After conjugation completed, the buffer of ADC was changed to storage buffer (20 mM Sodium acetate, pH 5.0) via ultrafiltration / diafiltration to afford final ADC (10.39 mg / mL, 2.89 mL). The drug-to-antibody ratio (DAR) value of final ADC is determined by hydrophobic interaction chromatography (HIC).Example 3: ADC analysis

[0158] The hydrophobic interaction chromatography (HIC) is currently considered the reference technique for the analysis of ADCs to determine drug load distribution (DLD) and average drug antibody ratio (DAR) for antibodies. TOSOH TSKgel Butyl-NPR column (Cat. No. 50-851-379; particle: 2.5 micrometer; 4.6mm I.D. x 3.5cm length) was applied. ADC was eluted with 1.5 M ammonium sulfate, 50 mM sodium phosphate and 50 mM sodium phosphate at pH 7.0 in 15% 2-propanol gradient with a flow rate of 0.8 mL / minute. The chromatograms was detected at 280 nm. 3-1. R4702 (anti-TROP2) ADC analysis

[0159] FIG.1A indicated the non-reducing and reducing PAGEs results of maleimide linker (LN0628-146) and 2SP linker (LN0760-010) R4702 (anti-TROP2) ADC. Furthermore, FIG. 1B indicated hydrophobic interaction chromatography (HIC) result of 2SP linker (LN0760-010) R4702 (anti-TROP2) ADC. The DAR distributions were DAR0 (3.79%), DAR2 (12.81%), DAR4 (41.58%), DAR6 (18.24%) and DAR8 (23.57%). The average DAR is 4.9. 3-2. 10K06 (anti-Nectin-4) ADC analysis

[0160] FIG. 2A indicated the non-reducing and reducing PAGEs results of 2SP linker (LN0819-022) 10K06 (anti-Nectin-4) ADC. Furthermore, FIG.2B indicated hydrophobic interaction chromatography (HIC) result of 2SP linker 10K06 (anti-Nectin-4) ADC. The DAR distribution is DAR8 (100%). Example 4: In vitro bio-activity comparison assay of the ADCs

[0161] Tumor cells (2 x 103cells / well) were seeded in 96 well plate and treated with ADCs for 6 days. CellTiter-Glo®Reagent (Cat. G7572, Promega) was prepared by adding CellTiter-Glo®Buffer into lyophilized CellTiter-Glo®Substrate. Reconstituted CellTiter-Glo®Reagent was added into the culture medium with cells at 1:1 ratio after treatment for six days. The plate was placed on an orbital shaker for 2 minutes to induce cell lysis and then incubated at room temperature for 10 minutes before recording theluminescent signals by Luminometer. Luminescence was determined using a microplate luminometer SpectraMax L (Molecular Devices, Sunnyvale, CA). Percentage of the cytotoxicity was calculated through dividing the non-treated cell luminescence minus experimental cell luminescence by the non-treated cell luminescence and multiplying by 100. IC50was determined by plotting x (concentration in nM) – y (drug cytotoxicity in %) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software. 4-1. In vitro cytotoxicity assay of R4702 (anti-TROP2) ADC analysis

[0162] The in vitro efficacy of R4702 (anti-TROP2) ADCs in human pancreatic carcinoma cell line (BxPC- 3) were evaluated by cytotoxicity assay (FIG. 3). The IC50of ADC-1 (anti-TROP2 ADC with a maleimide linker) was 0.294 nM and ADC-3 (anti-TROP2 ADC with a 2SP linker) was 0.333 nM. 4-2. In vitro cytotoxicity assay of 10K06 (anti-Nectin-4) ADC analysis

[0163] The in vitro efficacy of 10K06 and commercial anti-Nectin-4 ADCs in human lung cancer cell line (NCI-H460 / Nectin-4) were evaluated by cytotoxicity assay (FIG. 4B). The IC50 of ETx-22 (anti-Nectin-4 ADC with a glucuronide linker) was 0.56 nM, ADC-4 (anti-Nectin-4 ADC with a maleimide linker) was 0.49 nM and ADC-5 (anti-Nectin-4 ADC with a 2SP linker) was 0.48 nM. 4-3. In vitro Nectin-4 antigen binding assay of 10K06 (anti-Nectin-4) ADC analysis

[0164] The specific binding activity of 10K06 and commercial anti-Nectin-4 ADCs were measured using an ELISA. ADCs bound to the Nectin-4 antigen coated microplate were detected with peroxidase AffiniPure goat anti-human IgG antibody (Cat. No. 109-035-003, Jackson ImmunoResearch) and subsequently reacted with 3,3’,5,5’-tetramethylbenzidine (TMB). The color development was terminated with H2SO4 and the optical density were measured at 450 nm. The relative binding activity of test samples to the reference standard is calculated by EC50(half maximal effective concentration) on a 4-Parameter logistic regression (PL) curve fitting model via parallel-line analysis (PLA). The EC50 of ETx-22 (anti-Nectin-4 ADC with a glucuronide linker) was 18.41 ng / mL, ADC-4 (anti-Nectin-4 ADC with a maleimide linker) was 20.53 ng / mL and ADC- 5 (anti-Nectin-4 ADC with a 2SP linker) was 20.40 ng / mL (FIG. 4A). Example 5: In vivo tumor inhibition efficacy assay of the ADCs 5-1. In vivo tumor inhibition efficacy assay of R4702 (anti-TROP2) ADC analysis

[0165] 5-1-1. Test substances and dosing pattern in NCI-H1975 NSCLC CDX model (a) ADC-1 (R4702-MCCa-PEG24-VA-PAB-Exatecan): 3 mg / mL (b) ADC-2 (R4702-2SP-PEG24-VA-PAB-Exatecan): 3 mg / mL (c) ADC-3 (R4702-2SP-GGVA-PAB-PEG24-Exatecan): 3 mg / mL [00 G GG2: ADC-1 IV 3 5 6G3: ADC-2 IV 3 5 6 G In

[0167] 5-1-2. Test substances and dosing pattern in DLD-1 CRC CDX model (a) ADC-1: 10 mg / mL (b) ADC-2: 10 mg / mL (c) ADC-3: 10 mg / mL (d) Sac-TMT (sacituzumabtirumotecan): 10 mg / mL

[0168] Table 18. Study design and sampling R t D f V l f G G G G G G In

[0169] 5-1-3. Cell line: NCI-H1975 (Cat. No. CRL-5908, American Type Culture Collection) and DLD-1 cells (Cat. No. 60132, Bioresource Collection and Research Center) (High TROP2 expressed cancer cells)

[0170] 5-1-4. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: seven weeks (f) Body weight range at start of study: 15-25 g (g) Animal Grouping: The mice were divided into five groups and each group contained six mice. A total of thirty mice were involved in study of NCI-H1975 CDX model. And 5 groups in study of DLD-1 CDX model, a total of 30 mice were involved.

[0171] 5-1-5. Equipment and material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0172] 5-1-6. Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 5x106NCI-H1975 and DLD-1 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article: The first dosing day was denoted as Day 1 when average tumor volume reaches 150-200 mm3in NCI-H1975 CDX model, and 350-400 mm3in DLD-1 CDX model. All test articles (test item a to g) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 3 mg / kg for NCI-H1975 CDX model and 10 mg / kg for DLD-1 CDX model, and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 37 in NCI- H1975 CDX model and Day 43 in DLD-1 CDX model individually). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (d) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0173] 5-1-7. Result

[0174] FIG. 5A indicated the in-vivo efficacy result in NCI-H1975 NSCLC CDX model. The mean^SEM of tumor volume to vehicle group was 1457^210.36 mm3on Day 23. The mean^SEM of tumor volume to treated groups were 970.23^110.84 to ADC-1; 960.21^153.17 to ADC-2 and 646.05^44.43 to ADC-3 on Day 37. It demonstrated high tumor inhibitions efficacy of the two cysteine-reactive groups (MCCa and 2SP).

[0175] FIG.5B indicated the in-vivo efficacy result in DLD-1 CRC CDX model. The mean^SEM of tumor volume to vehicle group was 1291.93^187.95 mm3on Day 15. The mean^SEM of tumor volume to treated groups were 1319.13^159.71 to sac-TMT (sacituzumabtirumotecan) on Day 29; 889.83^157.83 to ADC-1 on Day 29; 753.69^122.08 to ADC-3 on Day 43; 584.04^160.50 to ADC-2. It also demonstrated high tumor inhibition efficacy of the two cysteine-reactive groups (MCCa and 2SP), even better than sac-TMT approved by national medical products administration (NMPA) in China. 5-2. In vivo tumor inhibition efficacy assay of 10K06 (anti-Nectin-4) ADC analysis

[0176] 5-2-1. Test substances and dosing pattern (a) ADC-4 (10K06-MCCa-GGVA-PAB-PEG24-Exatecan): 6 mg / mL (b) ETx-22 (LY4101174): 6 mg / mL (c) EV (Enfortumab Vedotin): 6 mg / mL (d) ADC-5 (10K06-2SP-GGVA-PAB-PEG24-Exatecan): 6 mg / mL

[0177] Table 19. Study Design and sampling Route and Dose of Volume of G l G G G G G In

[0178] 5-2-2. Cell line: Fadu cells (Cat. No. 60214, Bioresource Collection and Research Center) (Mid Nectin-4 expressed cancer cell)

[0179] 5-2-3. Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal grouping: The mice were divided into five groups and each group contained six mice. A total of thirty mice were involved in the study.

[0180] 5-2-4. Equipment and material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0181] 5-2-5. Method (a) Establishment of xenograft mouse modelSubcutaneous inoculation of tumor cells: 3x106Fadu cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article: The first dosing day was denoted as Day 1 when average tumor volume reaches 200-250 mm3. All test articles (test item a to g) or reference item (Sodium citrate solution) were intravenously administered to the mice on Day 1. The injection was performed using insulin syringe with the dosage 3 mg / kg and the injection volume was 5 mL / kg. (c) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 23 or less). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (d) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0182] 5-2-6 Result

[0183] FIG. 6 indicated the in-vivo efficacy result in Fadu human hypopharyngeal squamous cell cancer CDX model. The mean^SEM of tumor volume to vehicle group was 1436.46^170.21 mm3on Day 15. The mean^SEM of tumor volume to treated groups were 679.26^101.79 mm3to ADC-5 on Day 29 and 692.60^117.65 mm3ADC-4 on Day 23; 1079.48^200.34 mm3to ETx-22 (LY4101174) on Day 29 and 1434.08^266.49 mm3to EV (Enfortumab Vedotin) was on Day 19. It demonstrated high tumor inhibitions efficacy of the two cysteine-reactive groups (MCCa and 2SP) ), even better than other competitor’s ADCs (LY4101174 of Eli Lilly and Enfortumab Vedotin of Astellas).

[0184] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0185] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. Noadmission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

Claims

Claims 1. A linker-payload having a structure of Formula (II): E C - L - P (II); wherein: E is a hydrophilic moiety comprising polyethylene glycol (PEG), polysarcosine (pSar), poly lactic- co-glycolic acid (PLGA), poly(glycerols) (PGs), poly(oxazolines) (POX), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2- hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloylmorpholine) (PAcM), saccharides, or any combination thereof; C is a cysteine-reactive group for cysteine conjugation to an antibody or an antigen-binding fragment thereof; L is a linker unit comprising a protease cleavable peptide, a glycosidase cleavable sugar or a sulfatase cleavable unit; and P is a payload selected from a drug unit or a probe unit. The linker-payload according to claim 1, wherein the structure of Formula (II) is further represented by a structure of Formula (IX):wherein: L1 and L2 are the same or different connector units, wherein the connector units comprise an amino acid or an aromatic tri-functional group and includesn2 are independently 0, 1 or 2, with the proviso that n1 and n2 are not 0 at the same time; and QCLis a cleavable unit, wherein the cleavable unit is a protease cleavable peptide, a glycosidase cleavable sugar or a sulfatase cleavable unit. The linker-payload according to claim 1 or 2, wherein the cysteine-reactive group C is selected from a 2-sulfonylpyrimidine group or an derivative thereof, a maleimide group or an derivative thereof, an iodoacetamide group or an derivative thereof, a vinylpyridine group or a derivativethereof, a cyclopentenone group or a derivative thereof, a chlorooxime group or a derivative thereof , a cyclopropenyl ketone group or a derivative thereof, a heteroaromatic sulfone group or a derivative thereof, a 2-cyanobenzothiazole group or a derivative thereof, a 2-formyl phenylboronic acid group or a derivative thereof, a 2,3-dibromomaleimide group or a derivative thereof, a vinyl ketone group or a derivative thereof, a vinyl sulfone group or a derivative thereof, an alfa-nitrile vinyl ketone group or a derivative thereof, a methanesulfonothioate group or a derivative thereof, a 5-methanesulfonyl-1,2,3,4-tetrazole group or a derivative thereof, a 2- methanesulfonyl-[1,3,4]oxadiazole group or a derivative thereof , a cyclic alkyne group, a ^,^- unsaturated carbonyl group, or a sulfonyl pyrimidine group.

4. The linker-payload according to claim 1 or 2, wherein the payload P is selected from a toxin, a cytokine, a growth factor, a radionuclide, a hormone, an anti-viral agent, an anti-bacterial agent, an immunoregulatory agent, a fluorophore, a dye, or a contrast agent.

5. The linker-payload according to claim 4, wherein the toxin is selected from a pyrrolobenzodiazepine or an derivative thereof, an auristatin or an derivative thereof , a maytansinoid or an derivative thereof, a duocarmycin or an derivative thereof, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor or an derivative thereof, a tubulysin or an derivative thereof, an enediyne compound or an derivative thereof, an anthracycline or an derivative thereof, a pyrrole-based kinesin spindle protein (KSP) inhibitor or an derivative thereof, a cryptophycin or an derivative thereof, an efflux pump inhibitor or an derivative thereof, a sandramycin compound or an derivative thereof, an amanitin compound or an derivative thereof, a camptothecin compound or an derivative thereof, an ataxia-telangiectasia mutated (ATM) kinase inhibitor or an derivative thereof, an ataxia telangiectasia and Rad3-related protein (ATR) kinase inhibitor or an derivative thereof, a Chk1 or Chk2 checkpoint kinase inhibitor or an derivative thereof, or an adavosertib Wee1 inhibitor or an derivative thereof.

6. The linker-payload according to claim 5, wherein the ataxia-telangiectasia mutated (ATM) kinase inhibitor is selected from caffeine, wortmannin, dactolisib, torin2, ETP-46464, NVP-BEZ235, CGK733, CP466722, GSK635416A, AZ31, AZ32, AZD0156, AZD1390, M4076, KU-55933, KU-60019 or KU-59403.

7. The linker-payload according to claim 5, wherein the ataxia telangiectasia and Rad3-related protein (ATR) kinase inhibitor is selected from berzosertib, ceralasertib, camonsertib, dactolisib, elimusertib, gartisertib, VE-821, AZD6738, M6620, BAY1895344 or M4344.

8. The linker-payload according to claim 2, wherein the structure of Formula (IX) is furtherrepresented by a structure of the following Formula:wherein z is an integer ranging from 6 to 48. A linker-payload having a structure of Formula (III):(III); wherein: E is a hydrophilic moiety; C is a cysteine-reactive group; P is a payload selected from a drug unit or a probe unit; QSPis a spacer unit comprising an aromatic group or amino methylene; QCLis a cleavable unit, wherein the cleavable unit is a protease cleavable peptide, a glycosidase cleavable sugar or a sulfatase cleavable unit; LPis a connector unit comprising an amino acid or an aromatic tri-functional group and includesThe linker-payload according to claim 9, wherein the hydrophilic moiety E has the formula of:, wherein the wavy line indicates the site of covalent attachment to the connector unit LP; R20is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or; R21is H, SO3H, PO3H2, a sugar derivative, C1-C10 (hetero) alkyl group, C3-C10 (hetero) cycloalkyl group, C2-C10 alkyl-NH2, C1-C10 alkyl-COOH, C2-C10 alkyl-NH(C1-C3 alkyl), C2-C10 alkyl-N (C1- C3 alkyl)2, or sarcosine; and n is an integer ranging from 2 to 72. The linker-payload according to claim 9, wherein the structure of Formula (III) is further represented by a structure of the following Formula:; wherein z is an integer ranging from 6 to 48. A linker-payload having a structure of Formula (VI): E C - LB- QCL- BP- P (VI);wherein: E is a hydrophilic moiety; C is a cysteine-reactive group; P is a payload selected from a drug unit or a probe unit; BPis a branch unit comprising an aromatic tri-functional group and includesorQCLis a cleavable unit, wherein the cleavable unit is a protease cleavable peptide, a glycosidase cleavable sugar or a sulfatase cleavable unit; LBis a bridge unit. The linker-payload according to claim 12, wherein the hydrophilic moiety E has the formula of:wherein the wavy line indicates the site of covalent attachment to the branch unit BP, R20is -C(O)-, -O-, -S-, -NH-, -C(O)O-, alkyl-C(O)-NH-, alkyl-NH-C(O)-, alkyl-CO2-, alkyl-S-, or, R21is H, SO3H, PO3H2, a sugar derivative, C1-C10(hetero) alkyl group, C3-C10(hetero) cycloalkyl group, C2-C10 alkyl-NH2, C1-C10 alkyl-COOH, C2-C10 alkyl-NH(C1-C3 alkyl), C2-C10 alkyl-N (C1- C3 alkyl)2, or sarcosine; and n is an integer ranging from 2 to 72. The linker-payload according to claim 12, wherein the structure of Formula (VI) is further represented by a structure of the following Formula:, wherein z is an integer ranging from 6 to 48.

15. The linker-payload according to claim 12, wherein the structure of Formula (VI) is further represented by a structure of the following Formula:wherein z is an integer ranging from 6 to 48.

16. An antibody conjugate prepared by conjugating the linker-payload according to any one of claims 1-15 with an antibody or an antigen-binding fragment thereof.

17. An antibody-drug conjugate (ADC) prepared by conjugating the linker-payload according to any one of claims 1-15 with an antibody or an antigen-binding fragment thereof, wherein the payload is the drug unit.

18. An antibody-drug conjugate (ADC) having a structure of Formula (I): Ab - (DL)n(I); wherein: Ab is an antibody or an antigen-binding fragment thereof capable of binding to one or more of tumor-associated antigens or cell-surface receptors; DL is the linker-payload according to any one of claims 1-15 where the payload is the drug unit; and n is a drug-to-antibody ratio (DAR) ranging from 1 to 20.

19. The ADC of claim 18, wherein the antibody or antigen-binding fragment thereof is monospecific or multispecific.

20. The ADC of claim 18, wherein the antibody or antigen-binding fragment thereof is capable of binding to DLL3, CEACAM-5, CEACAM-6, ROR1, NaPi2b, CLDN18.2, CLDN1, CLDN2,CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfr1, TNF alpha, tissue factor, folate receptor alpha, c-MET, HER3, EGFR, HER2, TROP2, or Nectin- 4.

21. The ADC of claim 18, wherein the antibody or antigen-binding fragment thereof is bispecific to HER2 and TROP2, c-Met and HER3, EGFR and HER3, or EGFR and c-Met.

22. A pharmaceutical composition comprising the ADC according to any one of claims 18-21 and a pharmaceutically acceptable carrier.

23. A method for treating a disease, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to claim 22.

24. The method of claim 23, wherein the disease is characterized by expressing DLL3, CEACAM-5, CEACAM-6, ROR1, NaPi2b, CLDN18.2, CLDN1, CLDN2, CDH6, CDH17, B7-H3, MUC-1, PD-1, PD-L1, CTLA-4, VEGF, BCMA, PSMA, CGRP, Tfr1, TNF alpha, tissue factor, folate receptor alpha, c-MET, HER3, EGFR, HER2, TROP2, or Nectin-4.

25. The method of claim 23, wherein the disease is a cancer selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

26. A method of selecting a subject for a cancer therapy by imaging, comprising: (a) administering to the subject an effective amount of the antibody conjugate according to claim 16 where the payload is the probe unit, wherein the probe unit is an imaging agent selected from a fluorophore, a dye, a contrast agent, or a radionuclide; (b) detecting visually or instrumentally a reporting signal of the imaging agent in the subject; and (c) identifying the subject as suitable for the cancer therapy when the reporting signal is detected.

27. The method of claim 26, wherein the subject has a cancer, and wherein the method further comprises detecting metastasis of the cancer.

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