Oxazaphosphine antibody drug conjugates and methods of use

Abstract

Disclosed herein are antibody-drug conjugates (ADCs) comprising antibodies conjugated to an oxazaphosphine drug moiety via a linker, and methods of using the antibody drug conjugates. Including methods of treating undesirable or abnormal cell proliferative or hyperproliferative disorders, such as non-metastatic and metastatic neoplasia, cancers, stem cells, and malignancies expressing targets that bind to such antibodies or antibody fragments, antibody heavy and light chains, or nanoantibodies.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 524,400, filed June 30, 2023, the entirety of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present invention relates generally to cancer therapy. The present invention relates specifically to antibody-drug conjugates (ADCs) for cancer immunotherapy. BACKGROUND

[0003] Human epidermal growth factor receptor 2 (HER2) is a well-recognized target in a variety of malignancies, for tumors that overexpress or amplify HER2 (e.g., breast cancer, gastric cancer, colorectal cancer, cervical cancer, endometrial cancer, bladder cancer, esophagogastric cancer, biliary tract cancer, salivary gland cancer) and for tumors that have HER2 mutations (e.g., NSCLC and over 20 other tumor types) Raghav KPS and Moasser MM, Clin Cancer Res. 2023 July 05; 29(13): 2351-2361. doi:10.1158 / 1078-0432.CCR-22-0283. Antibodies and ADCs that target HER2, most notably trastuzumab, trastuzumab-deruxtecan (T-DXd), and trastuzumab emtansine (T-DM1), are standard of care for HER2 breast and other tumor types and for HER2-mutated tumors (e.g., non-small cell lung cancer (NSCLC)). Despite the efficacy of ADCs, however, drug resistance develops in many patients. For example, resistance to T-DXd develops, and the most common cause appears to be resistance to the topoisomerase inhibitor payload, as HER2 expression loss occurs in only a small number of drug-resistant cases. Another approved ADC that uses a topoisomerase inhibitor is sacituzumab govitecan, which treats cancers that overexpress trophoblast cell surface antigen 2 (TROP2), such as breast cancer, bladder cancer. TROP2 is also expressed in a variety of malignancies, e.g., cervical cancer, colorectal cancer, ovarian cancer, prostate cancer, thyroid cancer, gastric cancer, brain cancer, esophageal cancer, head and neck cancer, pancreatic cancer, and endometrial cancer. Resistance to sacituzumab govitecan also stems in part from resistance to the topoisomerase inhibitor payload. Thus, there is an urgent need to develop ADCs that target HER2 and TROP2 using alternative agents as payloads. The present invention describes the use of oxazaphosphorines as alternative potent drug payloads to generate new ADCs. SUMMARY

[0004] In one aspect, the subject matter described herein relates to an antibody conjugated to an oxazaphosphorine drug via a linker, the antibody having the formula: Ab-(L-D)n wherein D is an oxazaphosphorine covalently bound to an antibody (Ab) or antibody fragment that can bind to a tumor antigen via a linker (L), n has a value of 2 to 20.

[0005] Another aspect of the subject matter described herein is an antibody that can be engineered to enhance its anti-tumor cytotoxicity. The antibody can also be designed to bind to one or more antigens on a target cell simultaneously. Another aspect of the subject matter described herein is an antibody fragment, nanobody, affibody, or heavy / light chain.

[0006] Another aspect of the subject matter described herein is an activated oxazaphosphorine selected from the group comprising active metabolites, analogs, or derivatives having direct anti-tumor cytotoxicity ("tumor cell killing activity"). Another aspect of the subject matter described herein is an active oxazaphosphorine selected from the group comprising active metabolites, analogs, or derivatives that stimulate the anti-tumor immune system by depleting or reducing the number of and / or inhibiting the function of immunosuppressive cells in the tumor microenvironment (TME), such as regulatory T cells (Tregs).

[0007] Another aspect of the subject matter described herein is a pharmaceutical composition comprising an oxazaphosphorine antibody-drug conjugate and one or more pharmaceutically acceptable excipients.

[0008] Another aspect of the subject matter described herein is the use of an antibody-oxazaphosphorine drug conjugate in a method of treating a patient by administering to the subject a pharmaceutical composition comprising an oxazaphosphorine antibody conjugate.

[0009] Another aspect of the subject matter described herein is a method of making an antibody-drug conjugate covalently linked to an oxazaphosphorine.

[0010] Another aspect of the subject matter described herein is an article of manufacture comprising a pharmaceutical composition comprising an antibody or fragment conjugated to an oxazaphosphorine, a container, and a package insert or label indicating that the pharmaceutical composition can be used to treat a patient.

[0011] In one aspect, the subject matter described herein relates to a composition comprising an antibody-drug conjugate of the formula Ab-(L-D)n, wherein Ab is an antibody, antibody fragment, antibody chain, affibody, aptamer, or nanobody; D is an active oxygen aza phosphine payload; L is a linker; and, n has a value of 2 to 20.

[0012] In another aspect, n has a value of 2-8. In another aspect, Ab binds to a tumor associated antigen from at least one of the group comprising: HER2, HER3, VEGF-A, VEGFR-2, CSF-1R, PD-L1, CEACAM5 or CEACAM6, ROR1, CD20, CD19, CD22, CD30, CD33, CD133, CD38, CD39, CD25, CD47, CD52, CD56, CD70, CD73, CD74, CD79b, CD155, CD166, FGF receptor, B7-H3, B7-H4, LIV1, PSMA, PSCA, MAGE-A4, EpCAM, IL1R, CCR8, CCR4, claudin, APPL2, BCMA, EGFR, DLL3 / 4, SSX-2, tissue factor, folate receptor, mesothelin receptor, NaPi2b, 5T4, Claudin-4, Claudin-2 (CD112), c-MeT, TROP2, Fibroblast Activation Protein (FAP), LHRH (GnRH) receptor, gonadotropin (LH / hCG, FSH) receptor, prolactin receptor, claudin; survivin, STEAP1, transferrin receptor 1, NRG1, EphB2, and Caveolin-1.

[0013] In another aspect, the oxygen aza phosphine payload is cytotoxic to T regulatory cells.

[0014] In another aspect, the oxygen aza phosphine payload is cytotoxic to cancer cells.

[0015] In another aspect, the oxygen aza phosphine payload is of the formula:

[0016] wherein at least one of R3, R4, R5, and R6 is CH2CH2Y; wherein Y is a halogen; and wherein the remaining R3, R4, R5, and R6 groups are hydrogen or lower alkyl groups.

[0017] In another aspect, the halogen is Cl or Br.

[0018] In another aspect, the hydrogen in R3 and R5 is replaced with deuterium to form CD2CH2Y, or with a methyl group (CH3) to form CH3CH2Y.

[0019] In another aspect, the structure of the oxazaphosphorine payload is selected from the group consisting of:

[0020] wherein R is an alkyl chain;

[0021] wherein X and Y are halogen leaving groups;

[0022] wherein X is a halogen leaving group; and

[0023] wherein D is deuterium.

[0024] In another aspect, the oxazaphosphorine payload is selected from 4- hydroxycyclophosphamide, aldo-phosphoramide, phosphoramide mustard, 3- hydroxypropionaldehyde, isophosphoramide mustard, 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyifosfamide, 4- hydroperoxyifosfamide, evofosfamide, marphosamide, glufosfamide, or triphosgene mustard.

[0025] In another aspect, the oxazaphosphorine payload is phosphoramide mustard.

[0026] In another aspect, the oxazaphosphorine payload is selected from an analog or derivative of 4-hydroxycyclophosphamide, aldo-phosphoramide, phosphoramide mustard, 3-hydroxypropionaldehyde, isophosphoramide mustard, 4- hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyifosfamide, 4-hydroperoxyifosfamide, evofosfamide, marphosamide, glufosfamide, or triphosgene mustard.

[0027] In another aspect, the derivative of 4-hydroperoxyifosfamide is 4- hydroxyifosfamide.

[0028] In another aspect, the analog or derivative of 4-hydroperoxyifosfamide (4-HO- ifosfamide) is deuterated (d4-hydroxyifosfamide).

[0029] In another aspect, the derivative of 4-hydroperoxycyclophosphamide is 4- hydroxycyclophosphamide.

[0030] In another aspect, the structure of the oxazaphosphorine payload is:

[0031] wherein X is CI or Br.

[0032] In another aspect, X is CI.

[0033] In another aspect, the structure of the oxazaphosphorine payload is:

[0034] wherein X and Y represent independent leaving groups.

[0035] In another aspect, the oxazaphosphorine payload metabolite is isophosphoramide mustard or an analog thereof.

[0036] In another aspect, the oxazaphosphorine payload is selected from dimethyl isophosphoramide mustard or an analog thereof, or a 4-hydroxy derivative (4-HO- ifosfamide) or an analog or derivative thereof.

[0037] In another aspect, the oxazaphosphorine payload metabolite is selected from bromo isophosphoramide mustard or an analog or derivative thereof, including ifosfamide or dimethyl isophosphoramide mustard.

[0038] In another aspect, the oxazaphosphorine payload is geranyloxy-isophosphoramide mustard metabolite or an analog or derivative thereof.

[0039] In another aspect, the oxazaphosphorine payload metabolite is marphosamide or an analog or derivative thereof.

[0040] In another aspect, the oxazaphosphorine payload metabolite is glufosfamide or an analog or derivative thereof.

[0041] In another aspect, the oxazaphosphorine payload metabolite is triphosphoramide mustard or an analog or derivative thereof.

[0042] In another aspect, the composition further comprises a therapeutic agent.

[0043] In another aspect, the composition further comprises an anti-TAM (tumor associated macrophage) agent.

[0044] In another aspect, the composition further comprises one or more antibodies that enhance anti-tumor immunity, the one or more antibodies selected from the group of: an anti-PD-1, an anti-PD-Ll, an anti-CTLA4, an anti-LAG3, an anti-GITR, an anti-TIM-3, an anti-TIGIT, an anti-CD96, an anti-CD226, an anti-CD155, an anti-CD47, an anti-CEACAMl, an anti-CEACAM5, an anti-CEACAM6, an anti- galectin-1, an anti-claudin, an anti-Siglec-15 antibody, an anti-VISTA, an anti-CD137, an anti-CCR4 antibody, an anti-CCR8 antibody, an anti-CD39 antibody, an anti-CD25 antibody, an anti-CD-73 antibody, and an anti-CSFRl. In another aspect, the composition further comprises one or more cytokines selected from the group consisting of IL-1 β, IL-2, IL-6, IL-7, IL-12, IL-15, IL-21, IL-23, IL-27, TNFα, IFNα, IFNγ, GM-CSF, anti-IL2R, Toll-like receptor (TLR) activators, and stimulator of interferon genes (STING).

[0045] In another aspect, the Toll-like receptor activator is poly(I:C) and CpG.

[0046] In another aspect, the composition further comprises one or more chemotherapeutic drugs selected from the group of 5-fluorouracil, 2’-deoxy-5-fluoridine, cytarabine, cladribine, fludarabine, pentostatine, gemcitabine, 6-thioguanine, melphalan and any derivatives thereof; and alkylating drugs chiorambucil, bendamustine, melphalan, alkylating agents or anthracyclines such as doxorubicin, epirubicin, daunorubicin; temozolomide, oxaliplatin, cisplatin, chiorambucil, mechlorethamine, mitoxantrone, pexidartinib, lenvatinib, trabectedin, HDAC inhibitors, anti-angiogenic drugs, bisphosphonates, taxanes, vinorelbine, ibrutinib, eribulin, resiquimod, gardiquimod or analogs thereof, anti-semaphorin 4D, CXCR2 blockers, axitinib, sorafenib, carbozantinib, sunitinib, multi-kinase inhibitors such as regorafenib, bifunctional PROTACs and molecular glue degraders (e.g. thalidomide, lenalidomide, pomalidomide, avadomide), vandetanib, cediranib or analogs thereof, anti-VEGF-A antibodies (bevacizumab), anti-VEGF-R2 antibodies (ramucirumab), TRL9 agonists, anti-CCR4 antibodies, PPAR gamma agonists, miRNAs, angiotensin receptor blockers, CXCR4 blockers, CD4 / 6 inhibitors, proteasome inhibitors, JAK1 / 2 inhibitors, Bruton’s kinase (BTK) inhibitors, kinase inhibitors, topoisomerase inhibitors, epigenetic inhibitors, DNMT, HMT, HDM inhibitors, PARP inhibitors, hormone antagonists, anti-prolactin, VEGI, osteopontin, maspin, canstatin, itraconazole, carboxyamidotriazole, suramin, thrombospondin, tetrathiomolybdate, linomide, tasquinimod, carfilzomib, sunitinib, pazobanib, everolimus;Anti-hormones: luteinizing hormone-releasing hormone (LHRH) antagonists, tamoxifen, corticosteroid analogs, steroid receptor modulators or antagonists, cancer metabolism inhibitors, radioisotopes, radiopharmaceuticals, vinca alkaloids, mTOR inhibitors, MEK inhibitors, BRAF inhibitors, MAPK and tyrosine kinase inhibitors, bortezomib, demethylating agents, bleomycin, alkylating agents, dacarbazine, temozolomide, CELLMOD, and targeted protein degradation agents.

[0047] In another aspect, the taxane is selected from the group consisting of docetaxel, paclitaxel, cabazitaxel, and 6-alpha-hydroxypaclitaxel.

[0048] In another aspect, the epigenetic inhibitor targets HDAC, DNMT, LSD1, DOT1L, BET, or EZH.

[0049] In another aspect, the hormone antagonist is lupron.

[0050] In another aspect, the PARP inhibitor is selected from the group consisting of 1- aminobenzamides, iniparib, BMN-573, olaparib, niraparib, talazoparib, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, and derivatives thereof.

[0051] In another aspect, the corticosteroid analog is selected from the group consisting of prednisone, dexamethasone; raloxifene, anastrozole, letrozole, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162, and any derivatives thereof.

[0052] In another aspect, the steroid receptor modulator or antagonist is selected from the group consisting of anti-estrogens, anti-gestagens, anti-androgens, anti-corticosteroids, and anti-thyroid hormones.

[0053] In another aspect, the cancer metabolism inhibitor is selected from the group consisting of pyruvate kinase inhibitors and isocitrate dehydrogenase inhibitors.

[0054] In another aspect, the radioisotope is radium Ra 223 dichloride, lutetium Lu 177, actinium 225, yttrium 90, technetium 99, or iodine 131.

[0055] In another aspect, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine, and any derivative thereof.

[0056] In another aspect, the protein degrader is a proteolysis targeting chimera (PROTAC) or a molecular glue.

[0057] In another aspect, the composition further comprises a tumor-targeting antibody.

[0058] In another aspect, the composition further comprises a cell therapy.

[0059] In another aspect, the composition further comprises a gene therapy.

[0060] In another aspect, the composition further comprises a cancer vaccine.

[0061] In another aspect, the composition further comprises an oncolytic virus.

[0062] In another aspect, the antibody is trastuzumab.

[0063] In another aspect, the antibody is sacituzumab.

[0064] In another aspect, the linker and active oxygen phosphine payload is RTX5007.

[0065] In another aspect, the antibody-drug conjugate is trastuzumab-RTX5007.

[0066] In another aspect, the antibody-drug conjugate is sacituzumab-RTX5007.

[0067] In another aspect, the oxygen phosphine payload is isophosphoramide mustard.

[0068] In one aspect, the subject matter described herein relates to a method of treating a cancer in a subject in need thereof, comprising administering to the subject a composition comprising an antibody-drug conjugate of the formula Ab-(L-D)n, wherein Ab is an antibody, antibody fragment, antibody chain, affibody, aptamer, or nanobody; D is an active oxygen phosphine payload; L is a linker; and n has a value of 2 to 20.

[0069] In another aspect, the antibody-drug conjugate is present in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers.

[0070] In another aspect, the method further comprises administering a therapeutic agent.

[0071] In another aspect, the method further comprises administering radiochemotherapy.

[0072] In another aspect, the method further comprises administering an anti-TAM (tumor associated macrophage) agent.

[0073] In another aspect, the method further comprises administering one or more antibodies that enhance anti-tumor immunity selected from the group of: anti-PD-1, anti-PD-L1, anti- CTLA4, anti-LAG3, anti-GITR, anti-TIM-3, anti-TIGIT, anti-CD96, anti-CD226, anti- CD155, anti-CD47, anti-CEACAM1, anti-CEACAM5, anti-CEACAM6, anti- Galectin-1, anti-Siglec-15 antibody, anti-VISTA, anti-CD137, anti-CCR4 antibody, anti- CD39 antibody, anti-CD25 antibody, anti-CD-73 antibody, and anti-CSFR1. In another aspect, the method further comprises administering one or more cytokines from the group of: IL-1 beta, IL-2, IL-6, IL-7, IL-12, IL-15, IL-21, IL-23, IL-27, TNF alpha, IFN alpha, IFN gamma, GM-CSF), anti-IL2R, Toll-like receptor (TLR) activators, and stimulator of interferon genes (STING).

[0074] In another aspect, the Toll-like receptor activator is poly(I:C) and CpG.

[0075] In another aspect, the method further comprises one or more chemotherapeutic drugs selected from the group of 5-fluorouracil, 2'-deoxy-5-fluorouridine, cytarabine, cladribine, fludarabine, pentostatin, gemcitabine, 6-thioguanine, melphalan and any derivatives thereof; and alkylating drugs chiorambucil, bendamustine, melphalan, alkylating agents or anthracyclines such as doxorubicin, epirubicin, daunorubicin; temozolomide, melphalan, oxaliplatin, cisplatin, chiorambucil, mechlorethamine, mitoxantrone, pexidartinib, rafavertinib, trabectdin, HDAC inhibitors, anti-angiogenic drugs, bisphosphonates, taxanes, vinorelbine, ibutinib, eribulin, rigosertib or analogs thereof, anti-VISTA antibodies, anti-signalsome 4D, CXCR2 blockers, axitinib, sorafenib, cabozantinib, sunitinib, regorafenib, thalidomide, lenalidomide, pomalidomide, avadomide, vandetanib, cediranib or analogs thereof, anti-VEGF-A antibodies (bevacizumab), anti-VEGF-R2 antibodies (ramucirumab), TRL9 agonists, PPAR gamma agonists, miRNAs, angiotensin receptor blockers, CXCR4 blockers, CD4 / 6 inhibitors, proteasome inhibitors, JAK1 / 2 inhibitors, Bruton's kinase (BTK) inhibitors, kinase inhibitors, topoisomerase inhibitors, epigenetic inhibitors, DNMT, HMT, HDM inhibitors, PARP inhibitors, hormone antagonists, anti-prolactin, VEGI, osteopontin, matriptase, itraconazole, carboxyamidotriazole, suramin, thrombospondin, tetrathiomolybdate, linoamides, tasquinimod, carfilzomib, sunitinib, pazopanib, everolimus; anti-hormones: luteinizing hormone-releasing hormone (LHRH) antagonists, tamoxifen, corticosteroid analogs, steroid receptor modulators or antagonists, cancer metabolism inhibitors, radioisotopes, radiopharmaceuticals, vinca alkaloids, mTOR inhibitors, MEK inhibitors, BRAF inhibitors, MAPK and tyrosine kinase inhibitors, bortezomib, demethylating agents, bleomycin, alkylating agents, dacarbazine, temozolomide, and CELLMOD.

[0076] In another aspect, the taxane is selected from the group consisting of docetaxel, paclitaxel, cabazitaxel, and 6-alpha-hydroxypaclitaxel.

[0077] In another aspect, the epigenetic inhibitor targets HDAC, DNMT, LSD1, DOT1L, BET, or EZH.

[0078] In another aspect, the hormone antagonist is leuprolide.

[0079] In another aspect, the PARP inhibitor is selected from the group of 1-aminobenzamides, iniparib, BMN-573, olaparib, niraparib, talazoparib, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, and derivatives thereof.

[0080] In another aspect, the corticosteroid analogue is selected from the group of prednisone, dexamethasone; raloxifene, anastrozole, letrozole, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162, and any derivatives thereof.

[0081] In another aspect, the steroid receptor modulator or antagonist is selected from the group consisting of antiestrogens, antiprogestins, antiandrogens, anticorticoids, and antithyroid hormones.

[0082] In another aspect, the cancer metabolism inhibitor is selected from the group consisting of pyruvate kinase inhibitors and isocitrate dehydrogenase inhibitors.

[0083] In another aspect, the radioisotope is radium Ra 223 dichloride, lutetium Lu 177, actinium 225, yttrium 90, technetium 99, or iodine 131.

[0084] In another aspect, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine, and any derivatives thereof.

[0085] In another aspect, the method further comprises administering a tumor-targeting antibody.

[0086] In another aspect, the method further comprises administering a cell therapy.

[0087] In another aspect, the method further comprises administering a gene therapy.

[0088] In another aspect, the method further comprises administering a cancer vaccine.

[0089] In another aspect, the method further comprises administering radiotherapy.

[0090] In another aspect, the method further comprises administering phototherapy.

[0091] In another aspect, the method further comprises administering an oncolytic virus.

[0092] In another aspect, the antibody is trastuzumab.

[0093] In another aspect, the antibody is cetuximab.

[0094] In another aspect, the linker and active oxygen azaphosphane payload is RTX5007.

[0095] In another aspect, the antibody-drug conjugate is trastuzumab-RTX5007.

[0096] The antibody-drug conjugate is trastuzumab-RTX5007. BRIEF DESCRIPTION OF DRAWINGS

[0097] The present technology will be better understood by reading the following detailed description of non-limiting embodiments, taken in conjunction with the attached drawings, wherein: Figure 1 Metabolism of oxazaphosphorines and isophosphamidates is depicted (from Skarbek C. et al. 2015. J. Med. Chem. 58: 705-717); Figure 2 Conjugation of trastuzumab with oxazaphosphorine payload THLP-1 (SEC): 101921-10P is depicted; Figure 3 Drug antibody ratio (DAR) and concentration of 101921-10P determined by HIC and UV are depicted. DAR calculation from: UV: DAR = (ε p252nm- ε p280nm R) / (ε d280nmR- ε d252nm) (see reference: Clinical Cancer Research: 2004-10-7063~7070); Figure 4 Synthetic examples of linker-payload are depicted; Figure 5 Tregs in the tumor microenvironment and their key role in suppressing anti-tumor immunity are depicted (Dees, et al., Eur. J. Immunol. 2021. 51:280-291); Figure 6 Effects of Treg depletion on other immunosuppressive cells in the TME are depicted (adapted from Dees, et al., Eur. J. Immunol. 2021. 51:280-291); and Figure 7 Treg depletion and cancer cell cytotoxicity in the TME are depicted; and Figure 8 Examples of oxazaphosphorine structures and bioconversion pathways are depicted, from Anderson et al., Cancer Control: Journal of the Moffitt Cancer Center · January 2008.

[0098] Figure 9Cyclophosphamide metabolism is depicted. Gor et al. Breast Cancer Research 2010, 12:R26.

[0099] Figure 10 Synthesis of RTX5007 is depicted; Figure 11 Synthesis of RTX5014 is depicted; Figure 12 Synthesis of RTX5015 is depicted; Figure 13 Herceptin-RTX-5007 was assayed by SEC monomer; Figures 14A-14B Drug antibody ratio (DAR) of Herceptin-RTX-5007 was assayed by LC-MS; Figure 15 Concentration of Herceptin-RTX-5007 was assayed by measuring absorbance at 280 nm with an extinction coefficient of 14.8 on a nanodrop 2000C El%; Figure 16 Satumomab-RTX-5007 was assayed by SEC monomer; Figures 17A-17B DAR of Satumomab-RTX-5007 was assayed by LC-MS; Figure 18 Concentration of Satumomab-RTX-5007 was assayed by measuring absorbance at 280 nm with an extinction coefficient of 15.6 on a nanodrop 2000C El%; Figures 19A-19B Binding kinetics of Herceptin to HER2 are depicted; Figure 19B Individual concentration dependent calculations (local fit) are depicted; Figures 20A-20B Binding kinetics of Herceptin-ADC to HER2 are depicted; Figure 20B Individual concentration dependent calculations (local fit) are depicted; Figures 21A-21B Binding kinetics of Isotype to HER2 are depicted; Figure 21B Individual concentration dependent calculations (local fit) are depicted; Figures 22A-22B Binding kinetics of Satumomab to TROP2 are depicted; Figure 22B Individual concentration dependent calculations (local fit) are depicted; Figures 23A-23B Binding kinetics of Satumomab-ADC to TROP2 are depicted, Satumomab-ADC to TROP2 are depicted;Figure 23B Depicts individual concentration-dependent calculations (local fit); Figures 24A-24B Depicts binding kinetics of isotypes to TROP2; Figure 24B Depicts individual concentration-dependent calculations (local fit); Figures 25A-25B Depicts live cell imaging analysis (IncuCyte S3) evaluation of NCI-N87 cells across a range of seeding densities (Figure 25A for low density; Figure 25B for high density) in 96-well plates to monitor cell growth for up to 120 hours; Figure 26A depicts N87 cells treated with a cross-dose level of Herceptin-ADC (NJBP-0165-129) ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7 imaged every six hours for confluence % under phase contrast microscopy and apoptosis under 488 nm fluorescence to monitor, and Figure 26B-26E depict IC50 curves plotted at 24 hours (26B), 48 hours (26C), 72 hours (26D), and 96 hours (26E) of incubation; Figure 27A Depicts N87 cells treated with a cross-dose level of Herceptin-ADC (NJBP-0165-129) ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7 imaged every six hours for confluence % under phase contrast microscopy and apoptosis under 488 nm fluorescence to monitor, and Figure 26B-26E depict IC50 curves plotted at 24 hours (26B), 48 hours (26C), 72 hours (26D), and 96 hours (26E) of incubation; Figures 27B-27E Depicts IC50 curves plotted at 24 hours (27B), 48 hours (27C), 72 hours (27D), and 96 hours (27E) of incubation. DETAILED DESCRIPTION

[0100] All publications and patents cited in this disclosure are incorporated by reference in their entirety. In the event of inconsistencies between the disclosure of this specification and the materials incorporated by reference, the disclosure of this specification shall prevail. The citation of any reference in this disclosure is not an admission that such reference is prior art to the present disclosure. When expressing a range of values, it includes embodiments using any particular value within that range. Further, a reference to a value stated in a range includes each value within that range. All ranges include their endpoints and combinations. When a value is expressed as an approximation by use of the antecedent “about,” it is intended that the particular value forms another embodiment.

[0101] Various terms related to the aspects described are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise specified. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Techniques and procedures described or referenced herein are generally well understood and conventional methods are employed, such as, for example, widely used molecular cloning techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Unless otherwise indicated, procedures involving commercially available kits and reagents are carried out according to the manufacturer’s specified protocols and conditions, where appropriate.

[0102] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. The terms “comprises,” “comprising,” and similar terms are intended to cover a non-exclusive inclusion.

[0103] Unless otherwise indicated, the terms “at least,” “less than,” and “about” preceding a series of elements or a range of values are to be understood to refer to every element in the series or range of values. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

[0104] As used herein, the term “subject” refers to any animal, such as any mammal, including, but not limited to, humans, non-human primates, rodents, common pet mammal species (e.g., dogs and cats, etc.), livestock (e.g., cattle, sheep, goats, pigs, horses, camels, etc.), and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. The term “active oxygen aza phosphine payload” means that the oxygen aza phosphine is not a prodrug and does not need to be metabolized to be activated.

[0105] The abbreviation IPM stands for isophosphoramide mustard, and the abbreviation PM stands for phosphoramide mustard.

[0106] The drug antibody ratio (DAR) refers to the average number of drug molecules conjugated to an antibody.

[0107] Cancer remains a leading cause of death worldwide, with nearly 10 million deaths in 2020, nearly one in six of all deaths. According to the American Cancer Society, by 2040, population growth and aging alone will result in 27.5 million new cancer cases and 16.3 million cancer deaths globally.

[0108] New cancer therapies have made progress, such as targeted chemotherapy, immunotherapy such as immune checkpoint inhibitors (ICIs), cellular therapies, and antibody-drug conjugates (ADCs), which have revolutionized cancer treatment, improving patient survival. However, given that mortality rates remain unacceptably high, most patients do not respond to or develop resistance to existing treatments, and some patients cannot tolerate treatment due to toxicity, so there is a need to develop newer treatments.

[0109] Antibody drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of both antibodies and cytotoxic drugs to deliver toxic agents to tumor cells expressing antigens. They are typically designed to bind to target cancer cells, and release of the toxic agent can require internalization. Drug release occurs inside the cancer cell following cleavage of the linker at enzymatic or low pH. Alternatively, for ADCs that are not internalized, the toxic agent is released by cleavage of the linker at low oxygen or low pH within the tumor microenvironment (TME).

[0110] To date, clinically used ADCs consist of a targeting monoclonal antibody linked to a super-toxic agent such as a calicheamicin, auristatin, maytansinoid, or pyrrolobenzodiazepine. These agents are cytotoxic at extremely low (nanomolar or picomolar) concentrations. Upon delivery to the tumor, these highly toxic payloads re-enter the circulation and distribute to other organs, where they cause severe toxicities such as cardiotoxicity, neutropenia, anemia, fatigue, diarrhea, neurotoxicity, blindness, nephrotoxicity, or even death. Therefore, all of these ADCs have a “black box” warning on their labels. Black box warnings are the most serious warnings issued by the FDA for a prescription drug because such drugs can cause fatal, life-threatening, or permanently disabling adverse reactions. This means that such drugs must be limited and closely monitored during and after administration. The effectiveness of immunotherapy depends on conditions within the TME, including the dominance of immunosuppressive cells. Poor prognosis is associated with increased infiltration of immunosuppressive cells within the TME.

[0111] A current problem with cancer immunotherapy is that immunosuppressive T regulatory cells (Tregs) within the TME are a major obstacle to developing effective immunotherapy for cancer patients. Current methods of depleting Tregs lack distribution specificity because they also deplete Tregs in the blood circulation, disrupting systemic immunostasis, leading to autoimmunity, systemic inflammation, and off-target toxicity. The present disclosure describes a method of using ADCs to selectively deplete Tregs within the tumor microenvironment (TME) without disrupting systemic Treg homeostasis by delivering an oxazaphosphorine directly into the TME. Oxazaphosphorine metabolites are known to induce anti-tumor immunity through selective cytotoxicity against Tregs (Eid RA et al, 2016, Cancer Immunol Res ; 4(5);377-82. doi: 10.1158 / 2326-6066.CIR-16-0048; Traverso I et al. Human Immunology 73 (2012) 207-213 , doi:10.1016 / j.humimm.2011.12.020; Voelcker G 2018, Anti- Cancer Drugs 29:411-415; Heylmann D, Bauer M, Becker H, van Gool S, Bacher N, et al. (2013), PLoS ONE 8(12): e83384. doi:10.1371 / journal.pone.0083384). The ADCs target and bind to cancer cells to release a therapeutic oxazaphosphorine payload that depletes Tregs within the TME. The dual killing of cancer cells and Tregs triggers immune memory, thereby prolonging anti-tumor immunity. By increasing local delivery of the therapeutic payload within the TME, the therapeutic index is improved to minimize systemic toxicity and re-activate exhausted T cells to overcome immunotherapy resistance.

[0112] The present invention describes ADCs with superior safety profiles to approved ADCs because the drug payloads used are not super-toxic and drug-resistant cancer cells are responsive to the oxazaphosphorine payloads described above. The present invention relates to ADCs comprising a tumor-targeting antibody or antibody fragment conjugated to an oxazaphosphorine payload via a linker. ADCs comprising antibodies conjugated to active oxazaphosphorines have not been made previously.

[0113] Oxazaphosphorines include, but are not limited to, cyclophosphamide, ifosfamide, and trofosfamide. Oxazaphosphorine derivatives include, but are not limited to, mafosfamide, glufosfamide, beta-D-glucosylifosfamide mustard, aldo-phosphoramide dehydrothiazone, and aldo-phosphoramide thiazolidine. Cyclophosphamide and ifosfamide are prodrugs that require activation by cytochrome P450. Zhang, et al. (2005) Drug Metabolism Reviews, 37:4, 611-703. 4-hydroxycyclophosphamide is the major metabolite of cyclophosphamide. Figure 8Oxazaphosphorine structure and bioconversion pathway are depicted from Anderson et al., Cancer Control: Journal of the Moffitt Cancer Center · January 2008.

[0114] Oxazaphosphorines are alkylating agents that have been used for decades in routine clinical treatment of cancer. They are prodrugs that require bioactivation by cytochrome p450 in the liver to generate 4-hydroxy derivatives that are transported by blood circulation to tumors. They have both oncolytic and immunostimulatory properties. They directly kill cancer cells by causing immunogenic cell death (ICD). Their anti-tumor immunostimulatory properties occur because they selectively kill immunosuppressive cells such as regulatory T cells (Tregs), reactivating and proliferating tumor-specific cytotoxic T cells, NK cells, and macrophages that help kill cancer cells. The antibody portion of the ADC also directly kills cancer cells after binding to the tumor. Local delivery and release of oxazaphosphorine payloads within the tumor reduces systemic exposure, which is critical to minimize or reduce systemic toxicity and improve patient safety and tolerability. The ADCs can also be combined with established standard treatment therapies such as immune checkpoint inhibitors, chemotherapy, radiotherapy, phototherapy, gene therapy, or cell therapy to maximize patient treatment benefit.

[0115] Unlike previous ADCs that required highly potent toxic drug payloads, low doses (concentrations) of oxazaphosphorines are sufficient to exert their immunomodulatory effects. For example, it is well known that regular (metronomic) administration of low doses of cyclophosphamide to patients is sufficient to stimulate the immune system by depleting or reducing immunosuppressive regulatory T cells (Tregs). However, systemic toxicity still occurs due to drug distribution and exposure to normal tissues in the body from oral or systemic administration. Any toxicity of oxazaphosphorines is predictable and controllable based on decades of clinical experience relative to the potent payloads used in other ADCs.

[0116] Immune checkpoint inhibitors (ICIs) have also revolutionized cancer treatment. However, only 15-25% of patients respond to ICI therapy, while a large proportion of patients develop systemic immune-related toxicities, such as autoimmune diseases, cardiorespiratory complications, and most patients eventually develop resistance. Oxazaphosphorines, such as cyclophosphamide, can be effective partners for immune checkpoint inhibitors, as they can synergistically enhance antitumor immunity (George M et al., Cancer Res (2022) 82 (12_Supplement): 4167. https: / / doi.org / 10.1158 / 1538-7445.AM2022-4167). In a mouse melanoma model, the combination of cyclophosphamide with anti-CTLA-4 or anti-PD-1 antibodies showed to elicit potent tumor control. Clinically, the combination of ifosfamide, a cyclophosphamide derivative, with anti-CTLA-4 ipilimumab showed good clinical activity in 15 out of 22 patients, but drug-related toxicities were common (Hegde A, Jayaprakash P, Couillault CA, Piha-Paul S, Karp D, Rodon J, et al., Clin Cancer Res 2021; 27:3050-60). These results show that, while the combination of oxazaphosphorines with immune checkpoint inhibitors achieves synergistic effects, by localizing the drug action within the tumor microenvironment, it is possible to ameliorate dose-limiting toxicities caused by systemic exposure. A major mechanism leading to resistance of cancer to immune checkpoint inhibitor (ICI) treatment is the infiltration of immunosuppressive cells, such as T regulatory cells (Tregs), into the tumor microenvironment (TME). Tregs are a subset of CD4-positive T cells that play a central role in suppressing anti-tumor immunity by contributing to tumor cell evasion of immune surveillance (Lee, G. R., Phenotypic and functional properties of tumor-infiltrating regulatory T cells. Mediators Inflamm. 2017. 2017: 5458178.). They are the predominant immunosuppressive cells within the tumor microenvironment (TME), accounting for 10-50% of CD4 T cells in human tumors (Togashi, Y., Shitara, K. and Nishikawa, H., Regulatory T cells in cancer immunosuppression - implications for anticancer therapy. Nat. Rev. Clin. Oncol. 2019. 16: 356-371). In fact, high frequency of infiltrating Tregs in the TME is associated with poor prognosis and low survival rate in multiple types of solid tumors such as breast, prostate, lung, colorectal, ovarian, cervical, bladder, renal, pancreatic, hepatocellular, skin, brain, head and neck cancer (Tanaka, A.; Sakaguchi, S. Regulatory T Cells in Cancer Immunotherapy. Cell Res. 2017, 27, 109-118; Ménétrier-Caux, C.; Curiel, T.; Faget, J.; Manuel, M.; Caux, C.; Zou, W. Targeting Regulatory T Cells. Target. Oncol. 2012, 7, 15-28; Revilla SA et al., Colorectal Cancer-Infiltrating Regulatory T-Cells, Frontiers in Immunology 2022, 13: https: / / doi.org / 10.3389 / fimmu.2022.903564). Based on the key role of Tregs in suppressing anti-tumor immune responses, blocking their function and / or depleting them has become a viable strategy to enhance or stimulate anti-tumor immunity (Dees et al., Eur. J. Immunol. 2021. 51: 280-291). Multiple other approaches are currently being developed, including Treg cell depletion, inhibition of their activity, impeding their recruitment, and preventing their differentiation within the TME. The main challenge at present is how to specifically target Treg cells in the tumor site without affecting Tregs in the blood circulation. It is critical to avoid depleting Tregs in the blood circulation, as they play a key role in maintaining immune homeostasis to prevent the immune system from causing damage to self tissues. Depletion or inhibition of Tregs in the blood circulation can lead to unwanted immune-related side effects, such as autoimmunity or inflammation (Revilla SA et al., Colorectal Cancer-Infiltrating Regulatory T-Cells, Frontiers in Immunology 2022, 13: https: / / doi.org / 10.3389 / fimmu.2022.903564). In fact, current treatments that deplete or interfere with Treg function in the blood circulation also cause autoimmunity and other immune-related toxicities. To date, no specific treatment exists that specifically targets Tregs within the TME without affecting Tregs in the blood circulation.

[0117] Examples of commonly used oxazaphosphorines are cyclophosphamide, ifosfamide and trofosfamide. Trofosfamide is mainly metabolized to ifosfamide, with a small amount metabolized to cyclophosphamide. Cyclophosphamide and ifosfamide are prodrugs that must be metabolized by the cytochrome p450 enzyme pathway to produce active metabolites that are cytotoxic Figure 1). They undergo activation by 4-hydroxylation followed by spontaneous degradation into final active and inactive metabolites. Activation is mainly mediated by CYP2B6, leading to the formation of 4-hydroxycyclophosphamide, which is in equilibrium with the acyclic tautomers aldehyde phosphoramide and aldehyde ifosfamide (Jeswani G and Paul SD, Recent Advances in the Delivery of Chemotherapeutic Agents. In Nano- and Microscale Drug Delivery Systems, 2017). These metabolites then enter the blood circulation and distribute throughout the body before reaching the target cancer cells. Phosphoramide mustard and isophosphoramide mustard are the final active antitumor metabolites of cyclophosphamide and ifosfamide, respectively. Other byproducts of oxazaphosphorine metabolism are acrolein and chloroacetaldehyde, which cause known toxic effects (bladder hemorrhage, neurotoxicity, and urotoxicity are toxic effects of its precursors, as well as other dose-limiting toxicities including myelosuppression, cardiotoxicity, diarrhea, and fatigue). Phosphoramide mustard and isophosphoramide mustard do not cause urotoxicity or neurotoxicity because they do not contain acrolein or chloroacetaldehyde. Oxazaphosphorine derivatives or analogs that do not require liver CYP enzyme bioactivation have also been synthesized. They include mafosfamide, glufosfamide, and palifosfamide, evofosfamide, geranyloxy-isofosfamide, and analogs or derivatives thereof. Low doses of oxazaphosphorines have significant immunomodulatory activity, particularly the ability to suppress or deplete regulatory T cells (Tregs), thus countering immune suppression in the tumor microenvironment without affecting cytotoxic T cells or natural killer (NK) cells.

[0118] The approach of using ADCs to deliver oxazaphosphorines directly to the tumor microenvironment can mitigate or significantly reduce systemic toxicity. Local delivery of the ADC antibody moiety also contributes to the antitumor effect of the ADC. By engineering the Fc region, the optimal effector function of the ADC antibody moiety can be improved to increase its cytotoxic effect on cancer cells. By engineering the Fc region, modifications can improve its affinity / avidity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Glycosylation engineering, such as afucosylation and amino acid mutations, can be used. By modifying the antibody to bind to the neonatal Fc receptor, FcRn, pharmacokinetic properties can also be increased.

[0119] The rationale for using an azophosphine as a payload in an ADC is based on the existing literature reporting synergistic effects of azophosphines with antibodies, immune checkpoint inhibitors, or vaccines upon systemic administration. However, the toxicity of the combination limited its use. For example, cyclophosphamide has been shown to have a survival synergy when combined with mAbs such as rituximab (anti-CD20 mAb), trastuzumab (anti-HER2 mAb), cetuximab (anti-EGFR mAb), or alemtuzumab (anti-CD52 mAb) (Roghanian A et al 2019, Cancer Immunol Res 7: 1876-90; Nickenig C et al 2006, Cancer 107: 1014-22; Scott DW 2014, Nat Rev Cancer 14: 517-34; Keating MJ et al 2005, J Clin Oncol 23: 4079-88). Cyclophosphamide is also widely used as a component of combination chemotherapy (as part of a combination with other chemotherapy drugs for the treatment of breast cancer, leukemia, multiple myeloma, ovarian cancer, lymphoma, etc.). Despite the synergistic benefits of the combination, these combinations also have dose-limiting toxicities and are inconvenient. The combination therapy is time-consuming and inconvenient because each drug must be infused separately for an extended duration (2-4 hours each), with rest periods between infusions. This approach also reduces patient treatment compliance. The advantage of using an ADC is that it can combine the two drugs in one infusion, maximizing efficacy while minimizing systemic toxicity. ADCs can also be used in combination with other standard treatment therapies, including chemotherapy, immunotherapy, radiotherapy, phototherapy, gene therapy, cell therapy, oncolytic virus therapy, or vaccination.

[0120] The invention of the present disclosure includes antibodies, linkers, and payloads. In embodiments, the ADCs increase the accumulation of azophosphine payloads within the tumor microenvironment and reduce systemic exposure and toxicity. In embodiments, the payloads can selectively deplete immunosuppressive cells within the tumor microenvironment, such as Tregs; activate NK and effector T cells, reverse T cell exhaustion, alter the function of immune cells within the tumor microenvironment to more effectively target cancer cells. The azophosphine payloads also induce immunogenic cell death (ICD) of cancer cells and generate tumor-specific antigens, triggering long-lasting tumor-specific immune memory.

[0121] Advantages of the present disclosure include: applicability to both solid tumors and hematologic malignancies; use of a combination therapy that is clinically validated to be effective in cancer patients; novel immunomodulatory payloads and linkers; improved therapeutic index to eliminate or reduce systemic toxicity; ease of administration (one drug rather than two different modes of administration); and reduced development timeline from discovery to clinical trials.

[0122] The method can be used as a monotherapy or in combination with standard of care chemotherapy, radiotherapy, immunotherapy (including antibodies to immune checkpoint inhibitors), cellular therapy, gene therapy, oncolytic viral therapy, or vaccines. Herein, “immunotherapy” refers to the administration of antibodies, antigen binding fragments, antibody-drug conjugates (ADCs), and the like, which bind to one or more proteins in the body of a subject, thereby affecting a change in the function of the protein, which in turn improves the cancer in the subject. Radiotherapy can involve focusing a beam of radiation at the site of the cancer. Common radiotherapy regimens include treatment once per weekday for 6-12 weeks.

[0123] A clinician can select any one or a combination of these therapies based on one or more aspects of the subject’s cancer presentation, other aspects of the subject’s health, one or more demographic factors that can have medical significance, or one or more genetic markers of the subject, among other parameters. In embodiments, immunostimulatory antibody conjugates targeting proteins on cancer cells, such as HER2, EGFR, CD20, and TROP2, can be used to treat solid tumors or hematologic malignancies, including but not limited to colorectal cancer, breast cancer, lung cancer, gastric cancer, lymphoma, leukemia, sarcoma, or head and neck tumors, urothelial cancer, kidney cancer, brain cancer, uterine cancer, ovarian cancer, thyroid cancer, melanoma, liver cancer, pancreatic cancer, HNSCC.

[0124] In embodiments, the immunostimulatory ADC includes but is not limited to a monospecific, bispecific, trispecific, or tetraspecific monoclonal antibody.

[0125] Antibody-drug conjugates can be synthesized using a variety of methods. Yao et al., Int J Mol Sci. 2016 Feb; 17(2): 194.

[0126] The subject to be treated can be a mammal, such as a human, dog, cat, horse, or any other animal in need of cancer treatment.

[0127] In embodiments, the drug can be attached to the antibody or antibody fragment with a linker. In embodiments, the linker can be cleavable or non-cleavable. In embodiments, the linker can be cleaved by a variety of methods, including proteolysis, reduction or hydrolysis, hypoxia, or low pH.

[0128] In embodiments, RTX5007, RTX5014, and RTX5015 are linkers payloads.

[0129] In embodiments, the synthesis of RTX5007 is accomplished using the chemical methods exemplified in Example 2. Figure 10

[0130] In embodiments, the synthesis of RTX5014 is accomplished using the chemical methods exemplified in Example 3. Figure 11

[0131] In embodiments, the synthesis of RTX5015 is accomplished using the chemical methods exemplified in Example 4. Figure 12

[0132] In embodiments, the antibody-drug conjugate is Herceptin-RTX-5007. In embodiments, Herceptin-RTX-5007 is made as in Example 5.

[0133] RTX-5007 conjugation scaled up to a maximum of 7.4 mg, Herceptin was 4.9 mg / mL.

[0134] Herceptin was reduced at 37 °C using 6 eq of 10 mM TCEP (29.4 µL) for 1.5 h. After reduction, EDTA was added to the protein to 1 mM (taken from 250 mM stock, 6.1 µL). Prior to payload addition, the buffer composition was adjusted to 10% DMA with 150 µL of additional DMA. After addition of the organic solvent, 15 eq of RTX-5007 (73.5 µL of 10 mM RTX-5007 in DMA) was added to the mAb. The mAb solution was mixed at room temperature for 1.5 h, then checked for DAR. After confirming the targeted DAR, 30 eq of 10 mM N-acetylcysteine (147 µL) was added to the reaction to quench the RTX-5007. After conjugation, dialysis was performed using 1x PBS (pH 7.4) to remove excess payload. After 3 buffer exchanges over 12 hours, the final buffer exchange was to 1x PBS (pH 7.4).

[0135] ​​​After removal of the payload, 6.4 mg of ADC was recovered and the DAR was determined to be 7.8 by LC-MS. The monomer purity was determined to be 100% by SEC. Its concentration was determined to be 4.0 mg / mL by A280.

[0136] Figure 13 Hertcinitu-RTX-5007 monomer was profiled by SEC (20 pg conjugate injected onto TSKgel G3000SWXL with 1 x PBS with 10% IPA as the mobile phase).

[0137] Figures 14A-14B Hertcinitu-RTX-5007 drug to antibody ratio (DAR) was profiled by LC-MS (0.2 pg reduced material injected onto PLRP-S (1000 A, 8 pm, 2.1 mm x 50 mm)).

[0138] Figure 15 Hertcinitu-RTX-5007 concentration was profiled by A280 on a nanodrop 2000C El% with an extinction coefficient of 14.8.

[0139] In embodiments, the antibody-drug conjugate, gemtuzumab-RTX-5007 conjugate was made as in Example 6.

[0140] Prior to conjugation, gemtuzumab was dialyzed into 1 x PBS, pH 7.4. RTX-5007 conjugation was scaled up to a maximum of 10 mg reactions with gemtuzumab at 5.0 mg / mL.

[0141] Gemtuzumab was reduced at 37 °C using 6 equivalents of 10 mM TCEP (40 pL) for 1.5 h. After reduction, EDTA was added to the protein at 1 mM (taken from a 250 mM stock, 8.2 pL). Prior to the addition of the payload, the buffer composition was adjusted to 10% DMA with 200 pL of additional DMA. After the addition of the organic solvent, 20 equivalents of RTX-5007 (133.3 pL of 10 mM RTX-5007 in DMA) were added to the mAb. The mAb solution was mixed at room temperature for 1.5 h before checking the DAR. After confirming the targeted DAR, 40 equivalents of 10 mM N-acetyl cysteine (266.6 pL) were added to the reaction to quench the RTX-5007. After conjugation, dialysis was performed using 1 x PBS (pH 7.4) to remove excess payload. After 3 buffer exchanges over 12 hours, the final buffer exchange was to 1 x PBS (pH 7.4).

[0142] After removal of the payload, 5.4 mg of ADC were recovered and the DAR was determined by LC-MS to be 8.0. The monomer showed a purity of 100% by SEC. Its concentration was determined to be 3.0 mg / mL by A280.

[0143] Figure 16 Determination of the DAR of satumomab-RTX-5007 by LC-MS (0.2 pg of reduced material was injected on PLRP-S (1000 A, 8 pm, 2.1 mm x 50 mm)) is depicted.

[0144] Figures 17A-17B Determination of the DAR of satumomab-RTX-5007 by LC-MS (0.2 pg of reduced material was injected on PLRP-S (1000 A, 8 pm, 2.1 mm x 50 mm)) is depicted.

[0145] Figure 18 Determination of the concentration of satumomab-RTX-5007 by A280 with an extinction coefficient of 15.6 on a nanodrop 2000C El% is depicted.

[0146] In an embodiment, the binding kinetics of an antibody-drug conjugate using an anti-HER2 antibody (Herceptin) and an antibody-drug conjugate using an anti-TROP2 antibody (satumomab) were determined.

[0147] Different concentrations of Herceptin-ADC or satumomab-ADC were used to determine the interaction kinetics. Herceptin or satumomab were used as positive controls, human isotype IgG as negative control, with the same concentration dilution.

[0148] His-tagged human HER2 or His-tagged human Trop2 (~ 35 nM and 150 nM, respectively) were loaded on the Ni-NTA probe by immersing the probe in 180-300 seconds. The His-tagged receptor-loaded probe was immersed in different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) of Herceptin-ADC / satumomab-ADC or mAb for 300 seconds to determine the association kinetics. The His-tagged receptor probe was immersed in Q-buffer for 300 seconds to determine the dissociation kinetics of the bound ADC / mAb.

[0149] Figures 19A-19B Binding kinetics of Herceptin to HER2 are depicted.

[0150] Figures 20A-20B Binding kinetics of Herceptin-ADC to HER2 are depicted.

[0151] Figures 21A-21BBinding kinetics of isotype to HER2 are depicted.

[0152] Figures 22A-22B Binding kinetics of satumomab to TROP2 are depicted.

[0153] Figures 23A-23B Binding kinetics of satumomab-ADC to TROP2 are depicted.

[0154] Figures 24A-24B Binding kinetics of isotype to TROP2 are depicted.

[0155] Both Herceptin and its ADC derivative show comparable binding capacity to the HER-2 receptor (in the range of about 0.5-2 nM), indicating that conjugation has little effect on the receptor binding capacity of the mAb. Likewise, satumomab and its ADC show comparable binding capacity to the TROP-2 receptor (in the range of about 0.04-0.1 nM). In embodiments, in vitro cytotoxicity data for antibody-drug conjugates using an anti-HER2 antibody (Herceptin) and an anti-TROP2 antibody (satumomab) were determined.

[0156] Herceptin-ADC is an antibody-drug conjugate (ADC) targeting Her2 that delivers a small molecule (oxazaphosphorine) payload with anti-tumor activity. The N87 cell line was reported to express high levels of Her2 mRNA transcript and protein.

[0157] Satumomab-ADC is an antibody-drug conjugate (ADC) targeting Trop2 that delivers a small molecule (oxazaphosphorine) payload with anti-tumor activity. The N87 cell line was reported to express moderate levels of Trop2 mRNA transcript.

[0158] Figures 25A-25B Binding kinetics of satumomab to TROP2 are depicted. In embodiments, in vitro cytotoxicity studies were designed as follows. NCI-N87 cells were plated at 30,000 cells / well in 96-well plates and allowed to adhere overnight in normal growth media (RPMI-10% FBS). N87 cells were treated with cross-dose levels of trastuzumab-ADC (NJBP-0165-126) or herceptin-ADC (NJBP-0165-129) ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7, phase-contrast microscopy was imaged every six hours to monitor confluence, and apoptosis was monitored under 488 nm fluorescence. IC50 curves were plotted after 24, 48, 72-74, and 96 hours of incubation, respectively. In embodiments, cell density can vary depending on the cell line utilized.

[0159] Figure 26A depicts N87 cells treated with cross-dose levels of herceptin-ADC (NJBP-0165-129) ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7, phase-contrast microscopy was imaged every six hours to monitor confluence, and apoptosis was monitored under 488 nm fluorescence, and Figures 26B-26E depict IC50 curves plotted after 24 hours (26B), 48 hours (26C), 72 hours (26D), and 96 hours (26E) of incubation.

[0160] Figure 27A Figure 27A depicts N87 cells treated with cross-dose levels of trastuzumab-ADC (NJBP-0165-126) ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7, phase-contrast microscopy was imaged every six hours to monitor confluence, and apoptosis was monitored under 488 nm fluorescence; Figures 27B-27E Figures 27B-27E depict IC50 curves plotted after 24 hours (27B), 48 hours (27C), 72 hours (27D), and 96 hours (27E) of incubation.

[0161] Results show that the response to the ADCs is dose- and target-dependent. Results show that the cytotoxicity of both herceptin-ADC and trastuzumab-ADC is target (HER2 expression) and dose-dependent in N87 cells. N87 cells were more sensitive to herceptin-ADC (IC50 = 0.026 pg / mL) than to trastuzumab-ADC (IC50 = 0.440 pg / mL) at 96 hours.

[0162] In embodiments, the antibody in the ADC is trastuzumab (herceptin).

[0163] In embodiments, the antibody in the ADC is trastuzumab. In embodiments, the antibody in the ADC is trastuzumab.

[0164] In embodiments, the active oxazaphosphorine payload in the ADC is 10-oxa-4- azatricyclo[5.2.1.0,2.6]dec-8-ene-3,5-dione.

[0165] In embodiments, the linker and active oxazaphosphorine payload in the ADC is RTX5007.

[0166] In embodiments, the antibody-drug conjugate is trastuzumab-RTX5007.

[0167] In embodiments, the antibody-drug conjugate is trastuzumab-RTX5007.

[0168] In embodiments, the method of treating cancer comprises administering an ADC, wherein the antibody is trastuzumab.

[0169] In embodiments, the method of treating cancer comprises administering an ADC, wherein the antibody is trastuzumab.

[0170] In embodiments, the method of treating cancer comprises administering an ADC, wherein the active oxazaphosphorine payload is 10-oxa-4-azatricyclo[5.2.1.0,2.6]dec-8-ene-3,5-dione.

[0171] In embodiments, the method of treating cancer comprises administering an ADC, wherein the linker and active oxazaphosphorine payload is RTX5007.

[0172] In embodiments, the method of treating cancer comprises administering an ADC, wherein the antibody-drug conjugate is trastuzumab-RTX5007.

[0173] In embodiments, the method of treating cancer comprises administering an ADC, wherein the antibody-drug conjugate is trastuzumab-RTX5007.

[0174] The present disclosure also relates to pharmaceutical compositions that can include immunostimulatory ADCs that can be administered to a subject, with the aim including but not limited to treating cancer or other hyperproliferative diseases. Compositions including the ADCs can be optimized to be suitable for administration to a subject via intravenous, subcutaneous, intramuscular, intradermal, or intracerebral injection routes. The pharmaceutical compositions can be supplied in liquid solutions, suspensions, emulsions, or solid forms suitable for dissolution or suspension in a liquid prior to use.

[0175] The pharmaceutical composition can include a pharmaceutically acceptable carrier, that is, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is nontoxic to the subject to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include solutions of phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated in conventional ways and administered to a subject in a suitable dose. Preferably, the composition is sterile. These compositions also can contain adjuvants such as preservatives, emulsifying agents, and dispersing agents. Prevention of the growth of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents.

[0176] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippincott Williams & Wilkins (2005). Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic; of course, the formulation can also be hypertonic or hypotonic if desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffers such as Linger's solution and dextrose solution. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5. Certain carriers can be more preferred depending on, for example, the route of administration and the concentration of the composition being administered. Carriers suitable for direct delivery can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various Tween compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. More preferably, the composition is combined with saline, Linger's balanced salt solution (pH 7.4), and the like.

[0177] The pharmaceutical compositions disclosed herein can be formulated for administration. The amount of the pharmaceutical composition to be administered can be determined by standard procedures well known to those of ordinary skill in the art. The patient's physiological data (e.g., age, size, and weight) and the type and severity of the disease being treated must be considered in determining appropriate dosages.

[0178] The pharmaceutical composition can be formulated to be administered by injection, e.g., intravenous, intramuscular, subcutaneous, or intradermal injection. Formulations for injection can be presented in unit dosage form, e.g., in ampuls or multiple-dose containers. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Optionally, the pharmaceutical composition can be stored frozen and at any suitable temperature for storage. Gene therapy Gene therapy refers to the modification of a cell to insert a polynucleotide into the cell, e.g., as an episome and / or integrated into the genome, where the polynucleotide provides a therapeutic effect by encoding a polypeptide whose expression is desired, disrupting a genomic sequence that encodes a polypeptide whose silencing is desired, modulating transcription of an endogenous sequence that encodes a polypeptide, etc. Typically, such modification can be achieved by preparing a vector that includes the polynucleotide and using the vector to insert the polynucleotide into the nucleus of one or more cells.

[0179] Suitable gene therapy vectors are known in the art and include, e.g., AAV vectors and retroviral vectors (e.g., lentiviral vectors, gamma retroviral vectors).

[0180] Kit Also disclosed herein is a kit comprising an ADC described herein or a pharmaceutical composition thereof. The kit can be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, buffered solution, or other pharmaceutically acceptable sterile fluids. Alternatively, the complex can be lyophilized or dried; in such cases, the kit optionally further comprises a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.) in a container to reconstitute the complex to form a solution for injection purposes. The kit can further comprise a needle or syringe (preferably packaged in sterile form) for injection of the complex, and / or packaged alcohol swabs. Optionally included are instructions for administration of the composition by a clinician or patient.

[0181] Methods It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the application described herein are apparent and can be used without departing from the scope or spirit of the disclosure and that the scope of protection is only to be accorded the broadest interpretation of the appended claims to the fullest extent of the law. Having described certain compositions and methods in detail, these and other compositions and methods will be more clearly understood by reference to the following examples, which are presented by way of illustration and are not intended to be limiting.

[0182] Examples The following are examples of the methods and compositions of the present application. It is to be understood that various other embodiments can be practiced in view of the general description provided herein.

[0183] Example 1. Preparation of Trastuzumab-THLP1 as a prototype oxazaphosphorine-ADC using a hydrazone linker.

[0184] The preparation of Trastuzumab-THLP1 was done in two parts: first the synthesis of the linker payload and second the preparation of the corresponding ADC, Trastuzumab-THLP1. The synthesis of the linker payload was done as depicted in Figure 4

[0185] Reagents and reaction conditions: (a) tert-butyldimethylsilyl chloride, imidazole, DMF; (b) hydrazine hydrate, acetic acid, ethanol; (c) oxalyl chloride, triethylamine, DMF; (d) TBAF, acetic acid, THF; (e) POCl3, 2-chloroethylamine hydrochloride, triethylamine, DCM.

[0186] 1-[4-(hydroxymethyl)phenyl]ethan-1-one (1) was treated with tert-butyldimethylsilyl chloride and imidazole in DMF to give the protected alcohol 2 in 98% yield. This protected alcohol was treated with hydrazine hydrate and acetic acid in ethanol to give the desired hydrazone 3 in 86% yield. The 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid was activated with oxalyl chloride in dichloromethane using a catalytic amount of DMF to give the corresponding acid chloride which was coupled to the hydrazone 3 to give the acylhydrazone 5. Deprotection of the tert-butyldimethylsilyl group using TBAF buffered with acetic acid gave the alcohol 6. Finally, treatment with POCl3at -78 °C followed by 2-chloroethylamine hydrochloride and triethylamine in dichloromethane gave the target compound 7.

[0187] Using the linker payload, the target ADC was prepared following the procedure: a solution of TCEP in PBS buffer (156.7 µL, 0.5 mM, pH=7) was added to a solution of Trastuzumab (800.0 µL, 5 mg / mL), the resulting solution was gently shaken and incubated at 33 °C for 1 hour; the linker payload THLP-1 (66.0 µL, 2.5 mM in DMSO) was added, the solution was gently shaken and kept at 25 °C for 0.5 hour. The crude sample was purified through an Illustra NAP-10 column (GE Healthcare) and filtered through 0.25 µm microfiber to give the final ADC TmAb-THLP1, 1.75 mL, 1.77 mg / mL, DAR=4.73, UmAb%=3.33%, Agg%=0.80%.

[0188] ​Other linkers to conjugate antibodies with payloads are well known to the skilled person {Yao H et al. Int. J. Mol. Sci. (2016), 17, 194; doi:10.3390 / ijms17020194; Doronina SO et al. Nat. Biotechnol. 21 (2003) 778-784; Hamann PR et al. Chem. 13 (2002) 47-58; Backer BS et al. Tetrahedron Lett. (2020) 61(12): doi:10.1016 / j.tetlet.2; Tang H et al. Front. Pharmacol. (2019) 10:373. doi: 10.3389 / fphar.2019.00373}; Khongoruzul P et al. Mol Cancer Res 2020; 18:3-19 doi: 10.1158 / 1541-7786.MCR-19-0582; Baah S et al Molecules 2021, 26, 2943. https: / / doi.org / 10.3390 / molecules26102943.

[0189] Example 2. Synthesis of linker-payload RTX5007 (phosphoramide mustard).

[0190] The synthesis of RTX5007 was accomplished using the chemistry exemplified in Scheme 1. Figure 10 The synthesis of RTX5007 was accomplished using the chemistry exemplified in Scheme 1.

[0191] Reagents and reaction conditions: (a) methanol, sulfuric acid; (b) (3-bromopropoxy)-tert-butyldimethylsilane, potassium carbonate, DMF; (c) diisobutylaluminum hydride, THF; (d) lithium bis(trimethylsilyl)amide, bis(2-chloroethyl)phosphoramide dichloride, ammonia, THF; (e) hydrochloric acid, water, methanol; (f) carbon tetrabromide, triphenylphosphine, dichloromethane; (g) 10-oxa-4-azatricyclo[5.2.1.0,2.6]dec-8-ene-3,5-dione, potassium carbonate, DMF; (h) toluene. 2-Hydroxy-4-nitrobenzoic acid 1 was treated with sulfuric acid in methanol to give ester 2. Purification by silica gel column gave 76% yield; confirmed by 1H NMR (NMR purity > 95%). Ester 2 was treated with (3-bromopropoxy)-tert-butyldimethylsilane in the presence of potassium carbonate in DMF to give protected ester 3. Purification by silica gel column gave quantitative yield; confirmed by 1H NMR (NMR purity > 95%). Protected ester 3 was reduced with diisobutylaluminum hydride (DIBAL-H) in THF to give alcohol 4. Purification by silica gel column gave 87% yield; confirmed by 1H NMR (NMR purity > 95%). Alcohol 4 was treated with lithium bis(trimethylsilyl)amide at -78 °C, then bis(2-chloroethyl)phosphoramide dichloride, followed by ammonia in THF to give phosphoramide 5. Purification by silica gel column gave 79% yield; confirmed by 1H NMR (NMR purity > 95%). Phosphoramide 5 was deprotected with aqueous hydrochloric acid (1 M) in methanol to give alcohol 6. Purification by silica gel column gave 56% yield; confirmed by 1H NMR (NMR purity > 95%). Alcohol 6 was treated with carbon tetrabromide and triphenylphosphine in dichloromethane to give bromide 7. Purification by silica gel column gave 60% yield; confirmed by 1H NMR and MS ES+ = 492, 494 (NMR purity > 95%). Bromide 7 was substituted with 10-oxa-4-azatricyclo[5.2.1.0,2.6]dec-8-ene-3,5-dione in the presence of potassium carbonate in DMF to give protected phthalimide 8. Purification by silica gel column gave 57% yield; confirmed by 1H NMR and MS ES+ = 577 (NMR purity > 95%).

[0192] Finally, compound 8 underwent a reverse Diels-Alder in toluene at 120 °C to give target compound RTX5007. Crude RTX5007 was purified by silica gel column to give 55% yield. The product was confirmed by 1H NMR, 31P NMR, MS ES+ = 509, and HPLC (purity = 98.1%). 100 mg was prepared for biological testing.

[0193] Example 3. Synthesis of linker-payload RTX5014 (isophosphoramide mustard).

[0194] The synthesis of RTX5014 was accomplished in 5 steps from commercially available maleic anhydride 1 and furan 2. The synthesis of RTX5014 used Figure 11 The chemistry exemplified was accomplished.

[0195] Reagents & Conditions: a) diethyl ether, rt, 24 h; b) 3-amino-1-propanol, MeOH, 0 °C - rt, overnight; c) toluene, 130 °C, 6 h; f) MeOH, anodic oxidation; g) BF3.OEt2, DCM.

[0196] Procedure: Maleic anhydride (25 g, 253.5 mmol, 1 equiv) was reacted with furan (34.5 g, 507 mmol, 1 equiv) in diethyl ether (250 ml) at rt overnight to provide bicyclic Diels-Alder adduct 3 (21 g) as a white powder in 50% yield, which was filtered and washed with cold diethyl ether. The crude material was of high enough purity by NMR to be used in the next step without further purification. 3-Amino-1-propanol (4.5 g, 60 mmol, 1 equiv) was reacted with adduct 3 (10 g, 60 mmol, 1 equiv) in MeOH to provide compound 4 (5.6 g) as a white solid in 42% yield, which was used as such in the next step without further purification. Compound 4 (3 g, 13.44 mmol) was refluxed in toluene (10 ml) at 130 °C for 6 hours to provide the desired compound 5 (1.6 g) as a waxy solid in 75% yield (structure confirmed by1H NMR, 95% purity).

[0197] Commercially available isophosphoramide 6 (300 mg, 1.1 mmol, 1 equiv) was subjected to electrochemical anodic oxidation (J. Med. Chem. 2015, 58, 705 717) to provide compound 7 (300 mg, crude yield 96%) which was used immediately without any further purification due to its high instability. Formation of compound 7 was confirmed by TLC (DCM: acetone 1 : 1). Compound 7 (300 mg, 1.1 mmol, 1 eg.) was reacted with compound 5 (356 mg, 2.3 mmol, 2 eg.) in the presence of BF3.OEt2(152 mg, 1.1 mmol, 1 eg.) in DCM at -78 °C to provide crude RTX5014 (523 mg). Mass spectrometry analysis of the crude reaction mixture confirmed the formation of the product. The crude reaction mixture was purified by silica gel column chromatography.1H NMR of the isolated compound showed the desired product with a small amount of impurities (87 mg, 18%, ~85% purity by NMR). Sample NMR recorded after 4 days showed further decomposition. The bulk stored sample was purified again to remove the impurities to provide 27 mg of RTX5014 (~85% purity by NMR).

[0198] Example 4. Synthesis of linker-payload RTX5015.

[0199] Synthesis of RTX5015 was accomplished in 7 steps from commercially available maleic anhydride 1 and furan 2. The chemistry exemplified was used. Figure 12

[0200] Reagents & conditions: a) diethyl ether, rt, 24 h; b) 3-amino-1-propanol, MeOH, 0 °C - rt, overnight; c) PPh3, DIAD, 4-hydroxyacetophenone, 0 °C - rt, overnight; d) DCM:MeOH (3:1), NaBH4, rt, 4 h; e) toluene, 130 °C, 6 h; f) MeOH, anodic oxidation; g) BF3.OEt2, DCM.

[0201] ​Procedure: Maleic anhydride (25 g, 253.5 mmol, 1 equiv) was reacted with furan (34.5 g, 507 mmol, 1 equiv) in diethyl ether (250 ml) at room temperature overnight to provide bicyclic Diels-Alder adduct 3 (21 g) as a white powder, yield 50%, which was filtered and washed with cold diethyl ether. The crude material was pure enough by NMR to be used in the next step without further purification. 3-Amino-1-propanol (4.5 g, 60 mmol, 1 equiv) was reacted with adduct 3 (10 g, 60 mmol, 1 equiv) in MeOH to provide compound 4 (5.6 g) as a white solid, yield 42%, which was used as such in the next step without further purification. Compound 4 (6 g, 27 mmol, 1 equiv) was reacted with 4-hydroxyacetophenone (3.7 g, 27 mmol, 1 equiv), DIAD (6 g, 30 mmol, 1.1 equiv) and PPh3(7.7 g, 30 mmol, 1 equiv) in THF under Mitsunobu reaction conditions to provide compound 5 (5.2 g) after column chromatography, yield 58% (structure confirmed by1H NMR, purity 95%). Compound 5 (5 g, 14.6 mmol, 1 equiv) was reduced with NaBH4(1.1 g, 29.3 mmol, 2 equiv) in a mixture of DCM:MeOH (3:1) to provide the desired alcohol 6 (1 g) after column chromatography, yield 20% (structure confirmed by1H NMR). Compound 6 (175 mg, 0.5 mmol) was refluxed in toluene (3 ml) at 130 °C for 6 hours to provide the desired compound 7 (115 mg) as a waxy solid after column chromatography on silica gel, yield 82% (structure confirmed by1H NMR, purity 95%).

[0202] Commercially available cyclophosphamide 8 (300 mg, 1.1 mmol, 1 equiv) was subjected to electrochemical anodic oxidation (J. Med. Chem. 2015, 58, 705 717) to provide compound 9 (300 mg, crude yield 96%) which was used immediately without any further purification as compound 9 is reported to be very unstable. Formation of compound 9 was confirmed by TLC (DCM: Acetone 1:1). Compound 9 (300 mg, 1 mmol, 1 eq) was reacted with compound 7 (443 mg, 16 mmol, 1.5 eq) in the presence of BF3.OEt (152 mg, 1 mmol, 1 eq) in DCM at -78 °C to provide crude RTX5015 (533 mg). Mass spectrometry analysis of the crude reaction mixture confirmed the formation of the product. The crude reaction mixture was purified by silica gel column chromatography.1H NMR of the isolated compound showed that the desired product had multiple stereoisomeric products with a yield (38 mg, 7%, ~85% purity by NMR). However, the presence of multiple product stereoisomers made it difficult to calculate their exact ratio and total purity. The presence of characteristic product signals in1H NMR along with mass spectrometry data supported the formation of the product and total purity. The stability of the product was checked by NMR after 48 h and it was found to be stable at -20 °C.

[0203] Example 5. Preparation of antibody-drug conjugates using an anti-HER2 antibody (trastuzumab (Herceptin)).

[0204] Herceptin-RTX-5007 RTX-5007 conjugation scaled up to 7.4 mg with Herceptin at 4.9 mg / mL.

[0205] Herceptin was reduced at 37 °C using 6 eq of 10 mM TCEP (29.4 µL) for 1.5 h. After reduction, EDTA was added to the protein at 1 mM (taken from 250 mM stock, 6.1 µL). The buffer composition was adjusted to 10% DMA with 150 µL of additional DMA prior to the addition of the payload. After the addition of the organic solvent, 15 eq of RTX-5007 (73.5 µL of 10 mM RTX-5007 in DMA) was added to the mAb. The mAb solution was mixed at room temperature for 1.5 h before checking the DAR. After confirming the targeted DAR, 30 eq of 10 mM N-acetylcysteine (147 µL) was added to the reaction to quench the RTX-5007. After conjugation, dialysis was performed using 1x PBS (pH 7.4) to remove excess payload. After 3 buffer exchanges in 12 h, the final buffer exchange was done with 1x PBS (pH 7.4).

[0206] After removal of the payload, 6.4 mg of ADC was recovered and the DAR was determined to be 7.8 by LC-MS. The monomer purity was determined to be 100% by SEC. Its concentration was determined to be 4.0 mg / mL by A280.

[0207] Figure 13 Hertcinitu-RTX-5007 monomer was profiled by SEC (20 pg conjugate was injected onto a TSKgel G3000SWXL with a mobile phase of 1 x PBS with 10% IPA).

[0208] Figures 14A-14B Hertcinitu-RTX-5007 drug to antibody ratio (AR) was profiled by LC-MS (0.2 pg reduced material was injected onto a PLRP-S (1000 A, 8 pm, 2.1 mm x 50 mm)).

[0209] Figure 15 Hertcinitu-RTX-5007 concentration was determined by A280 on a nanodrop 2000C El% with an extinction coefficient of 14.8.

[0210] Example 6. Making antibody-drug conjugates using an anti-TROP2 antibody (satumuzumab).

[0211] Satumuzumab-RTX-5007 conjugate.

[0212] Satumuzumab was dialyzed into 1 x PBS, pH 7.4 prior to conjugation. RTX-5007 conjugation was scaled up to a maximum of 10 mg reactions with satumuzumab at 5.0 mg / mL.

[0213] Satumuzumab was reduced at 37 °C for 1.5 h using 6 equivalents of 10 mM TCEP (40 pL). After reduction, EDTA was added to the protein at 1 mM (taken from a 250 mM stock, 8.2 pL). The buffer composition was adjusted to 10% DMA with 200 pL of additional DMA prior to payload addition. After addition of the organic solvent, 20 equivalents of RTX-5007 were added to the mAb (133.3 pL of 10 mM RTX-5007 in DMA). The mAb solution was mixed at room temperature for 1.5 h, then the DAR was checked. After confirmation of the targeted DAR, 40 equivalents of 10 mM N-acetyl cysteine (266.6 pL) were added to the reaction to quench the RTX-5007. After conjugation, dialysis was performed using 1 x PBS (pH 7.4) to remove excess payload. After 3 buffer exchanges over 12 hours, the final buffer exchange was to 1 x PBS (pH 7.4).

[0214] After payload removal, 5.4 mg of ADC was recovered and the DAR was determined to be 8.0 by LC-MS. The monomer showed a purity of 100% by SEC. Its concentration was determined to be 3.0 mg / mL by A280.

[0215] Figure 16 Depiction of determination of satumomab-RTX-5007 monomer by SEC (20 pg of conjugate was injected into TSKgel G3000SWXL with 10% IPA in 1 x PBS as mobile phase).

[0216] Figures 17A-17B Depiction of determination of DAR of satumomab-RTX-5007 by LC-MS (0.2 pg of reduced material was injected into PLRP-S (1000 A, 8 pm, 2.1 mm x 50 mm)).

[0217] Figure 18 Depiction of determination of concentration of satumomab-RTX-5007 by A280 with an extinction coefficient of 15.6 on a nanodrop 2000C El%.

[0218] Example 7. Binding kinetics of antibody-drug conjugates using anti-HER2 antibody (Herceptin) and antibody-drug conjugates using anti-TROP2 antibody (satumomab).

[0219] Probes used: Ni-NTA; buffers: Q-buffer, K-buffer (Q-buffer: 0.2% BSA, 0.02% Tween-20, 0.05% Proclin 300 in PBS; K-buffer: 10% Q-buffer diluted in PBS) All receptors and mAbs were diluted in K-buffer, Q-buffer was used to initiate dissociation of the complex.

[0220] Loading concentrations of His-tagged human HER2 (2.5 pg / mL) or His-tagged human Trop2 (5 pg / mL) (receptors for ADCs) for Ni-NTA probe.

[0221] Different concentrations of Herceptin-ADC / satumomab-ADC were used to determine the interaction kinetics. Herceptin / satumomab were used as positive controls, human isotype IgG was used as negative control, with the same concentration dilution.

[0222] Human HER2 with His tag or human Trop2 with His tag (~35 nM and 150 nM, respectively) were loaded onto Ni-NTA probes by immersing the probes for 180–300 seconds; additional probes that were not loaded with His-tagged receptors in the association step but were used with ADC / mAbs were used for background correction.

[0223] The probe loaded with the His-tagged receptor was immersed in different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) of Herceptin-ADC / Saltuzumab-ADC or mAb for 300 seconds to determine the association kinetics.

[0224] The His-tagged receptor probe was immersed in Q buffer for 300 seconds to determine the dissociation kinetics of the bound ADC / mAb.

[0225] The data are presented as individual concentration-dependent calculations (local fitting).

[0226] Figures 19A-19B The binding kinetics of Herceptin to HER2 were depicted. Probes loaded with His-tagged receptors were immersed in different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) of Herceptin-ADC or mAb for 300 seconds to determine the association kinetics. Figure 19B The calculation of individual concentration dependence (local fitting) is described.

[0227] Figures 20A-20B The binding kinetics of Herceptin-ADC to HER2 were depicted. Probes loaded with His-tagged receptors were immersed in different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) of Herceptin-ADC for 300 seconds to determine the association kinetics. Figure 20B The calculation of individual concentration dependence (local fitting) is described.

[0228] Figures 21A-21B The binding kinetics between the isotype and HER2 were depicted. Probes loaded with the His-tagged receptor were immersed in different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) of Herceptin-ADC for 300 seconds to determine the association kinetics. Figure 21B The calculation of individual concentration dependence (local fitting) is described.

[0229] Figures 22A-22B The binding kinetics of sastuzumab to TROP2 were depicted. Probes loaded with His-tagged receptors were immersed in sastuzumab-ADCs at different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) for 300 seconds to determine the association kinetics. Figure 22BThe calculation of individual concentration dependence (local fitting) is described.

[0230] Figures 23A-23B The binding kinetics of sastuzumab-ADC to TROP2 were depicted. Probes loaded with His-tagged receptors were immersed in sastuzumab-ADC at different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) for 300 seconds to determine the association kinetics. Figure 23B The calculation of individual concentration dependence (local fitting) is described.

[0231] Figures 24A-24B The binding kinetics between the isotype and TROP2 were depicted. Probes loaded with the His-tagged receptor were immersed in mAbs of different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) for 300 seconds to determine the association kinetics. Figure 24B The calculation of individual concentration dependence (local fitting) is described.

[0232] Herceptin and its ADC derivatives both showed comparable binding affinity to the HER-2 receptor (approximately 0.5–2 nM), indicating that conjugation has little effect on the receptor binding affinity of mAbs.

[0233] Similarly, sastuzumab and its ADC have comparable binding affinity to the TROP-2 receptor (approximately 0.04–0.1 nM).

[0234] Human isotype IgG (negative control) does not bind to any receptor.

[0235] Example 8. In vitro cytotoxicity data of antibody-drug conjugates using anti-HER2 antibody (Herceptin) and antibody-drug conjugates using anti-TROP2 antibody (Saltuzumab).

[0236] Herceptin-ADC is an antibody-drug conjugate (ADC) that targets Her2 and delivers a small molecule (oxazine) with antitumor activity.

[0237] It has been reported that the N87 cell line expresses high levels of Her2 mRNA transcript (https: / / www.proteinatlas.org / ENSG00000141736-ERBB2 / cell+line) and protein (Cancer Cell International. 2014: https: / / cancerci.biomedcentral.com / articles / 10.1186 / 1475-2867-14-10).

[0238] Satuzumab-ADC is an antibody-drug conjugate (ADC) that targets Trop2 and delivers a small molecule (oxazine) payload with antitumor activity.

[0239] It has been reported that the N87 cell line expresses moderate levels of Trop2 mRNA transcript (https: / / www.proteinatlas.org / ENSG00000184292-TACSTD2 / cell+line).

[0240] Figures 25A-25B Live-cell imaging analysis (IncuCyte S3) of NCI-N87 cells at a range of seeding densities (Fig. 25A for low density; Fig. 25B for high density) in 96-well plates was performed to monitor cell growth for up to 120 hours.

[0241] In this implementation, the in vitro cytotoxicity study was designed as follows. The NCI-N87 cell line was purchased from ATCC and expanded before use in the in vitro assay. First, cells across a range of seeding densities were assessed in 96-well plates using live-cell imaging analysis (IncuCyte S3) to monitor cell growth for up to 120 hours. For the dose-response study, a seeding density of 30,000 cells per well was selected. NCI-N87 cells were seeded at 30,000 cells / well in 96-well plates and allowed to adhere overnight in normal growth medium (RPMI-10% FBS). N87 cells were treated with sastuzumab-ADC (NJBP-0165-126) or Herceptin-ADC (NJBP-0165-129) at dose levels ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7 (CellEvent™ Green Reagent, Invitrogen). Phase-contrast microscopy was used to monitor confluence percentages every six hours, and apoptosis was monitored under 488 nm fluorescence. IC50 curves were plotted after 24, 48, 72–74, and 96 hours of culture. Figure 26A depicts N87 cells treated with Herceptin-ADC (NJBP-0165-129) at dose levels ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7 (CellEvent™ Green Reagent, Invitrogen), with phase-contrast microscopy imaging every six hours to monitor confluence % and cell apoptosis monitored under 488 nm fluorescence. Figures 26B-26E depict IC50 curves plotted at 24 hours (26B), 48 hours (26C), 72 hours (26D), and 96 hours (26E) of culture.

[0242] Figure 27A The study described the treatment of N87 cells with sastuzumab-ADC (NJBP-0165-126) at dose levels ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7 (CellEvent™ Green Reagent, Invitrogen), with phase contrast microscopy imaging every six hours to monitor confluence % and cell apoptosis monitored under 488 nm fluorescence. Figures 27B-27E IC50 curves were plotted for 24 hours (27B), 48 hours (27C), 72 hours (27D), and 96 hours (27E).

[0243] The results showed that the response to ADCs was dose- and target-dependent. Specifically, in N87 cells, the cytotoxicity of both Herceptin-ADC and Satuzumab-ADC was target- (HER2 expression) and dose-dependent. At 96 hours, N87 cells were more sensitive to Herceptin-ADC (IC50 = 0.026 μg / mL) than to Satuzumab-ADC (IC50 = 0.440 μg / mL).

Claims

1. A composition comprising the formula Ab-(LD) n Antibody-drug conjugates, in which Ab can be an antibody, antibody fragment, antibody chain, affinity, aptamer, or nanobody; D represents the effective load of reactive oxygen-nitrophosphine; L stands for connector; and n can have values ​​from 2 to 20.

2. The composition according to claim 1, wherein, n can have values ​​ranging from 2 to 8.

3. The composition according to claim 1, wherein, The antibody is trastuzumab.

4. The composition according to claim 1, wherein, The antibody is sastuzumab.

5. The composition according to claim 1, wherein, The connector and the active oxygen phosphine payload are RTX5007.

6. The composition according to claim 1, wherein, The antibody-drug conjugate is trastuzumab-RTX5007.

7. The composition according to claim 1, wherein, The antibody-drug conjugate is saltuzumab-RTX5007.

8. The composition according to claim 1, wherein, The Ab binds to a tumor-associated antigen from at least one of the following groups: HER2, HER3, VEGF-A, VEGFR-2, CSF-1R, PD-L1, CEACAM5 or CEACAM6, ROR1, CD20, CD19, CD22, CD30, CD33, CD133, CD38, CD39, CD25, CD47, CD52, CD56, CD70, CD73, CD74, CD79b, CD155, CD166, FGF receptor, B7-H3, B7-H4, LIV1, PSMA, PSCA, MAGE-A4, EpCAM, IL1R, CCR8, CCR4, sealing protein, APPL2, BCMA, EGFR, DLL3 / 4, SSX-2, tissue factor, folate receptor, mesothelin receptor, NaPi2b, 5T4, connexin-4, connexin-2. (CD112), c-MeT, Trop-2, LHRH (GnRH) receptor, gonadotropin (LH / hCG, FSH) receptor, prolactin receptor, sealing protein; survival protein, STEAP1, transferrin receptor 1, NRG1, EphB2 and caverin-1.

9. The composition according to claim 1, wherein, The oxazine payload is cytotoxic to immunosuppressive T regulatory cells.

10. The composition according to claim 1, wherein, The oxazine payload exhibits cytotoxicity against cancer cells.

11. The composition according to claim 1, wherein, The effective load of the oxynitrophosphine has the following formula: At least one of R3, R4, R5 and R6 is CH2CH2Y; Where Y is a halogen; and The remaining R3, R4, R5, and R6 groups are hydrogen or lower alkyl groups.

12. The composition according to claim 11, wherein, The halogen is Cl or Br.

13. The composition according to claim 11, wherein, The hydrogen in R3 and R5 is replaced by deuterium to form CD2CH2Y, or by methyl (CH3) to form CH3CH2Y.

14. The composition according to claim 11, wherein, The structure of the oxynitrophosphine payload is selected from the group consisting of: Where R is an alkyl chain; Where X and Y are halogen leaving groups; Where X is a halogen leaving group; and Where D represents deuterium.

15. The composition according to claim 11, wherein, The effective load of the oxazine is selected from 4-Hydroxycyclophosphamide, aldehyde phosphoramide, phosphoramide nitrogen mustard, 3-hydroxypropional, isophosphamide nitrogen mustard, 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyisocyclophosphamide, 4-hydroperoxyisocyclophosphamide, evaphosphamide, maphosphamide, glucosylphosphamide, or triphosphamide nitrogen mustard.

16. The composition according to claim 11, wherein, The effective load of the oxazine is selected from the following analogues or derivatives: 4-hydroxycyclophosphamide, aldehyde phosphoramide, phosphoramide mustard, 3-hydroxypropional, isophosphamide mustard, 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyisocyclophosphamide, 4-hydroperoxyisocyclophosphamide, evaphosphamide, maphosphamide, glucosylphosphamide, or triphosphamide mustard.

17. The composition according to claim 16, wherein, The effective payload is phosphoramide nitrogen mustard.

18. The composition according to claim 16, wherein, The derivative of the 4-hydroperoxyisocyclophosphamide is 4-hydroxyisocyclophosphamide.

19. The composition according to claim 16, wherein, Analogs or derivatives of 4-hydroperoxyisocyclophosphamide (4-HO-isocyclophosphamide) are deuterated (d4-hydroxyisocyclophosphamide).

20. The composition according to claim 16, wherein, The 4-hydroperoxycyclophosphamide derivative is 4-hydroxycyclophosphamide.

21. The composition according to claim 11, wherein, The oxynitrophosphine payload has the following structure: Where X is Cl or Br.

22. The composition according to claim 20, wherein, X is Cl.

23. The composition according to claim 11, wherein, The oxynitrophosphine payload has the following structure: Where X and Y represent independent leaving groups.

24. The composition according to claim 11, wherein, The effective load metabolite of the oxazine is isophosphoramide nitrogen mustard or an analogue thereof.

25. The composition according to claim 11, wherein, The effective load of the oxyphosphonium is selected from dimethylisophosphoramide nitrogen mustard or its analogues, or 4-hydroxy derivatives (4-HO-isocyclophosphamide) or their analogues or derivatives.

26. The composition according to claim 11, wherein, The oxazine payload metabolite is selected from bromoisophosphoramide nitrogen mustard or its analogues or derivatives, including evaphosphoramide or dimethylisophosphoramide nitrogen mustard.

27. The composition according to claim 11, wherein, The effective load of the oxyphosphonate is geraniol isophosphoramide nitrogen mustard metabolite or its analogue or derivative.

28. The composition according to claim 11, wherein, The effective load metabolite of the oxazine is malphosphamide or its analogues or derivatives.

29. The composition according to claim 11, wherein, The effective load metabolite of the oxazine is glucosamine or its analogues or derivatives.

30. The composition according to claim 11, wherein, The effective load metabolite of the oxazine is triphosphatidylcholine mustard or its analogues or derivatives.

31. The composition according to claim 1, further comprising a therapeutic agent.

32. The composition according to claim 1, further comprising an anti-TAM (tumor-associated macrophage) drug.

33. The composition of claim 1, further comprising one or more antibodies that enhance anti-tumor immunity, said one or more antibodies being selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA4, anti-LAG3, anti-GITR, anti-TIM-3, anti-TIGIT, anti-CD96, anti-CD226, anti-CD155, anti-CD47, anti-CEACAM1, anti-CEACAM5, anti-CEACAM6, anti-galactoglobulin-1, anti-sealing protein, anti-Siglec-15 antibody, anti-VISTA, anti-CD137, anti-CCR4 antibody, anti-CCR8 antibody, anti-CD39 antibody, anti-CD25 antibody, anti-CD-73 antibody, and anti-CSFR1.

34. The composition according to claim 1, further comprising one or more cytokines selected from: IL-1β, IL-2, IL-6, IL-7, IL-12, IL-15, IL-21, IL-23, IL-27, TNFα, IFNα, IFNγ, GM-CSF, anti-IL2R, Toll-like receptor (TLR) activator, and interferon gene stimulating factor (STING).

35. The composition according to claim 34, wherein, The Toll-like receptor activators are poly(I:C) and CpG.

36. The composition of claim 1, further comprising one or more chemotherapeutic agents selected from the group consisting of: 5-fluorouracil, 2'-deoxy-5-fluorouridine, cytarabine, claribine, fludarabine, pentostatin, gemcitabine, and 6-thioguanine, melphalan, and any derivative thereof; and alkylating agents such as chlorambucil, bendamustine, melphalan, alkylating agents, or anthracyclines such as doxorubicin, epirubicin, daunorubicin; temozolomide, oxaliplatin, cisplatin, chlorambucil, dichloroethylmethylamine, mitoxantrone, pyxitinib, lenvastatin. Trabectedin, HDAC inhibitors, anti-angiogenic drugs, bisphosphonates, taxanes, vinorelbine, ibrutinib, eribulin, retinotimod, gademod or their analogues, anti-signaling 4D, CXCR2 blockers, axitinib, sorafenib, cabozantinib, sunitinib, regorafenib, thalidomide, lenalidomide, pomalidomide, avalidomide, vandetanib, siddinib or their analogues, anti-VEGF-A antibody (bevacizumab), anti-VEGF-R2 antibody (ramucirumab), TRL9 agonists, anti-CCR4 antibodies PPARγ agonists, miRNAs, angiotensin receptor blockers, CXCR4 blockers, CD4 / 6 inhibitors, proteasome inhibitors, JAK1 / 2 inhibitors, Bruton's kinase (BTK) inhibitors, kinase inhibitors, topoisomerase inhibitors, epigenetic inhibitors, DNMT, HMT, HDM inhibitors, PARP inhibitors, hormone antagonists, antiprolactin, VEGI, osteopontin, mammary filamentin, angiotensin II, itraconazole, carboxytriazole, suramin, platelet-reactive protein, tetrathiomolybdate. Linolamide, taquimod, carfilzomib, sunitinib, pazopanib, everolimus; anti-hormones: luteinizing hormone-releasing hormone (LHRH) antagonists, tamoxifen, cortisol analogs, steroid receptor modulators or antagonists, cancer metabolism inhibitors, radioisotopes, radiopharmaceuticals, vinca alkaloids, mTOR inhibitors, MEK inhibitors, BRAF inhibitors, MAPK and tyrosine kinase inhibitors, bortezomib, demethylating agents, bleomycin, alkylating agents, dacarbazine, temozolomide, CELLMOD, and targeted protein degraders.

37. The composition according to claim 36, wherein, The taxane is selected from the group consisting of: docetaxel, paclitaxel, cabazitaxel and 6-α-hydroxypaclitaxel.

38. The composition according to claim 36, wherein, The epigenetic inhibitors target HDAC, DNMT, LSD1, DOT1L, BET, or EZH.

39. The composition according to claim 36, wherein, The hormone antagonist is leuprorelin.

40. The composition according to claim 36, wherein, The PARP inhibitors are selected from the group consisting of: 1-aminobenzamide, iniparib, BMN-573, olaparib, niraparib, tapazoli, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290 and their derivatives.

41. The composition according to claim 36, wherein, The cortisol analogues are selected from the group consisting of: prednisone, dexamethasone; raloxifene, anarodazole, lerozolol, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162 and any derivative thereof.

42. The composition according to claim 36, wherein, The steroid receptor modulator or antagonist is selected from the group consisting of: anti-estrogens, anti-progestins, anti-androgens, anti-corticosteroids, and anti-thyroid hormones.

43. The composition according to claim 36, wherein, The cancer metabolic inhibitors are selected from the group consisting of: pyruvate kinase inhibitors and isocitrate dehydrogenase inhibitors.

44. The composition according to claim 36, wherein, The radioactive isotopes are radium dichloride Ra 223, lutetium Lu 177, actinium 225, yttrium 90, technetium 99, or iodine 131.

45. The composition according to claim 36, wherein, The vinca alkaloids are selected from the group consisting of: vincristine, vinblastine, vindesine and vinorelbine and any of their derivatives.

46. ​​The composition according to claim 36, wherein, The protein degrading agent is a protein hydrolysis targeted chimera (PROTAC) or a molecular gel.

47. The composition according to claim 1, further comprising a tumor-targeting antibody.

48. The composition of claim 1, further comprising cell therapy.

49. The composition according to claim 1, further comprising gene therapy.

50. The composition according to claim 1, further comprising a cancer vaccine.

51. The composition according to claim 1, further comprising an oncolytic virus.

52. A method of treating cancer in a subject with this need, comprising administering to said subject an injection of formula Ab-(LD) n A composition of antibody-drug conjugates, wherein Ab can be an antibody, antibody fragment, antibody chain, affinity, aptamer, or nanobody; D represents the effective load of reactive oxygen-nitrophosphine; L stands for connector; and n can have values ​​from 2 to 20.

53. The method for treating cancer according to claim 52, wherein, The antibody is trastuzumab.

54. The method for treating cancer according to claim 52, wherein, The antibody is sastuzumab.

55. The method for treating cancer according to claim 52, wherein, The connector and the active oxygen phosphine payload are RTX5007.

56. The method for treating cancer according to claim 52, wherein, The antibody-drug conjugate is trastuzumab-RTX5007.

57. The method for treating cancer according to claim 52, wherein, The antibody-drug conjugate is saltuzumab-RTX5007.

58. The method for treating cancer according to claim 52, wherein, The effective load of the oxyphosphonium is phosphoramide nitrogen mustard.

59. The method according to claim 52, wherein, The antibody-drug conjugate is present in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers.

60. The method of claim 52, further comprising administering a therapeutic agent.

61. The method of claim 52, further comprising administering radiotherapy and chemotherapy.

62. The method of claim 52, further comprising administering an anti-TAM (tumor-associated macrophage) drug.

63. The method of claim 52, further comprising administering one or more antibodies that enhance antitumor immunity, said one or more antibodies being selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA4, anti-LAG3, anti-GITR, anti-TIM-3, anti-TIGIT, anti-CD96, anti-CD226, anti-CD155, anti-CD47, anti-CEACAM1, anti-CEACAM5, anti-CEACAM6, anti-galactoglobulin-1, anti-Siglec-15 antibody, anti-VISTA, anti-CD137, anti-CCR4 antibody, anti-CCR8 antibody, anti-CD39 antibody, anti-CD25 antibody, anti-CD-73 antibody, and anti-CSFR1.

64. The method of claim 52, further comprising administering one or more cytokines from the group consisting of: IL-1β, IL-2, IL-6, IL-7, IL-12, IL-15, IL-21, IL-23, IL-27, TNFα, IFNα, IFNγ, GM-CSF, anti-IL2R, Toll-like receptor (TLR) activator, and interferon gene stimulating factor (STING).

65. The method according to claim 64, wherein, The Toll-like receptor activators are poly(I:C) and CpG.

66. The method of claim 52, further comprising one or more chemotherapeutic agents selected from the group consisting of: 5-fluorouracil, 2'-deoxy-5-fluorouridine, cytarabine, claribine, fludarabine, pentostatin, gemcitabine, and 6-thioguanine, melphalan, and any derivative thereof; and alkylating agents such as chlorambucil, bendamustine, melphalan, alkylating agents, or anthracyclines such as doxorubicin, epirubicin, daunorubicin; temozolomide, melphalan, oxaliplatin, cisplatin, chlorambucil, dichloroethylmethylamine, mitoxantrone, and pyxitinib. Lenvatinib, Trabectedin, HDAC inhibitors, anti-angiogenic drugs, bisphosphonates, taxanes, vinorelbine, ibrutinib, eribulin, retinotimod, gademod or their analogues, anti-VISTA antibody, anti-signaling 4D, CXCR2 blockers, axitinib, sorafenib, cabozantinib, sunitinib, regorafenib, thalidomide, lenalidomide, pomalidomide, avalidomide, vandetanib, sildenafil or their analogues, anti-VEGF-A antibody (bevacizumab), anti-VEGF-R2 antibody (ramucirumab). TRL9 agonists, PPARγ agonists, miRNAs, angiotensin receptor blockers, CXCR4 blockers, CD4 / 6 inhibitors, proteasome inhibitors, JAK1 / 2 inhibitors, Bruton's kinase (BTK) inhibitors, kinase inhibitors, topoisomerase inhibitors, epigenetic inhibitors, DNMT, HMT, HDM inhibitors, PARP inhibitors, hormone antagonists, antiprolactin, VEGI, osteopontin, mammary filamentin, angiotensin, itraconazole, carboxytriazole, suramin, platelet-reactive protein. Tetrathiomolybdate, Linolamine, Taquimod, Carfilzomib, Sunitinib, Pazopanib, Everolimus; Antihormones: Luteinizing Hormone-Releasing Hormone (LHRH) antagonists, Tamoxifen, Cortisol analogs, Steroid receptor modulators or antagonists, Cancer Metabolic Inhibitors, Radioisotopes, Radiopharmaceuticals, Vinca alkaloids, mTOR inhibitors, MEK inhibitors, BRAF inhibitors, MAPK and tyrosine kinase inhibitors, Bortezomib, Demethylating agents, Bleomycin, Alkylating agents, Dacarbazine, Temozolomide, and CELLMOD.

67. The method according to claim 66, wherein, The taxane is selected from the group consisting of: docetaxel, paclitaxel, cabazitaxel and 6-α-hydroxypaclitaxel.

68. The method according to claim 66, wherein, The epigenetic inhibitors target HDAC, DNMT, LSD1, DOT1L, BET, or EZH.

69. The method according to claim 66, wherein, The hormone antagonist is leuprorelin.

70. The method of claim 66, wherein, The PARP inhibitors are selected from 1-aminobenzamide, iniparib, BMN-573, olaparib, niraparib, tapazoparib, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290 and their derivatives.

71. The method according to claim 66, wherein, The cortisol analogues are selected from the group consisting of: prednisone, dexamethasone; raloxifene, anarodazole, lerozolol, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162 and any derivative thereof.

72. The method according to claim 66, wherein, The steroid receptor modulator or antagonist is selected from the group consisting of: anti-estrogens, anti-progestins, anti-androgens, anti-corticosteroids, and anti-thyroid hormones.

73. The method according to claim 66, wherein, The cancer metabolic inhibitors are selected from the group consisting of: pyruvate kinase inhibitors and isocitrate dehydrogenase inhibitors.

74. The method according to claim 66, wherein, The radioactive isotopes are radium dichloride Ra 223, lutetium Lu 177, actinium 225, yttrium 90, technetium 99, or iodine 131.

75. The method according to claim 66, wherein, The vinca alkaloids are selected from the group consisting of: vincristine, vinblastine, vindesine and vinorelbine and any of their derivatives.

76. The method of claim 52, further comprising administering a tumor-targeting antibody.

77. The method of claim 52, further comprising administering cell therapy.

78. The method of claim 52, further comprising administering gene therapy.

79. The method of claim 52, further comprising administering a cancer vaccine.

80. The method of claim 52, further comprising administering radiotherapy.

81. The method of claim 52, further comprising applying phototherapy.

82. The method of claim 52, further comprising administering an oncolytic virus.

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