Oxazaphosphorine 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

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480055913.5 (22) Application Date 2024.06.28 (30) Priority Data 63 / 524,400 2023.06.30 US (85) PCT International Application Entering National Phase Date 2026.02.28 (86) PCT International Application Application Data PCT / US2024 / 036135 2024.06.28 (87) PCT International Application Publication Data WO2025 / 006970 EN 2025.01.02 (71) Applicant LA Life Products LLC Address Texas, USA (72) Inventor Hector Allila (74) Patent Agency Chengdu Fandian Intellectual Property Agency Co., Ltd. 51258 Patent Attorney Lu Xingwang (51) Int.Cl. A61K 47 / 55 (2006.01) A61K 31 / 664 (2006.01) A61K 39 / 44 (2006.01) A61K 47 / 68 (2006.01) C07F 9 / 24 (2006.01) C07K 16 / 22 (2006.01) A61P 35 / 00 (2006.01) (54) Invention Title Oxazepam Antibody-Drug Conjugate and Method of Use (57) Abstract This document discloses an antibody-drug conjugate (ADC) comprising an antibody partially conjugated to an oxazepam drug via a linker, and a method of using the antibody-drug conjugate. Methods for treating undesirable or abnormal cell proliferation or hyperproliferative disorders, such as non-metastatic and metastatic tumor formation, cancer, stem cells, and malignant tumors expressing targets that bind to such antibodies or antibody fragments, antibody heavy and light chains, or nanobodies. Claims 7 pages, Description 24 pages, Drawings 41 pages. CN 121752295 A 2026.03.27 CN 1 21 75 22 95 A 1. A composition comprising an antibody-drug conjugate of the formula Ab-(L-D)n, wherein Ab is an antibody, antibody fragment, antibody chain, affinity, aptamer, or nanobody; D is a reactive oxygen species (ROS) phosphonium payload; L is a linker; and n has a value of 2 to 20. 2. The composition of claim 1, wherein n has a value of 2 to 8. 3. The composition of claim 1, wherein the antibody is trastuzumab. 4. The composition of claim 1, wherein the antibody is sastuzumab. 5. The composition according to claim 1, wherein the connector and the active oxygen zeolite payload are RTX5007.6. The composition of claim 1, wherein the antibody-drug conjugate is trastuzumab-RTX5007. 7. The composition of claim 1, wherein the antibody-drug conjugate is sastuzumab-RTX5007. 8. The composition according to claim 1, wherein the Ab binds to a tumor-associated antigen from at least one of the following: 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 caveolin-1. 9. The composition of claim 1, wherein the oxazine payload is cytotoxic to immunosuppressive T regulatory cells. 10. The composition of claim 1, wherein the oxazine payload is cytotoxic to cancer cells. 11. The composition of claim 1, wherein the oxazine payload has the following formula: where at least one of R3, R4, R5, and R6 is CH2CH2Y; where Y is a halogen; and where the remaining R3, R4, R5, and R6 groups are hydrogen or lower alkyl groups. 12. The composition of claim 11, wherein the halogen is Cl or Br. 13. The composition of claim 11, wherein the hydrogen in R3 and R5 is replaced with deuterium to form CD2CH2Y, or with methyl (CH3) to form CH3CH2Y. 14. The composition of claim 11, wherein the structure of the oxyzeline loading is selected from the group consisting of: (Claims 1 / 7 page 2 CN 121752295 A) where R is an alkyl chain; where X and Y are halogen leaving groups; where X is a halogen leaving group; and where D is deuterium. 15. The composition of claim 11, wherein the oxyzeline loading is selected from...16. The composition according to claim 11, wherein the oxyphosphazene effective load is selected from the analogues or derivatives of the following: 4-hydroxycyclophosphamide, aldehyde phosphoramide, phosphoramide mustard, 3-hydroxypropanal, isophosphazene mustard, 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyifocyclophosphamide, 4-hydroperoxyifocyclophosphamide, levamisole, glucosylphosphazene, or triphosphazene mustard. 17. The composition according to claim 16, wherein the effective load is phosphoramide mustard. 18. The composition of claim 16, wherein the derivative of the 4-hydroperoxyisocyclophosphamide is 4-hydroxyisocyclophosphamide. 19. The composition of claim 16, wherein an analog or derivative of 4-hydroperoxyisocyclophosphamide (4-HO-isocyclophosphamide) is deuterated (d4-hydroxyisocyclophosphamide). 20. The composition of claim 16, wherein the derivative of the 4-hydroperoxyisocyclophosphamide is 4-hydroxycyclophosphamide. 21. The composition of claim 11, wherein the oxazine payload has the following structure: wherein X is Cl or Br. 22. The composition of claim 20, wherein X is Cl. 23. The composition of claim 11, wherein the oxazine payload has the following structure: wherein X and Y represent independent leaving groups. 24. The composition of claim 11, wherein the oxazine payload metabolite is isophosphoramide mustard or an analog of it. 25. The composition of claim 11, wherein the oxazine load is selected from dimethylisophosphoramide mustard or its analogues, or a 4-hydroxy derivative (4-HO-isocyclophosphamide) or its analogues or derivatives. 26. The composition of claim 11, wherein the oxazine load metabolite is selected from bromoisophosphoramide mustard or its analogues or derivatives, including evaphosphoramide or dimethylisophosphoramide mustard. 27. The composition of claim 11, wherein the oxazine load is a geraniol isophosphoramide mustard metabolite or its analogues or derivatives. 28. The composition of claim 11, wherein the oxazine load metabolite is malphosphoramide or its analogues or derivatives. 29. The composition of claim 11, wherein the oxazine load metabolite is glucosylphosphonamide or phosphonamide. (Claims 3 / 7, page 4, CN 121752295 A)30. The composition of claim 11, wherein the oxazine payload metabolite is triphosphatamide mustard or its analogue or derivative. 31. The composition of claim 1, further comprising a therapeutic agent. 32. The composition of claim 1, further comprising an anti-TAM (tumor-associated macrophage) drug. 33. The composition according to 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 of 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 of claim 34, wherein the Toll-like receptor activator is 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, lenvatinib, trabectedin, HDAC inhibitors, anti-angiogenic agents, bisphosphonates, taxanes, vinorelbine, ibrutinib, eribulin, requimod, gademod, or analogues thereof, and anti-signaling 4D. CXCR2 blockers, axitinib, sorafenib, cabozantinib, sunitinib, regorafenib, thalidomide, lenalidomide, pomalidomide, avalidomide, vandetanib, siddinib or its analogues, anti-VEGF-A antibody (bevacizumab), anti-VEGF-R2 antibody (ramucirumab), TRL9 agonists, anti-CCR4 antibodies, PPARγ agonists, miRNA, angiotensin receptor blockersAnti-steroids, 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, mammary filamentin, angiotensin, itraconazole, carboxytriazole, suramin, platelet-reactive protein, tetrathiomolybdate, linolamine, 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, 37. The composition of claim 36, wherein the taxane is selected from the group consisting of: docetaxel, paclitaxel, cabazitaxel, and 6-α-hydroxypaclitaxel. 38. The composition of claim 36, wherein the epigenetic inhibitor targets HDAC, DNMT, LSD1, DOT1L, BET, or EZH. 39. The composition of claim 36, wherein the hormone antagonist is leuprorelin. 40. The composition of claim 36, wherein the PARP inhibitor is selected from the group consisting of: 1-aminobenzamide, iniparib, BMN-573, olaparib, niraparib, tapazolidin, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, and derivatives thereof. Claims 4 / 7, page 5, CN 121752295 A 41. The composition of claim 36, wherein the cortisol analogue is selected from the group consisting of: prednisone, dexamethasone; raloxifene, analoxazole, lenoxazole, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162, and any derivative thereof. 42. The composition of 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 of claim 36, wherein the cancer metabolism inhibitor is selected from the group consisting of: pyruvate kinase inhibitors and isocitrate dehydrogenase inhibitors. 44. The composition according to claim 36, wherein the radioactive isotope is radium dichloride Ra 223, lutetium Lu 177, actinium 225, yttrium 90, technetium 99 or iodine 131.45. The composition of claim 36, wherein the vinca alkaloid is selected from the group consisting of: vincristine, vinblastine, vindesine, and vinorelbine and any derivative thereof. 46. The composition of claim 36, wherein the protein degrading agent is a protein hydrolysis-targeted chimeric conjugate (PROTAC) or a molecular gel. 47. The composition of claim 1, further comprising a tumor-targeting antibody. 48. The composition of claim 1, further comprising cell therapy. 49. The composition of claim 1, further comprising gene therapy. 50. The composition of claim 1, further comprising a cancer vaccine. 51. The composition of claim 1, further comprising an oncolytic virus. 52. A method of treating cancer in a subject with this need, 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, affinity, aptamer, or nanobody; D is a reactive oxygen species (ROS) phosphonate payload; L is a linker; and n has a value of 2 to 20. 53. The method of treating cancer according to claim 52, wherein the antibody is trastuzumab. 54. The method of treating cancer according to claim 52, wherein the antibody is sastuzumab. 55. The method of treating cancer according to claim 52, wherein the linker and reactive oxazine payload are RTX5007. 56. The method of treating cancer according to claim 52, wherein the antibody-drug conjugate is trastuzumab-RTX5007. 57. The method of treating cancer according to claim 52, wherein the antibody-drug conjugate is sastuzumab-RTX5007. 58. The method of treating cancer according to claim 52, wherein the oxazine payload is phosphoramide mustard. 59. The method of 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 or chemotherapy. 62. The method of claim 52, further comprising administering an anti-TAM (tumor-associated macrophage) drug. Claims 5 / 7, page 6, CN 121752295 A 63. The method of claim 52, further comprising administering 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-galactagogue-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 of claim 64, wherein the Toll-like receptor activator is 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, pyxitinib, lenvatinib, trabectedin, HDAC inhibitors, and anti-angiogenic agents. Bisphosphonates, taxanes, vinorelbine, ibrutinib, eribulin, requimod, 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 γ-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, linolenic acid, taquimod, carfilzomib, sunitinib, pazopanib, everolimus; anti-hormonal agents: luteinizing hormone-releasing hormone (LHRH) antagonists, tamoxifen, cortisol analogs67. The method of claim 66, wherein the taxane is selected from the group consisting of docetaxel, paclitaxel, cabazitaxel, and 6-α-hydroxypaclitaxel. 68. The method of claim 66, wherein the epigenetic inhibitor targets HDAC, DNMT, LSD1, DOT1L, BET, or EZH. 69. The method of claim 66, wherein the hormone antagonist is leuprorelin. 70. The method of claim 66, wherein the PARP inhibitor is selected from 1-aminobenzamide, iniparib, BMN-573, olaparib, niraparib, tapolazoparib, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, and derivatives thereof. 71. The method of claim 66, wherein the cortisol analog is selected from the group consisting of: prednisone, dexamethasone; raloxifene, analoxazole, lenoxazole, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162, and any derivatives thereof. 72. The method of 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. Claims 6 / 7, page 7, CN 121752295 A 73. The method of claim 66, wherein the cancer metabolic inhibitor is selected from the group consisting of: pyruvate kinase inhibitors and isocitrate dehydrogenase inhibitors. 74. The method of claim 66, wherein the radioactive isotope is radium dichloride Ra-223, lutetium Lu-177, actinium-225, yttrium-90, technetium-99, or iodine-131. 75. The method of claim 66, wherein the vinca alkaloid is selected from the group consisting of: vincaine, vincristine, vindesine, and vinorelbine and any derivative thereof. 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. Claims 7 / 7 Page 8 CN 121752295 A Cross-Reference to Related Applications Regarding Oxazine Antibody-Drug Conjugates and Methods of Use

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 524,400, filed June 30, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention generally relates to cancer treatment. Specifically, the present invention relates to antibody-drug conjugates (ADCs) for cancer immunotherapy. Background Art

[0003] Human epidermal growth factor receptor 2 (HER2) is a recognized target in a variety of malignant tumors. Tumors that overexpress or amplify HER2 (e.g., breast cancer, gastric cancer, colorectal cancer, cervical cancer, endometrial cancer, bladder cancer, esophageal and gastric cancer, biliary tract cancer, salivary gland cancer) and tumors with HER2 mutations (e.g., NSCLC and more than 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. The most prominent HER2-targeting antibodies and adjuvants (ADCs) are trastuzumab, trastuzumab-deruxtecan (T-DXd), and trastuzumab emtansine (T-DM1), which are the standard of care for HER2-positive breast and other tumor types, as well as HER2-mutant tumors such as non-small cell lung cancer (NSCLC). Despite the efficacy of ADCs, many patients develop resistance. For example, resistance to T-DXd can occur, and the most common cause appears to be resistance to topoisomerase inhibitor payloads, as HER2 expression loss occurs only in a minority of resistant cases. Another approved ADC using a topoisomerase inhibitor is sacituzumab govitecan, used to treat cancers with overexpression of trophoblast surface antigen 2 (TROP2), such as breast and bladder cancer. TROP2 is also expressed in a variety of malignant tumors, such as 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. Part of the reason for resistance to goxatozumab is resistance to topoisomerase inhibitor payloads. Therefore, there is an urgent need to develop ADCs targeting HER2 and TROP2 using alternative payloads. This invention describes the use of oxazine.(oxazaphosphorine) as an alternative potent drug payload to generate new ADCs. Summary of the Invention

[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 where D is oxazaphosphorine, covalently bound to the antibody (Ab) or antibody fragment via a linker (L), the antibody or antibody fragment being capable of binding to a tumor antigen, and n having 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 antitumor cytotoxicity. The antibody can also be designed to bind to one or more antigens on target cells simultaneously. Another aspect of the subject matter described herein is an antibody fragment, nanobody, affinity compound, or heavy / light chain.

[0006] Another aspect of the subject matter described herein is an activated oxazine, selected from the group comprising active metabolites, analogues, or derivatives having direct antitumor cytotoxic (“tumor-killing activity”). Another aspect of the subject matter described herein is an active oxazine, selected from the group comprising active metabolites, analogues, or derivatives that stimulate the antitumor immune system by consuming or reducing the number and / or inhibiting the function of immunosuppressive cells (such as regulatory T cells (Tregs)) in the tumor microenvironment (TME).

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

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

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

[0010] Another aspect of the subject matter described herein is an article comprising a pharmaceutical composition, a container, and a package insert or label, said pharmaceutical composition comprising an antibody or fragment conjugated to oxazine, said package insert or label indicating that the pharmaceutical composition may 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, affinity, aptamer, or nanobody; D is an active oxazine payload; L is a linker; and n has a value of 2 to 20.

[0012] In another aspect, n has a value of 2 to 8.On the other hand, Ab binds to tumor-associated antigens 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, TROP2, fibroblast activation protein (FAP), LHRH (GnRH) receptor, gonadotropin (LH / hCG, FSH) receptor, prolactin receptor, sealing protein; survival protein, STEAP1, transferrin receptor 1, NRG1, EphB2 and caveolin-1.

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

[0014] In another aspect, the oxazine payload is cytotoxic to cancer cells.

[0015] In another aspect, the oxazine payload is of the following 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 methyl (CH3) to form CH3CH2Y.

[0019] In another aspect, the structure of the oxyzeline payload is selected from the group consisting of: Specification 2 / 24 pages 10 CN 121752295 A

[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] On the other hand, the effective load of oxazine is selected from 4-hydroxycyclophosphamide, aldehyde phosphoramide, phosphoramide mustard, 3-hydroxypropionaldehyde, isophosphamide mustard, 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyisocyclophosphamide, 4-hydroperoxyisocyclophosphamide, evofosfamide, horsephosphamide, and glucosamine.Amide or triphosphatamide nitrogen mustard.

[0025] In another aspect, the oxyphosphatamide nitrogen mustard is a phosphoramide nitrogen mustard. Specification 3 / 24 pages 11 CN 121752295 A

[0026] In another aspect, the oxyphosphatamide nitrogen mustard is selected from the analogues or derivatives of: 4-hydroxycyclophosphamide, aldehyde phosphoramide, phosphoramide nitrogen mustard, 3-hydroxypropional, isophosphatamide nitrogen mustard, 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyisophosphamide, 4-hydroperoxyisophosphamide, evaphosphamide, maphosphamide, glucosylphosphamide or triphosphatamide nitrogen mustard.

[0027] In another aspect, a derivative of 4-hydroperoxyisophosphamide is 4-hydroxyisophosphamide.

[0028] In another aspect, an analogue or derivative of 4-hydroperoxyisophosphamide (4-H0-isophosphamide) is deuterated (d4-hydroxyisophosphamide).

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

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

[0031] where X is Cl or Br.

[0032] In another aspect, X is Cl.

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

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

[0035] In another aspect, the metabolite of the oxazine payload is isophosphoramide mustard or its analogue.

[0036] In another aspect, the oxazine payload is selected from dimethyl isophosphoramide mustard or its analogue, or a 4-hydroxy derivative (4-H0-isocyclophosphamide) or its analogue or derivative.

[0037] In another aspect, the metabolite of the oxazine payload is selected from bromoisophosphoramide mustard or its analogue or derivative, including evaphosphoramide or dimethyl isophosphoramide mustard.

[0038] In another aspect, the oxazine payload is a geranyloxy-isophosphoramide nitrogen mustard metabolite or an analogue or derivative thereof.

[0039] In another aspect, the oxazine payload metabolite is horsephosphoramide or an analogue or derivative thereof.

[0040] In another aspect, the oxazine payload metabolite is glucosamine or an analogue or derivative thereof.

[0041] In another aspect, the oxazine payload metabolite is triphosphoramide nitrogen mustard or an analogue 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) drug.

[0044] In another aspect, the composition further comprises one or more antibodies that enhance anti-tumor immunity, as described in the specification 4 / 24 pages 12 CN 121752295 A.One or more antibodies are selected from the following group: 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. In another aspect, the composition further comprises one or more cytokines selected 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).

[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 agents selected from the group consisting of: 5-fluorouracil, 2'-deoxy-5-fluorouridine, cytarabine, cladribine, fludarabine, pentostatine, gemcitabine, 6-thioguanine, Melphalan and any of its derivatives; as well as alkylating agents such as chlorambucil, bendamustine, melphalan, alkylating agents or anthracyclines such as doxorubicin, epirubicin, daunorubicin; temozolomide, oxaliplatin, cisplatin, chlorambucil, mechlorethamine, mitoxantrone, pexidartinib, lenvatinib, trabectedin, HDAC inhibitors, anti-angiogenic drugs, bisphosphonates, taxanes, vinorelbine, ibrutinib, eribulin, requimod, gademod or their analogues, and anti-signaling 4D. CXCR2 blockers, axitinib, sorafenib, carbozantinib, sunitinib, multi-kinase inhibitors such as regorafenib, bifunctional protacoders, and molecular gel degraders (such as thalidomide, lenalidomide, pomalidomide, and avalidomide)(avadomide), vandetanib, cediranib or their analogues, anti-VEGF-A antibody (bevacizumab), anti-VEGF-R2 antibody (ramucirumab), TRL9 agonist, anti-CCR4 antibody, PPARγ agonist, miRNA, angiotensin receptor blocker, CXCR4 blocker, CD4 / 6 inhibitor, proteasome inhibitor, JAK1 / 2 inhibitor, Bruton's kinase (BTK) inhibitor, kinase inhibitor, topoisomerase inhibitor, Epigenetic inhibitors, DNMT, HMT, HDM inhibitors, PARP inhibitors, hormone antagonists, antiprolactin, VEGI, osteopontin, mastin, canstatin, itraconazole, carboxytriazole, suramin, platelet-reactive protein, tetrathiomolybdate, linomide, tasquinimod, carfilzomib, sunitinib, pazopanib Obanib), everolimus; anti-hormonal agents: 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 degrading agents.

[0047] In another aspect, taxanes are selected from the group consisting of: docetaxel, paclitaxel, cabazitaxel and 6-α-hydroxypaclitaxel.

[0048] In another aspect, epigenetic inhibitors target HDAC, DNMT, LSD1, DOT1L, BET or EZH.

[0049] In another aspect, hormone antagonists are leuprorelin.

[0050] On the other hand, PARP inhibitors are selected from the group consisting of: 1-aminobenzamide, iniparib, BMN-573, olaparib, niraparib, talazoparib, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290 and their derivatives. Specification 5 / 24 pages 13 CN 121752295 A

[0051] On the other hand, cortisol analogues are selected from the group consisting of: prednisone, dexamethasone; raloxifene, anastrozole, letrozole, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162 and any derivative thereof.

[0052] On the other hand, steroid receptor modulators or antagonists are selected from the group consisting of: anti-estrogens, anti-progestins, anti-androgens, anti-corticosteroids and anti-thyroid hormones.

[0053] On the other hand, cancer metabolism inhibitors are selected from the group consisting of: pyruvate kinase inhibitors and isocitrate dehydrogenase inhibitors.

[0054] On the other hand, the radioactive isotope is radium dichloride Ra 223, lutetium Lu 177, actinium-225, yttrium-90, technetium-99 or iodine-131.

[0055] In another aspect, the vinca alkaloids are selected from the group consisting of: vincaine, vincristine, vindesine, and vinorelbine and any derivative thereof.

[0056] In another aspect, the protein degrading agent is a protein hydrolysis-targeting chimeric (PROTAC) or a molecular gel.

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

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

[0059] In another aspect, the composition further comprises 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 reactive oxygen species (ROS) phosphonate payload is RTX5007.

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

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

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

[0068] In one aspect, the subject matter described herein relates to a method of treating cancer in a subject with this need, 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, affinity, aptamer, or nanobody; D is an active oxazine 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 includes administering a therapeutic agent.

[0071] In another aspect, the method further includes administering radiotherapy or chemotherapy.

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

[0073] In another aspect, the method further includes administering 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-galactogenin-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 includes administering one or more cytokines from the group consisting of: IL-1 (instructions 6 / 24 page 14 CN 121752295 A), 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).

[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 agents selected from the group consisting of: 5-fluorouracil, 2'-deoxy-5-fluorouridine, cytarabine, claribine, fludarabine, pentostatin, gemcitabine, 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, pyxitinib, lenvatinib, trabectedin, HDAC inhibitors, anti-angiogenic agents, bisphosphonates, taxanes, vinorelbine, ibrutinib, eribulin, requimod, gademod or analogues thereof, and anti-VISTA antibodies. Anti-Signal 4D, CXCR2 blockers, axitinib, sorafenib, cabozantinib, sunitinib, regorafenib, thalidomide, lenalidomide, pomalidomide, avalidomide, vandetanib, siddinib or its analogues, anti-VEGF-A antibody (bevacizumab), anti-VEGF-R2 antibody (ramucirumab), TRL9 agonist, PPARγ agonist, miRNA, angiotensin receptor blockerAnti-hormonal agents: 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; Anti-hormonal agents: 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, and CELLMOD.

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

[0077] In another aspect, epigenetic inhibitors target HDAC, DNMT, LSD1, DOT1L, BET, or EZH.

[0078] In another aspect, hormone antagonists are leuprorelin.

[0079] In another aspect, PARP inhibitors are selected from 1-aminobenzamide, iniparib, BMN-573, olaparib, niraparib, tapazoparib, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, and their derivatives.

[0080] In another aspect, the cortisol analogue is selected from the group consisting of: prednisone, dexamethasone; raloxifene, anaroxazole, lerozolol, exemestane, spironolactone, cyproterone acetate, bicalutamide, RU53063, thiohydantoin, RD162 and any derivative thereof.

[0081] In another aspect, 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.

[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 radioactive isotope is radium dichloride Ra 223, lutetium Lu 177, actinium-225, yttrium-90, technetium-99 or iodine-131.

[0084] In another aspect, the vinca alkaloids are selected from the group consisting of: vincristine, vinblastine, vindesine, and vinorelbine and any derivative thereof.

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

[0086] In another aspect, the method further includes administering cell therapy. Specification 7 / 24 pages 15 CN121752295 A

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

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

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

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

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

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

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

[0094] In another aspect, the linker and reactive oxygen species (ROS) payload are RTX5007.

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

[0096] The antibody-drug conjugate is sastuzumab-RTX5007.

[0097] The present invention will be better understood by reading the following detailed description of non-limiting embodiments and by examining the accompanying drawings, in which: Figure 1 depicts the metabolism of oxazine and ifosfamide (from Skarbek C. et al. 2015. J. Med. Chem. 58: 705-717); Figure 2 depicts the conjugation (SEC) of trastuzumab with the oxazine payload THLP-1: 101921-10P; Figure 3 depicts the drug-antibody ratio (DAR) and the concentration of 101921-10P as determined by HIC and UV. DAR calculations are from: UV: DAR = (εp252nm-εp280nm R) / (εd280nmR-εd252nm) (see reference: Clinical Cancer Research: 2004-10-7063~7070); Figure 4 depicts an example of the synthesis of the adapter payload; Figure 5 depicts Tregs in the tumor microenvironment and their key role in suppressing antitumor immunity (Dees, et al., Eur. J. Immunol. 2021. 51:280-291); Figure 6 depicts the effect of Treg depletion on other immunosuppressive cells in the TME (adapted from Dees, et al., Eur. J. Immunol. 2021. 51:280-291); Figure 7 depicts Treg depletion and cancer cell cytotoxicity in the TME; and Figure 8 depicts an example of the structure and biotransformation pathway of oxazine, from Anderson et al., Cancer Control: Journal of the Moffitt Cancer Center, January 2008.

[0098] Figure 9 depicts cyclophosphamide metabolism. Gor et al.Breast Cancer Research 2010, 12:R26.

[0099] Figure 10 depicts the synthesis of RTX5007; Figure 11 depicts the synthesis of RTX5014; Figure 12 depicts the synthesis of RTX5015; Figure 13 depicts the determination of Herceptin-RTX-5007 by SEC monomer; Figures 14A-14B depict the determination of the drug-antibody ratio (DAR) of Herceptin-RTX-5007 by LC-MS; Figure 15 depicts the determination of the concentration of Herceptin-RTX-5007 by measuring absorbance at 280 nm on nanodrop 2000C E1% with an extinction coefficient of 14.8; Figure 16 depicts the determination of saltuzumab-RTX-5007 by SEC monomer; Figures 17A-17B depict the determination of the DAR of saltuzumab-RTX-5007 by LC-MS; Figure 18 depicts the determination of the concentration of saltuzumab-RTX-5007 by nanodrop 2000C E1%; The absorbance at 280 nm above E1% was measured with an extinction coefficient of 15.6. (See page 8 / 24 of the instruction manual, CN 121752295 A for details). The concentration of sastuzumab-RTX-5007 was determined. Figures 19A-19B depict the binding kinetics of Herceptin with HER2; Figure 19B depicts the concentration-dependent calculation (local fitting). Figures 20A-20B depict the binding kinetics of Herceptin-ADC with HER2; Figure 20B depicts the concentration-dependent calculation (local fitting). Figures 21A-21B depict the binding kinetics of the homotype with HER2; Figure 21B depicts the concentration-dependent calculation (local fitting). Figures 22A-22B depict the binding kinetics of sastuzumab with TROP2; Figure 22B depicts the concentration-dependent calculation (local fitting). Figures 23A-23B depict the binding kinetics of sastuzumab-ADC with TROP2; Figure 23B depicts the concentration-dependent calculation (local fitting); Figures 24A-24B depict the binding kinetics of the isoform with TROP2; Figure 24B depicts the concentration-dependent calculation (local fitting); Figures 25A-25B depict the live-cell imaging analysis (IncuCyte S3) assessment of NCI-N87 cells at a range of seeding densities (low density in Figure 25A; high density in Figure 25B) in 96-well plates to monitor cell growth for up to 120 hours; Figure 26A depicts the use of Herceptin-ADC at a range of dose levels from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7. N87 cells were treated with (NJBP-0165-129), and phase-contrast microscopy was performed every six hours to monitor confluence percentage, with a peak at 488.Cell apoptosis was monitored under 488 nm fluorescence. Figures 26B-26E depict IC50 curves plotted after 24 hours (26B), 48 hours (26C), 72 hours (26D), and 96 hours (26E) of culture. Figure 27A depicts N87 cells treated 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, with phase-contrast microscopy imaging every six hours to monitor confluence percentage and cell apoptosis monitored under 488 nm fluorescence. Figures 27B-27E depict IC50 curves plotted after 24 hours (27B), 48 hours (27C), 72 hours (27D), and 96 hours (27E) of culture. Detailed Description

[0100] All publications and patents referenced in this disclosure are incorporated herein by reference in their entirety. Where any material incorporated by reference contradicts or is inconsistent with this specification, this specification shall supersede any such material. No reference cited herein is intended to imply that such references are prior art to this disclosure. When expressing a numerical range, it includes embodiments using any particular value within that range. Further, references to values ​​stated within a range include every value within that range. All ranges encompass their endpoints and combinations. When a value is expressed as an approximation using the antecedent “about,” it is meant that the particular value forms another embodiment.

[0101] Various terms related to the described aspects are used throughout the specification and claims. Unless otherwise stated, these terms will be given their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein. The techniques and procedures described or cited herein are generally well known to those skilled in the art and are typically applied using conventional methods, such as, for example, the widely used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, unless otherwise stated, procedures involving the use of commercially available kits and reagents are generally performed according to the manufacturer's prescribed protocols and conditions.

[0102] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include the plural forms. Unless otherwise clearly stated, the terms “comprising,” “such as,” etc., are intended to convey an unrestricted inclusion.

[0103] Unless otherwise stated, the terms “at least,” “less than,” and “about,” or similar terms preceding a series of elements or ranges, are understood to refer to each element in the series or range. Those skilled in the art will recognize or be able to determine many equivalents of a particular embodiment of the invention using experiments that do not exceed the norm. Such equivalents are intended to be covered in the following claims.

[0104] As used herein, the term “subject” means any animal, such as any mammal, including but not limited to humans, non-human primates, rodents, common pet mammals (e.g., dogs and cats), livestock (e.g., cattle, sheep, goats, pigs, horses, and camels), etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. The term “reactive oxazine payload” means that oxazine is not a prodrug and can be activated without metabolism.

[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 with the antibody.

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

[0108] Advances in novel cancer therapies, such as targeted chemotherapy, immunotherapies (such as immune checkpoint inhibitors (ICIs)), cell therapies, and antibody-drug conjugates (ADCs), have revolutionized cancer treatment and improved patient survival. However, given the still high mortality rates, the fact that most patients do not respond to existing treatments or develop resistance, and the inability to tolerate treatment due to toxicity, there is a need to develop newer therapies.

[0109] Antibody-drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of antibodies and cytotoxic drugs to deliver the toxic agent to tumor cells expressing antigens. They are typically designed to bind to target cancer cells, and the release of the toxic agent may require internalization. Drug release occurs inside cancer cells after cleavage by enzymes or low-pH linkers. Alternatively, for non-internalized ADCs, the toxic agent is released via cleavage by low-oxygen or low-pH linkers within the tumor microenvironment (TME).

[0110] To date, clinically used ADCs consist of targeting monoclonal antibodies linked to highly toxic agents such as chalcogenide, auristatin, maytansinoids, or pyrrolobenzodiazepines. These agents produce cytotoxicity at extremely low (nanomolar or picomolar) concentrations. Upon delivery to the tumor, these highly toxic agents...The payload can re-enter circulation and distribute to other organs, where it can cause severe toxicities such as cardiotoxicity, neutropenia, anemia, fatigue, diarrhea, neurotoxicity, blindness, nephrotoxicity, or even death. Therefore, these ADCs are labeled with a “black box” warning. A black box warning is the most severe warning the FDA can issue for prescription drugs because such drugs can be fatal, life-threatening, or cause disabling adverse effects in patients. This means that such drugs must be restricted 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 challenge in cancer immunotherapy is that immunosuppressive regulatory T cells (Tregs) within the TME are a major obstacle to developing effective immunotherapies for cancer patients. Current methods for depleting Tregs lack distribution specificity because they also deplete circulating Tregs, disrupting systemic immune homeostasis and leading to autoimmunity, systemic inflammation, and off-target toxicity. This disclosure describes a method for selectively depleting Tregs within the tumor microenvironment (TME) using an ADC, which delivers oxazine directly into the TME without disrupting systemic Treg immune homeostasis. It is known that oxazine metabolites induce antitumor 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 ADC targets and binds to cancer cells to release a therapeutic oxazine payload, which depletes Tregs within the TME. The dual killing effect of cancer cells and Tregs triggers immune memory, thereby prolonging anti-tumor immunity. By increasing the local delivery of the therapeutic payload within the TME, the therapeutic index is improved to minimize systemic toxicity and reactivate exhausted T cells to overcome immunotherapy resistance.

[0112] This invention describes an ADC with a safety profile superior to approved ADCs because the drug payload used is not supertoxic and drug-resistant cancer cells respond to the aforementioned oxazine payload. This invention relates to ADCs comprising tumor-targeting antibodies or antibody fragments conjugated to an oxazine payload via a linker. ADCs comprising antibodies conjugated to active oxazine have not previously been developed.

[0113] Oxazine includes, but is not limited to, cyclophosphamide, ifosfamide, and trefophosphamide. Oxazine derivatives include, but are not limited to, malphosphamide, glucosfamide, β-D-glucosylisophosphamide nitrogen mustard, aldehydephosphamide perhydrothiazide, and aldehydephosphamide thiazolidinyl. Cyclophosphamide and ifosfamide are prodrugs that require hepatic cytochrome P450 activation. Zhang, et al. (2005) Drug Metabolism Reviews, 37:4, 611-703. 4-Hydroxycyclophosphamide is the major metabolite of cyclophosphamide. Figure 8 illustrates the structure and biotransformation pathway of oxazine, from Anderson et al., Cancer Control: Journal of the Moffitt Cancer Center, January 2008.

[0114] Oxazines are alkylating agents that have been used for decades in routine clinical treatment of cancer. They are prodrugs that require bioactivation of cytochrome p450 in the liver to generate 4-hydroxy derivatives, which are then transported to the tumor via the bloodstream. They possess tumor-killing and immunostimulatory properties. They kill cancer cells directly by inducing immunogenic cell death (ICD). Their antitumor immunostimulatory properties occur because they selectively kill immunosuppressive cells, such as regulatory T cells (Tregs), thereby reactivating and proliferating tumor-specific cytotoxic T cells, NK cells, and macrophages, which help kill cancer cells. The antibody portion of the ADC can also directly kill cancer cells after binding to the tumor. Local delivery and release of oxazine payloads within the tumor reduces systemic exposure, which is crucial for minimizing or reducing systemic toxicity and improving patient safety and tolerability. ADCs can also be combined with established standard treatments, such as immune checkpoint inhibitors, chemotherapy, radiotherapy, phototherapy, gene therapy, or cell therapy, to maximize patient benefit.

[0115] Unlike previous ADCs that required highly potent and toxic drug payloads, low doses (concentrations) of oxazine are sufficient to exert their immunomodulatory effects. For example, it is well known that regular (rhythmic) 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, due to the fact that oral or systemic administration can...This leads to drug distribution and exposure to normal tissues in the body, thus systemic toxicity can still occur. Based on decades of clinical experience, any toxicity of oxazine is predictable and manageable compared 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, and a large proportion experience systemic immune-related toxicities such as autoimmune diseases, cardiopulmonary complications, and most eventually develop resistance. Oxazine chemotherapeutic agents, such as cyclophosphamide, can serve as effective partners to immune checkpoint inhibitors because they can synergistically enhance antitumor immunity (George M et al., Cancer Manual 11 / 24 pages 19 CN 121752295 A Res (2022) 82 (12_Supplement): 4167. https: / / doi.org / 10.1158 / 1538-7445.AM2022-4167). In a mouse melanoma model, the combined use of cyclophosphamide with anti-CTLA-4 or anti-PD-1 antibodies demonstrated potent tumor control. Clinically, the combination of the ifosfamide derivative evaphosphamide with the anti-CTLA-4 ipilimumab showed good clinical activity in 15 of 22 patients, but drug-related toxicities were relatively 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 suggest that while oxazine achieves synergistic effects when combined with immune checkpoint inhibitors, dose-limiting toxicities from systemic exposure can be mitigated by confining drug action to the tumor microenvironment. The main mechanism leading to cancer resistance to immune checkpoint inhibitor (ICI) therapy is the infiltration of immunosuppressive cells, such as regulatory T cells (Tregs), into the tumor microenvironment (TME). Treg cells are a subset of CD4-positive T cells that play a central role in suppressing antitumor immunity by helping tumor cells evade immune surveillance (Lee, G.R., Phenotypic and functional properties of tumor-infiltrating regulatory T cells. Mediators Inflamm. 2017. 2017: 5458178.). They are the main immunosuppressive cells in 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, the high frequency of invasive Tregs in the TME is associated with poor prognosis and low survival in many types of solid tumors, such as breast cancer, prostate cancer, lung cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, hepatocellular carcinoma, skin cancer, brain cancer, and 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 crucial role of Tregs in suppressing anti-tumor immune responses, blocking their function and / or depleting them has become a viable strategy for enhancing or stimulating anti-tumor immunity (Dees et al., Eur. J. Immunol. 2021. 51: 280–291). Several other approaches are currently being developed, including Treg cell depletion, inhibition of their activity, inhibition of their recruitment, and prevention of their differentiation within the tumor microenvironment (TME). The main challenge currently is how to specifically target Treg cells in tumor sites without affecting circulating Tregs. Avoiding the depletion of circulating Tregs is crucial because they play a key role in maintaining immune homeostasis and preventing the immune system from damaging its own tissues. Depletion or inhibition of circulating Tregs 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 the function of Tregs in the bloodstream can also lead to autoimmune and other immune-related toxicities. To date, there is no specific treatment that targets Tregs within the TME without affecting Tregs in the bloodstream.

[0117] Commonly used examples of oxazines are cyclophosphamide, ifosfamide, and trefophosphamide. Trefophosphamide is mainly metabolized to ifosfamide, with a small amount metabolized to cyclophosphamide. Cyclophosphamide and ifosfamide are prodrugs that must be metabolized via the hepatic cytochrome p450 enzyme pathway to produce cytotoxic active metabolites (Figure 1). They undergo activation via 4-hydroxylation, followed by spontaneous degradation into the final active and inactive metabolites. Activation is primarily mediated by CYP2B6, leading to the formation of 4-hydroxycyclophosphamide, which is in equilibrium with the acyclic tautomers aldehydephosphamide and aldehyde isocyclophosphamide (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 bloodstream and distribute throughout the body before reaching target cancer cells. Oxazine mustard and isophosphamide mustard are the final active antitumor metabolites of cyclophosphamide and isocyclophosphamide, respectively. Other byproducts of oxazine metabolism are acrolein and chloroacetaldehyde, which have known toxic effects (bladder bleeding, neurotoxicity, and urinary tract toxicity are the toxic effects of their precursors, and other dose-limiting toxicities include myelosuppression, cardiotoxicity, diarrhea, and fatigue). Oxazine mustard and isophosphamide mustard do not cause urinary tract toxicity or neurotoxicity because they do not contain acrolein or chloroacetaldehyde. Oxazine derivatives or analogs that do not require hepatic CYP enzyme bioactivation have also been synthesized. These include phosphatidylcholine, glucosfamide, palifosfamide, evaphosphatidylcholine, geranyloxy-isofosfamide, and their analogues or derivatives. Low doses of oxazine exhibit significant immunomodulatory activity, particularly in inhibiting or depleting regulatory T cells (Tregs), thereby counteracting immunosuppression in the tumor microenvironment without affecting cytotoxic T cells or natural killer (NK) cells.

[0118] The method of directly delivering oxazine to the tumor microenvironment using ADCs can mitigate or significantly reduce systemic toxicity. Local delivery of the antibody portion of an ADC also contributes to the antitumor effect of the ADC. By engineering the Fc region, the optimal effector function of the antibody portion of the ADC can be improved to increase its cytotoxic effect on cancer cells. By engineering and modifying the Fc region, its affinity / affinity, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) can be improved. Glycosylation engineering, such as defucosylation and amino acid mutation, can be used. Pharmacokinetic properties can also be improved by modifying the antibody to bind to the neonatal Fc receptor FcRn.

[0119] The theoretical basis for using oxazine as the payload in an ADC is based on existing literature reporting that oxazine has a synergistic effect with antibodies, immune checkpoint inhibitors, or vaccines after systemic administration. However, the toxicity of combined administration limits its use. For example, cyclophosphamide has been shown to have a survival synergistic effect 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 in combination chemotherapy (as part of a combination with other chemotherapeutic agents for the treatment of breast cancer, leukemia, multiple myeloma, ovarian cancer, lymphoma, etc.). While combination therapy offers synergistic benefits, it also presents dose-limiting toxicities and inconveniences. Combination therapy is time-consuming and inconvenient because each drug must be infused separately for an extended duration (2-4 hours per infusion), with rest periods required between infusions. This approach also reduces patient adherence. The advantage of using ADCs is that they combine two drugs in a single 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 disclosed herein includes antibodies, adapters, and payloads. In embodiments, ADCs increase the tumor microenvironment...The accumulation of oxazine payload and reduction of systemic exposure and toxicity. In embodiments, the payload selectively depletes immunosuppressive cells, such as Tregs, within the tumor microenvironment; activates NK and effector T cells, reverses T cell exhaustion, and alters the function of immune cells within the tumor microenvironment to more effectively target cancer cells. The oxazine payload also induces immunogenic cell death (ICD) in cancer cells and generates tumor-specific antigens, thereby triggering durable tumor-specific immune responses.

[0121] The advantages of this disclosure include: applicability to solid tumors and hematologic malignancies; use of clinically validated combination therapies effective in cancer patients; novel immunomodulatory payloads and connectors; improved therapeutic index to eliminate or reduce systemic toxicity; convenient administration (one drug rather than two different administration routes); and reduced development timeline from discovery to clinical trials.

[0122] This method can be used as a monotherapy or in combination with standard treatments such as chemotherapy, radiotherapy, immunotherapy (including antibodies against immune checkpoint inhibitors), cell therapy, gene therapy, oncolytic virus therapy, or vaccines. In this context, “immunotherapy” refers to the administration of antibodies, antigen-binding fragments, antibody-drug conjugates (ADCs), etc., which bind to one or more proteins in the subject’s body, thereby affecting protein function and thus improving the subject’s cancer. Radiotherapy may involve focusing a beam of radiation onto the cancer site. Common radiotherapy regimens involve treatment once a week for 6–12 weeks.

[0123] Clinicians may 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 may be medically significant, or one or more genetic markers of the subject. 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 carcinoma, kidney cancer, brain cancer, uterine cancer, ovarian cancer, thyroid cancer, melanoma, liver cancer, pancreatic cancer, and HNSCC.

[0124] In embodiments, immunostimulatory ADCs include, but are not limited to, monospecific, bispecific, trispecific, or tetraspecific monoclonal antibodies.

[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 requiring cancer treatment.

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

[0128] In an embodiment, RTX5007, RTX5014, and RTX5015 are linker payloads.

[0129] In an embodiment, the synthesis of RTX5007 is carried out using the chemical methods illustrated in FIG. 10 and Example 2.

[0130] In an embodiment, the synthesis of RTX5014 is carried out using the chemical methods illustrated in FIG. 11 and Example 3.

[0131] In an embodiment, the synthesis of RTX5015 is carried out using the chemical methods illustrated in FIG. 12 and Example 4.

[0132] In an embodiment, the antibody-drug conjugate is Herceptin-RTX-5007. In an embodiment, Herceptin-RTX-5007 is prepared as in Example 5.

[0133] RTX-5007 conjugation amplified the reaction up to 7.4 mg, with Herceptin at 4.9 mg / mL.

[0134] Herceptin was reduced for 1.5 h at 37°C using 6 equivalents of 10 mM TCEP (29.4 µL). After reduction, 1 mM EDTA (from a 250 mM stock solution, 6.1 µL) was added to the protein. Before adding the payload, the buffer composition was adjusted to 10% DMA with an additional 150 µL of DMA. After adding the organic solvent, 15 equivalents of RTX-5007 (73.5 µL of 10 mM RTX-5007 in DMA) were added to the mAb. The mAb solution was mixed at room temperature for 1.5 h, and then the DAR was checked. After confirming the target DAR, 30 equivalents of 10 mM N-acetylcysteine ​​(147 µL) were added to the reaction to quench RTX-5007. Following conjugation, the mixture was dialyzed with 1×PBS (pH 7.4) to remove excess payload. After three buffer exchanges over 12 hours, the final buffer was replaced with 1×PBS (pH 7.4).

[0135] After payload removal, 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 1-3 depicts the determination of Herceptin-RTX-5007 monomer by SEC (20 µg of conjugate injected into a TSKgel G3000SWXL, mobile phase: 10% IPA in 1×PBS).

[0137] Figures 14A-14B depict the determination of the drug-antibody ratio (DAR) of Herceptin-RTX-5007 by LC-MS (0.2 µg of the reduced substance was injected into a PLRP-S (1000 Å, 8 μm, 2.1 mm x 50 mm)).

[0138] Figure 15 depicts the determination of the concentration of Herceptin-RTX-5007 by A280 at an extinction coefficient of 14.8 on a nanodrop 2000C E1%.

[0139] In this embodiment, the antibody-drug conjugate saltuzumab-RTX-5007 conjugate was prepared as in Example 6.

[0140] Prior to conjugation, saltuzumab was dialyzed into 1×PBS at pH 7.4. The RTX-5007 conjugate amplifies the reaction up to 10 mg, wherein saltuzumab is 5.0 mg / mL.

[0141] Sastuzumab was reduced for 1.5 h at 37°C using 6 equivalents of 10 mM TCEP (40 µL). After reduction, 1 mM EDTA (8.2 µL from 250 mM stock solution) was added to the protein. Before adding the payload, the buffer composition was adjusted to 10% DMA with an additional 200 µL of DMA. After adding the organic solvent, 20 equivalents of RTX-5007 (133.3 µL of 10 mM RTX-5007 in DMA) were added to the mAb. The mAb solution was mixed at room temperature for 1.5 h, and then the DAR was checked. After confirming the targeting of the DAR, 40 equivalents of 10 mM N-acetylcysteine ​​(266.6 µL) were added to the reaction to quench RTX-5007. After conjugation, the protein was dialyzed with 1×PBS (pH 7.4) to remove excess payload. After three buffer exchanges over 12 hours, the final buffer was replaced with 1×PBS (pH 7.4).

[0142] After removing the payload, 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.

[0143] Figure 16 depicts the determination of saltuzumab-RTX-5007 monomer by SEC (20 µg of conjugate was injected into a TSKgel G3000SWXL, with 10% IPA in 1×PBS as the mobile phase).

[0144] Figures 17A-17B depict the determination of saltuzumab-RTX-5007 DAR by LC-MS (0.2 µg of reduced material was injected into a PLRP-S (1000 Å, 8 μm, 2.1 mm x 50 mm)).

[0145] Figure 18 depicts the results measured on nanodrop 2000C E1% by A280 at an extinction coefficient of 15.6.The concentration of saltuzumab-RTX-5007.

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

[0147] Different concentrations of Herceptin-ADC or saltuzumab-ADC were used to determine the interaction kinetics. Herceptin or saltuzumab was used as a positive control, and human isotype IgG was used as a negative control at the same concentration dilution.

[0148] His-tagged human HER2 or His-tagged human Trop2 (~35 nM and 150 nM, respectively) were loaded onto the Ni-NTA probe by immersing the probe in the probe for 180–300 seconds. 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 / Sastuzumab-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 depict the binding kinetics of Herceptin with HER2.

[0150] Figures 20A-20B depict the binding kinetics of Herceptin-ADC with HER2.

[0151] Figures 21A-21B depict the binding kinetics of the isoform with HER2.

[0152] Figures 22A-22B depict the binding kinetics of Sastuzumab with TROP2.

[0153] Figures 23A-23B depict the binding kinetics of sastuzumab-ADC with TROP2.

[0154] Figures 24A-24B depict the binding kinetics of the isoform with TROP2.

[0155] Both Herceptin and its ADC derivatives showed comparable binding ability to the HER-2 receptor (approximately 0.5-2 nM), indicating that the conjugation has little effect on the receptor binding ability of the mAb. Similarly, sastuzumab and its ADC showed comparable binding ability to the TROP-2 receptor (approximately 0.04-0.1 nM). In this embodiment, in vitro cytotoxicity data were determined for the antibody-drug conjugate using an anti-HER2 antibody (Herceptin) and the antibody-drug conjugate using an anti-TROP2 antibody (sastuzumab).

[0156] Herceptin-ADC is an antibody-drug conjugate (ADC) targeting Her2, which delivers a small molecule (oxazine) with antitumor activity. It has been reported that the N87 cell line expresses high levels of Her2 mRNA transcripts and proteins.

[0157] Satuzumab-ADC is an antibody-drug conjugate (ADC) targeting Trop2, which delivers small molecules (oxygen)The azirmonium (azirmonium) payload has antitumor activity. The N87 cell line has been reported to express moderate levels of Trop2 mRNA transcripts.

[0158] Figures 25A-25B depict live-cell imaging analysis (IncuCyte S3) of NCI-N87 cells at 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. In this embodiment, the in vitro cytotoxicity study was designed as follows: 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. 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. In this embodiment, cell density may be varied depending on the cell line used.

[0159] 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, 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.

[0160] Figure 27A depicts N87 cells treated 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, with phase-contrast microscopy imaging every six hours to monitor confluence percentage and cell apoptosis monitored under 488 nm fluorescence; Figures 27B-27E depict IC50 curves plotted at 24 hours (27B), 48 hours (27C), 72 hours (27D), and 96 hours (27E) of culture.

[0161] The results showed that the response to the ADC was dose- and target-dependent. The results also showed that the cytotoxicity of both Herceptin-ADC and sastuzumab-ADC in N87 cells was target-dependent (HER2 expression) and dose-dependent. At 96 hours, N87 cells were more sensitive to Herceptin-ADC (IC50 = 0.026 μg / mL) than to sastuzumab-ADC (IC50 = 0.440 μg / mL).

[0162] In one embodiment, the antibody in the ADC is trastuzumab (Herceptin).

[0163] In one embodiment, the antibody in the ADC is sastuzumab. Specification 16 / 24 pages 24 CN 121752295 A

[0164] In one embodiment, the reactive oxygen species (ROS) phosphine payload in the ADC is 10-oxa-4-azatricyclo[5.2.1.0, 2,6]dec-8-en-3,5-dione.

[0165] In one embodiment, the linker and ROS phosphine payload in the ADC are RTX5007.

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

[0167] In one embodiment, the antibody-drug conjugate is sastuzumab-RTX5007.

[0168] In one embodiment, a method of treating cancer includes administering an ADC, wherein the antibody is trastuzumab.

[0169] In an embodiment, a method of treating cancer includes administering an ADC, wherein the antibody is sastuzumab.

[0170] In an embodiment, a method of treating cancer includes administering an ADC, wherein the reactive oxygen species (ROS) loading is 10-oxa-4-azatricyclo[5.2.1.0,2.6]dec-8-en-3,5-dione.

[0171] In an embodiment, a method of treating cancer includes administering an ADC, wherein the linker and the ROS loading are RTX5007.

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

[0173] In an embodiment, a method of treating cancer includes administering an ADC, wherein the antibody-drug conjugate is sastuzumab-RTX5007.

[0174] This disclosure also relates to pharmaceutical compositions that may include an immunostimulatory ADC, which may be administered to a subject for purposes including, but not limited to, treating cancer or other hyperproliferative diseases. Compositions including the ADC can be optimized to make them suitable for administration to subjects via intravenous, subcutaneous, intramuscular, intradermal, or intracerebral injection routes. The pharmaceutical composition can be supplied in liquid solution, suspension, emulsion, or solid form, suitable for dissolving or suspending in a liquid prior to use.

[0175] The pharmaceutical composition may include a pharmaceutically acceptable carrier, that is, any carrier that does not interfere with the effectiveness of the bioactivity of the component and is non-toxic to the subject to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and administered to subjects at appropriate doses. Preferably, the composition is sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersants. By including various antibacterial agents andAntifungal agents ensure protection against microbial activity.

[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 pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic; of course, the formulation may also be hypertonic or hypotonic if desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffers such as Ringer's solution and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to 7.5. Certain carriers may be preferred, depending, for example, on the route of administration and the concentration of the composition being administered. Carriers suitable for direct delivery can be administered without excessive 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. This may include pharmaceutically acceptable salts, such as mineral salts (e.g., hydrobromide, phosphate, sulfate, etc.); and organic salts such as acetate, propionate, malonate, benzoate, etc. More preferably, the composition is combined with saline, Ringer's balanced salt solution (pH 7.4), etc.

[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 skilled in the art. The patient's physiological data (e.g., age, body size, and weight) and the type and severity of the disease being treated must be considered to determine the appropriate dosage.

[0178] The pharmaceutical composition can be formulated for administration by injection, such as intravenous injection, intramuscular injection, subcutaneous injection, or intradermal injection. The injectable formulation can be in unit dosage form, such as in ampoules or multi-dose containers. The composition may take the form of a suspension, solution, or emulsion in an oily or aqueous solvent and may contain formulation agents such as suspensions, stabilizers, and / or dispersants. Alternatively, the active ingredient may be in powder form for mixing with a suitable carrier, such as sterile, pyrogen-free water, prior to use. Optionally, the pharmaceutical composition may be frozen and stored at any suitable storage temperature. Gene therapy refers to the modification of cells to insert polynucleotides into the cells, for example, as episomes inserted and / or integrated into the genome, wherein the polynucleotide provides therapeutic effects by encoding a desired expressed polypeptide, disrupting the genomic sequence encoding a desired silenced polypeptide, regulating the transcription of an endogenous sequence encoding the polypeptide, etc. Typically, such modification can be achieved by preparing a vector comprising the polynucleotide and using the vector to insert the polynucleotide into one or more cells.The gene therapy vector is implemented in the cell nucleus.

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

[0180] Kits Kits comprising the ADC described herein or pharmaceutical compositions thereof are also disclosed herein. The kit may be in a pharmaceutically acceptable solution form, for example, conjugated with sterile saline, glucose solution, buffer solution or other pharmaceutically acceptable sterile fluid. Alternatively, the complex may be lyophilized or dried; in this case, the kit optionally further includes a pharmaceutically acceptable solution (e.g., saline, glucose solution, etc.) in a container to reconstitute the complex into a solution for injection purposes. The kit may further include a needle or syringe (preferably packaged in a sterile form) for injecting the complex, and / or packaged alcohol swabs. Instructions for administering the composition by a clinician or patient may optionally be included.

[0181] Methods It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope or implementation of this disclosure. Certain compositions and methods have been described in detail, and these compositions and methods will be more clearly understood by referring to the following examples, which are introduced for illustrative purposes only and are not intended to be limiting.

[0182] Examples The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced in light of the general description provided herein.

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

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

[0185] Reagents and reaction conditions: (a) tert-butyldimethylchlorosilane, 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]ethyl-1-one (1) was treated with tert-butyldimethylchlorosilane and imidazole in DMF to give a protective alcohol 2 in 98% yield. The protective alcohol was treated with hydrazine hydrate and acetic acid in ethanol to give the desired hydrazone 3 in 86% yield. 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid was activated with oxalyl chloride in dichloromethane using a catalytic amount of DMF to give the corresponding acyl chloride, which was coupled with hydrazone 3 to give acylhydrazone 5. TBAF buffered with acetic acid was used to treat tert-butyldiphenyl ether.The methylsilyl group was deprotected to give alcohol 6. Finally, it was treated with POCl3 in dichloromethane at -78°C, and then treated with 2-chloroethylamine hydrochloride and triethylamine to give target compound 7.

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

[0188] Other adapters for conjugating antibodies to payloads are well known to those skilled in the art {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 alMolecules2021, 26, 2943. https: / / doi.org / 10.3390 / molecules26102943.

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

[0190] The synthesis of RTX5007 from commercially available 2-hydroxy-4-nitrobenzoic acid 1 was completed in 8 steps. The synthesis of RTX5007 was completed using the chemical method illustrated in Figure 10.

[0191] Reagents and reaction conditions: (a) methanol, sulfuric acid; (b) (3-bromopropoxy)tert-butyldimethylsilane, potassium carbonate, DMF; (c) diisobutylaluminum hydride, THF; (d) bis(trimethylsilyl)aminolithium, bis(2-chloroethyl)phosphamide 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-en-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. The yield was 76% after purification by silica gel column chromatography; the purity was confirmed by 1H NMR (NMR purity > 95%). Ester 2 was treated with (3-bromopropoxy)-tert-butyldimethylsilane in DMF in the presence of potassium carbonate to give protected ester 3. A quantitative yield was obtained after purification by silica gel column chromatography; the purity was confirmed by 1H NMR (NMR purity > 95%). Protected ester 3 was reduced in THF with diisobutylaluminum hydride (DIBAL-H) ​​to give alcohol 4. The yield was 87% after purification by silica gel column chromatography; the purity was confirmed by 1H NMR (NMR purity > 95%). Alcohol 4 was treated with bis(trimethylsilyl)aminolithium at -78°C, followed by treatment with bis(2-chloroethyl)phosphoramide dichloride, and then with ammonia in THF to give phosphoramide 5. The yield was 79% after purification by silica gel column chromatography; the purity was confirmed by 1H NMR (NMR purity > 95%). Phosphoramide 5 was deprotected in methanol with aqueous hydrochloric acid (1M) to give alcohol 6. Purified by silica gel column chromatography, the yield was 56%; confirmed by 1H NMR (NMR purity > 95%). Alcohol 6 was treated with carbon tetrabromide and triphenylphosphine in dichloromethane to give bromide 7. Purified by silica gel column chromatography, the yield was 60%; 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-en-3,5-dione in DMF in the presence of potassium carbonate to give protected phthalimide 8. Purified by silica gel column chromatography, the yield was 57%; confirmed by 1H NMR and MS ES+ = 577 (NMR purity > 95%).

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

[0193] Example 3. Synthesis of connector-payload RTX5014 (isophosphoramide mustard). Specification 19 / 24 pages 27 CN 121752295 A

[0194] The synthesis of RTX5014 was carried out in 5 steps from commercially available maleic anhydride 1 and furan 2. The synthesis of RTX5014 was carried out using the chemical method illustrated in Figure 11.

[0195] Reagents & conditions: a) diethyl ether, room temperature, 24 h; b) 3-amino-1-propanol, MeOH, 0℃-room temperature, overnight; c) toluene, 130℃, 6 h; f) MeOH, anodizing; g) BF3.OEt2, DCM.

[0196] Procedure: Maleic anhydride (25 g, 253.5 mmol, 1 equivalent) was reacted with furan (34.5 g, 507 mmol, 1 equivalent) in diethyl ether (250 ml) overnight at room temperature, yielding a bicyclic Diels-Alder adduct 3 (21 g) in 50% yield as a white powder, which was filtered and washed with cold diethyl ether. The crude substance was of sufficiently high purity by NMR and could be used in the next step without further purification. 3-Amino-1-propanol (4.5 g, 60 mmol, 1 equivalent) was reacted with adduct 3 (10 g, 60 mmol, 1 equivalent) in MeOH, yielding compound 4 (5.6 g) as a white solid in 42% yield, which could also be used 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 h, followed by silica gel column chromatography purification to yield the desired compound 5 (1.6 g) in 75% yield as a waxy solid (structure confirmed by 1H NMR, purity 95%).

[0197] Commercially available ifosfamide 6 (300 mg, 1.1 mmol, 1 equivalent) was subjected to electrochemical anodic oxidation (J. Med. Chem. 2015, 58, 705 717) to yield compound 7 (300 mg, crude yield 96%), which was used immediately without any further purification due to its high instability. The formation of compound 7 was confirmed by TLC (DCM:acetone 1:1). In DCM, at -78 °C, in the presence of BF3·OEt (152 mg, 1.1 mmol, 1 equate), compound 7 (300 mg, 1.1 mmol, 1 equate) reacted with compound 5 (356 mg, 2.3 mmol, 2 equates) to provide the crude product 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 compounds showed that the desired product contained minor impurities and the yield (87%) was [not specified].mg, 18%, purity ~85% by NMR). NMR of the sample recorded 4 days later showed further decomposition. The bulk-stored sample was purified again to remove impurities to provide 27 mg of RTX5014 (purity ~85% by NMR).

[0198] Example 4. Synthesis of the connector-payload RTX5015.

[0199] The synthesis of RTX5015 was carried out in 7 steps from commercially available maleic anhydride 1 and furan 2. The synthesis of RTX5015 was carried out using the chemical method illustrated in Figure 12.

[0200] Reagents & Conditions: a) Diethyl ether, room temperature, 24 h; b) 3-amino-1-propanol, MeOH, 0℃-room temperature, overnight; c) PPh3, DIAD, 4-hydroxyacetophenone, 0℃-room temperature, overnight; d) DCM:MeOH (3:1), NaBH4, room temperature, 4 h; e) Toluene, 130℃, 6 h; f) MeOH, anodizing; g) BF3.OEt2, DCM.

[0201] Procedure: Maleic anhydride (25 g, 253.5 mmol, 1 equivalent) was reacted with furan (34.5 g, 507 mmol, 1 equivalent) in diethyl ether (250 ml) overnight at room temperature, yielding a bicyclic Diels-Alder adduct 3 (21 g) in 50% yield as a white powder, which was filtered and washed with cold diethyl ether. The crude compound was of sufficiently high purity by NMR and could be used in the next step without further purification. The reaction of 3-amino-1-propanol (4.5 g, 60 mmol, 1 equivalent) with adduct 3 (10 g, 60 mmol, 1 equivalent) in MeOH yielded compound 4 (5.6 g) as a white solid in 42% yield, which could also be used in the next step without further purification. Compound 4 (6 g, 27 mmol, 1 equivalent) was reacted with 4-hydroxyacetophenone (3.7 g, 27 mmol, 1 equivalent), DIAD (6 g, 30 mmol, 1.1 equivalent), and PPh3 (7.7 g, 30 mmol, 1 equivalent) in THF under Mitsunobu conditions. Column chromatography purification yielded compound 5 (5.2 g) in 58% yield (structure confirmed by 1H NMR, purity 95%). Compound 5 (5 g, 14.6 mmol, 1 equivalent) was reduced with NaBH4 (1.1 g, 29.3 mmol, 2 equivalent) in a DCM:MeOH (3:1) mixture, and purified by column chromatography to give the desired alcohol 6 (1 g) in 20% yield (structure confirmed by 1H NMR). Compound 6 (175 mg, 0.5 mmol) was refluxed in toluene (3 ml) at 130 °C for 6 h, and purified by silica gel column chromatography to give the desired compound 7 (115 mg, 1 g) in 20% yield.The yield was 82%, and the product was a waxy solid (page 20 / 24, CN 121752295 A, structure confirmed by 1H NMR, purity 95%).

[0202] Commercially available cyclophosphamide 8 (300 mg, 1.1 mmol, 1 equivalent) was subjected to electrochemical anodization (J. Med. Chem. 2015, 58, 705 717) to provide compound 9 (300 mg, crude yield 96%). Since compound 9 was reported to be very unstable, it was used immediately without any further purification. The formation of compound 9 was confirmed by TLC (DCM:acetone 1:1). In a DCM at -78 °C, compound 9 (300 mg, 1 mmol, 1 equivalent) reacted with compound 7 (443 mg, 16 mmol, 1.5 equivalent) in the presence of BF3.OEt (152 mg, 1 mmol, 1 equivalent) to provide the crude product RTX5015 (533 mg). Mass spectrometry analysis of the crude reaction mixture confirmed product formation. The crude reaction mixture was purified by silica gel column chromatography. 1H NMR analysis of the isolated compounds revealed multiple stereoisomers of the desired product in yield (38 mg, 7%, NMR purity ~85%). However, the presence of multiple product stereoisomers made it difficult to calculate their precise ratios and total purity. The characteristic product signals present in 1H NMR and the mass spectrometry data supported product formation and total purity. After 48 h, the stability of the product was examined by NMR and found to be stable at -20 °C.

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

[0204] Herceptin-RTX-5007 RTX-5007 conjugates were expanded to a maximum of 7.4 mg, with Herceptin at 4.9 mg / mL.

[0205] Herceptin was reduced at 37°C for 1.5 h with 6 equivalents of 10 mM TCEP (29.4 µL). After reduction, 1 mM EDTA (from 250 mM stock solution, 6.1 µL) was added to the protein. Before adding the payload, the buffer composition was adjusted to 10% DMA with an additional 150 µL of DMA. After adding the organic solvent, 15 equivalents of RTX-5007 (73.5 µL of 10 mM RTX-5007 in DMA) were added to the mAb. The mAb solution was mixed at room temperature for 1.5 h, and then the DAR was checked. After confirming DAR targeting, 30 equivalents of 10 mM N-acetylcysteine ​​(147 µL) were added to the reaction to quench RTX-5007. Following conjugation, dialysis with 1×PBS (pH 7.4) was performed to remove excess payload. The reaction was carried out over 12 hours.After three buffer exchanges, the final buffer was replaced with 1×PBS (pH 7.4).

[0206] After removing 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 depicts the determination of Herceptin-RTX-5007 monomer by SEC (20 µg of conjugate was injected into a TSKgel G3000SWXL, and the mobile phase was 10% IPA in 1×PBS).

[0208] Figures 14A-14B depict the determination of Herceptin-RTX-5007 drug-antibody ratio (AR) by LC-MS (0.2 µg of reduced material was injected into a PLRP-S (1000 Å, 8 μm, 2.1 mm x 50 mm)).

[0209] Figure 15 depicts the concentration of Herceptin-RTX-5007 determined by A280 at an extinction coefficient of 14.8 on a nanodrop 2000C E1%.

[0210] Example 6. Preparation of antibody-drug conjugates using anti-TROP2 antibody (saltuzumab).

[0211] Saltuzumab-RTX-5007 conjugate.

[0212] Before conjugation, satuzumab was dialyzed into 1×PBS at pH 7.4. The RTX-5007 conjugation was amplified to a maximum of 10 mg, with satuzumab at 5.0 mg / mL.

[0213] Saltuzumab was reduced at 37°C for 1.5 h using 6 equivalents of 10 mM TCEP (40 µL). After reduction, 1 mM EDTA (taken from 250 mM stock solution, 8.2 µL) was added to the protein. Before adding the payload, the buffer composition was adjusted to 10% DMA with an additional 200 µL of DMA. After adding the organic solvent, 20 equivalents of RTX-5007 (133.3 µL of 10 mM RTX-5007 in DMA) were added to the mAb. The mAb solution was mixed at room temperature for 1.5 h, and then the DAR was checked. After confirming the targeting DAR, 40 equivalents of 10 mM N-acetylcysteine ​​(266.6 µL) were added to the reaction to quench RTX-5007. After conjugation, the excess payload was removed by dialysis with 1×PBS (pH 7.4). After 3 buffer exchanges over 12 hours, the final buffer was replaced with 1×PBS (pH 7.4).

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

[0215] Figure 16 depicts the determination of saltuzumab-RTX-5007 monomer by SEC (20 µg of conjugate was injected into a TSKgel G3000SWXL, mobile phase was 10% IPA in 1×PBS).

[0216] Figures 17A-17B depict the DAR of saltuzumab-RTX-5007 by LC-MS (0.2 µg of reduced material was injected into a PLRP-S (1000 Å, 8 μm, 2.1 mm x 50 mm)).

[0217] Figure 18 depicts the concentration of saltuzumab-RTX-5007 determined by A280 at an extinction coefficient of 15.6 on a nanodrop 2000C E1%.

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

[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, and Q buffer was used to initiate the dissociation of the complex.

[0220] Loading concentrations of His-tagged human HER2 (2.5 μg / mL) or His-tagged human Trop2 (5 μg / mL) (receptor for ADC) used for the Ni-NTA probe.

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

[0222] His-tagged human HER2 or His-tagged human Trop2 (~35 nM and 150 nM, respectively) were loaded onto Ni-NTA probes by immersing the probes for 180–300 seconds; additional probes without His-tagged receptors but using ADC / mAbs in the association step were used for background correction.

[0223] His-tagged receptor-loaded probes were 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 association kinetics.

[0224] His-tagged receptor probes were immersed in Q buffer for 300 seconds to determine the dissociation kinetics of bound ADC / mAbs.

[0225] Data are presented as concentration-dependent calculations (local fitting).

[0226] Figures 19A-19B depict the binding kinetics of Herceptin with HER2. Probes loaded with the His-tagged receptor 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 depicts the concentration-dependent calculations (local fitting).

[0227] Figures 20A-20B depict the binding kinetics of Herceptin-ADC with HER2. 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 20B depicts the concentration-dependent calculations (local fitting).

[0228] Figures 21A-21B depict the binding kinetics of the isotype with HER2. 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 for 300 seconds to determine the association kinetics. Figure 21B depicts the concentration-dependent calculations (local fitting) alone.

[0229] Figures 22A-22B depict the binding kinetics of sautuzumab with TROP2. The probe loaded with the His-tagged receptor was immersed in different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) of sautuzumab-ADC for 300 seconds to determine the association kinetics. Figure 22B depicts the concentration-dependent calculations (local fitting) alone. Specification 22 / 24 pages 30 CN 121752295 A

[0230] Figures 23A-23B depict the binding kinetics of sautuzumab-ADC with TROP2. The probes loaded with the His-tagged receptor 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 depicts the concentration-dependent calculations (local fitting) alone.

[0231] Figures 24A-24B depict the binding kinetics of the isoform with TROP2. The probes loaded with the His-tagged receptor were immersed in mAbs at different concentrations (500 nM, 250 nM, 125 nM, 62.5 nM) for 300 seconds to determine the association kinetics. Figure 24B depicts the concentration-dependent calculations (local fitting) alone.

[0232] Both Herceptin and the ADC derivative showed comparable binding abilities to the HER-2 receptor (approximately 0.5-2 nM range), indicating that conjugation has little effect on the receptor binding ability of the mAb.

[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 (satruzumab).

[0236] Herceptin-ADC is an antibody-drug conjugate (ADC) targeting Her2, which delivers a small molecule (oxazine) with antitumor activity.

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

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

[0239] The N87 cell line has been reported to express moderate levels of Trop2 mRNA transcripts (https: / / www.proteinatlas.org / ENSG00000184292-TACSTD2 / cell+line).

[0240] Figures 25A-25B depict live-cell imaging analysis (IncuCyte S3) of NCI-N87 cells at 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.

[0241] In this embodiment, the in vitro cytotoxicity study was designed as follows. The NCI-N87 cell line was purchased from ATCC and expanded in vitro before use. 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 dose-response studies, 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). Saltozumab-ADC was administered at dose levels ranging from 30 mg / mL to 0.03 mg / mL in the presence of caspase 3 / 7 (CellEvent™ Green Reagent, Invitrogen).N87 cells were treated with either Herceptin-ADC (NJBP-0165-129) or NJBP-0165-129, with phase-contrast microscopy imaging every six hours to monitor confluence percentage and apoptosis monitored at 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 percentage and apoptosis monitored at 488 nm fluorescence. Figures 26B–26E depict IC50 curves plotted after 24 hours (26B), 48 hours (26C), 72 hours (26D), and 96 hours (26E). Instructions for Use, pages 23 / 24, 31 CN 121752295 A

[0242] Figure 27A depicts N87 cells treated 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 depict IC50 curves plotted at 24 hours (27B), 48 hours (27C), 72 hours (27D), and 96 hours (27E) of culture.

[0243] The results showed that the response to ADC was dose- and target-dependent. The results showed that the cytotoxicity of both Herceptin-ADC and sastuzumab-ADC in N87 cells 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 Saltuzumab-ADC (IC50 = 0.440 μg / mL). [Instructions for Use, Page 24 / 24, 32, CN 121752295 A, Figure 1; Instructions for Use, Figure 1 / 41, Page 33, CN 121752295 A, Figure 2; Instructions for Use, Figure 2 / 41, Page 34, CN 121752295 A, Figure 3; Instructions for Use, Figure 3 / 41, Page 35, CN 121752295 A, Figure 4; Instructions for Use, Figure 4 / 41, Page 36, CN 121752295 A, Figure 5; Instructions for Use, Figure 5 / 41, Page 37, CN 121752295 A, Figure 6] [Note: The last line appears to be a continuation of the previous paragraph and is not directly related to the preceding text.]Figure 7 of the instruction manual (Figure 6 / 41, page 38, CN 121752295 A); Figure 8 of the instruction manual (Figure 8 / 41, page 40, CN 121752295 A); Figure 9 of the instruction manual (Figure 9 / 41, page 41, CN 121752295 A); Figure 10 of the instruction manual (Figure 11); Figure 12 of the instruction manual (Figure 10 / 41, page 42, CN 121752295 A); Figure 13 of the instruction manual (Figure 13 / 41, page 44, CN 121752295 A); Figure 14A of the instruction manual (Figure 13 / 41, page 45, CN 121752295 A); Figure 14B of the instruction manual (Figure 14 / 41, page 46, CN 121752295 A); Figure 15 of the instruction manual (Figure 15 / 41, page 47, CN 121752295 A). 121752295 A Figure 16, Instruction Manual Appendix 16 / 41, Page 48 CN 121752295 A Figure 17A, Instruction Manual Appendix 17 / 41, Page 49 CN 121752295 A Figure 17B, Instruction Manual Appendix 18 / 41, Page 50 CN 121752295 A Figure 18, Instruction Manual Appendix 19 / 41, Page 51 CN 121752295 A Figure 19A, Instruction Manual Appendix 20 / 41, Page 52 CN 121752295 A Figure 19A Figure 19B, Instruction Manual Appendix 21 / 41, Page 53 CN 121752295 A Figure 20A, Instruction Manual Appendix 22 / 41, Page 54 CN 121752295 A Figure 20A Figure 20B, Instruction Manual Appendix 23 / 41, Page 55 CN 121752295 A Figure 21A, Instruction Manual Appendix 24 / 41 Page 56 CN 121752295 A Figure 21A Figure 21B Description Drawings 25 / 41 Page 57 CN 121752295 A Figure 22A Description Drawings 26 / 41 Page 58 CN 121752295 A Figure 22A Description Drawings 27 / 41 Page 59 CN 121752295 A Figure 22B Description Drawings 28 / 41 Page 60 CN 121752295 A Figure 23A Description Drawings 29 / 41 Page 61 CN 121752295 A Figure 23A Description Drawings 30 / 4162 CN 121752295 A Figure 23B Instruction Manual Drawings, Page 31 / 41 63 CN 121752295 A Figure 24A Instruction Manual Drawings, Page 32 / 41 64 CN 121752295 A Figure 24A Instruction Manual Drawings, Page 33 / 41 65 CN 121752295 A Figure 24B Figures 25A-25B Instruction Manual Drawings, Page 34 / 41 66 CN 121752295 A Figures 26A-26B Instruction Manual Drawings, Page 35 / 41 67 CN 121752295 A Figures 26C-26D Instruction Manual Drawings, Page 36 / 41 68 CN 121752295 A Figure 26E Instruction Manual Drawings, Page 37 / 41 69 CN 121752295 A Figure 27A Instruction Manual Drawings, Page 38 / 41 70 CN 121752295 Figure 27B, Instruction Manual Drawings, Page 39 / 41, CN 121752295 Figure 27C-27D, Instruction Manual Drawings, Page 40 / 41, CN 121752295 Figure 27E, Instruction Manual Drawings, Page 41 / 41, CN 121752295

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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