Increasing immune activity through modulation of post-cellular signaling factors

文档序号：788154 发布日期：2021-04-09 浏览：36次中文

阅读说明：本技术 通过后细胞信号传导因子的调节来增加免疫活性 (Increasing immune activity through modulation of post-cellular signaling factors ) 是由 A·M·巴索蒂 A·M·坎特莱 J·帕克于 2019-06-14 设计创作，主要内容包括：本发明提供了通过诱导铁依赖性细胞拆解来增加免疫应答的方法。免疫应答的增加可用于治疗例如感染或癌症。本发明还提供了筛选测定,用于鉴定诱导铁依赖性细胞拆解并且也是免疫刺激剂的化合物。本发明还提供了鉴定由经历铁依赖性细胞拆解的细胞产生的免疫刺激剂的方法。(The present invention provides methods for increasing immune responses by inducing iron-dependent cell disassembly. The increase in the immune response can be used to treat, for example, an infection or cancer. The invention also provides screening assays for identifying compounds that induce iron-dependent cell disassembly and that are also immunostimulants. The invention also provides methods of identifying an immunostimulant produced by a cell undergoing iron-dependent cell disassembly.)

1. A method of increasing immune activity in an immune cell, the method comprising:

(i) contacting a target cell with an agent that induces disassembly of iron-dependent cells and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase immune activity in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent, wherein the agent that induces disassembly of iron-dependent cells is selected from the group consisting of: antiporter system Xc ^-An inhibitor of GPX4, and a statin.

2. A method of increasing the level or activity of NFkB in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cellular disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of NFkB in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent, wherein the agent that induces iron-dependent cellular disassembly is selected from the group consisting of: antiporter system Xc^-An inhibitor of GPX4, and a statin.

3. A method of increasing the level or activity of an Interferon Regulatory Factor (IRF) or an interferon gene Stimulator (STING) in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cell disassembly and (ii) exposing the immune cell to an amount of a composition comprising(ii) target cells contacted with the agent or post-cellular signaling factors produced by target cells that have been contacted with the agent, in an amount sufficient to increase the level or activity of IRF or STING in the immune cells relative to immune cells in the absence of contacting the target cells with the agent, wherein the agent that induces iron-dependent cellular disassembly is selected from the group consisting of: antiporter system Xc ^-An inhibitor of GPX4, and a statin.

4. A method of increasing the level or activity of a pro-immune cytokine in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cellular disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of a proammunocytokine in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent, wherein the agent that induces iron-dependent cellular disassembly is selected from the group consisting of: antiporter system Xc^-An inhibitor of GPX4, and a statin.

5. The method of any one of claims 1 to 4, wherein the iron-dependent cell disassembly is iron death.

6. The method of any one of claims 1-4, wherein the antiporter system Xc^-The inhibitor of (a) is elastine or a derivative or analogue thereof.

7. The method of claim 6, wherein the elapsin or a derivative or analogue thereof has the formula:

Or a pharmaceutically acceptable salt or ester thereof, wherein

R₁Selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, hydroxy and halogen;

R₂selected from the group consisting of: H. halo and C_1-4An alkyl group;

R₃selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, 5-7 membered heterocycloalkyl, and 5-6 membered heteroaryl;

R₄selected from the group consisting of: h and C_1-4An alkyl group;

R₅is halo;

optionally substituted with ═ O; and is

n is an integer of 0 to 4.

8. The method of claim 6, wherein the analogue of elastine is PE or IKE.

9. The method of any one of claims 1 to 4, wherein the inhibitor of GPX4 is selected from the group consisting of: (1S,3R) -RSL3 or a derivative or analog thereof, ML162, DPI compound 7, DPI compound 10, DPI compound 12, DPI compound 13, DPI compound 17, DPI compound 18, DPI compound 19, FIN56, and FINO 2.

10. The method of claim 9, wherein the RSL3 derivative or analog is a compound represented by structural formula (I):

or an enantiomer, optical isomer, diastereoisomer, N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof, wherein

R₁、R₂、R₃And R₆Independently selected from H, C_1-8Alkyl radical, C_1-8Alkoxy radical, C_1-8Aralkyl, 3-to 8-membered carbocycle, 3-to 8-membered heterocycle, 3-to 8-membered aryl or 3-to 8-membered heteroaryl, acyl, alkylsulfonyl and arylsulfonyl wherein each of alkyl, alkoxy, aralkyl, carbocycle, heterocycle, aryl, heteroaryl, acyl, alkylsulfonyl and arylsulfonyl is optionally substituted with at least one substituent;

R₄and R₅Independently selected from H₁ C_1-8Alkyl radical, C_1-8Alkoxy, 3-to 8-membered carbocyclic ring, 3-to 8-membered heterocyclic ring, 3-to 8-membered aryl or 3-to 8-membered heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid, acyl, alkoxy-substituted acyl, sugar alcohol, NR⁷R⁸、OC(R⁷)₂COOH、SC(R⁷)₂COOH、NHCHR⁷COOH、COR⁸、CO₂R⁸Sulfate, sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein each alkyl, alkoxy, carbocycle, heterocycle, aryl, heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid, acyl, alkoxy-substituted acyl, sugar alcohol, NR⁷R⁸、OC(R⁷)₂COOH、SC(R⁷)₂COOH、NHCHR⁷COOH、COR⁸、CO₂R⁸Sulfate, sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether are optionally substituted with at least one substituent;

R⁷selected from H, C_1-8Alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle, wherein each alkyl, carbocycle, aryl, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, and alkylheterocycle may be optionally substituted with at least one substituent;

R⁸Selected from H, C_1-8Alkyl radical, C_1-8Alkenyl radical, C_1-8Alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle and heteroaromatic, wherein each alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle and heteroaromatic may be optionally substituted with at least one substituent; and is

X is 0 to 4 substituents on the ring to which it is attached.

11. The method of claim 9, wherein the RSL3 derivative or analog is a compound represented by structural formula (II):

or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; wherein:

R₁selected from the group consisting of: H. OH and- (OCH)₂CH₂)_xOH；

X is an integer from 1 to 6; and is

R₂、R₂'、R₃And R₃' is independently selected from the group consisting of: H. c_3-8Cycloalkyl and combinations thereof, or R₂And R₂' may be joined together to form a pyridyl or pyranyl group, and R₃And R₃' may be combined together to form a pyridyl or pyranyl group.

12. The method of claim 9, wherein the RSL3 derivative or analog is a compound represented by structural formula (III):

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; wherein:

n is 2,3 or 4; and R is substituted or unsubstituted C₁-C₆Alkyl radical, substituted or unsubstituted C₃-C₁₀Cycloalkyl radical, substituted or unsubstituted C₂-C₈Heterocycloalkyl radical, substituted or unsubstituted C₆-C₁₀Aromatic ring group or substituted or unsubstituted C₃-C₈A heteroaryl ring group; wherein said substitution means that one or more hydrogen atoms in each group are substituted by a group selected from the group consisting of: halogen, cyano, nitro, hydroxy, C₁-C₆Alkyl, halo C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halo C₁-C₆Alkoxy, COOH (carboxyl), COOC₁-C₆Alkyl, OCOC₁-C₆An alkyl group.

13. The method of any one of claims 1 to 4, wherein the statin is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, cerivastatin and simvastatin.

14. The method of any one of claims 1-4, wherein the immune cell is a macrophage, monocyte, dendritic cell, T cell, CD4+ cell, CD8+ cell, or CD3+ cell.

15. The method of any one of claims 1 to 4, wherein the immune cell is a THP-1 cell.

16. The method of any one of claims 1 to 15, wherein the method is performed in vitro or ex vivo.

17. The method of any one of claims 1 to 15, wherein the method is performed in vivo.

18. The method of any one of claims 1 to 15, wherein step (i) is performed in vitro and step (ii) is performed in vivo.

19. A method of increasing immune activity in a cell, tissue, or subject, the method comprising administering to the cell, tissue, or subject an agent that induces disassembly of iron-dependent cells in an amount sufficient to increase immune activity relative to a cell, tissue, or subject not treated with the agent that induces disassembly of iron-dependent cells.

20. The method of claim 19, wherein the subject is in need of increased immune activity.

21. The method of claim 19 or 20, wherein the agent that induces iron-dependent cellular disassembly is administered in an amount sufficient to increase one or more of the following in the cell, tissue, or subject: the level or activity of NFkB, the level or activity of Interferon Regulatory Factor (IRF) or interferon gene Stimulator (STING), the level or activity of macrophage, the level or activity of monocyte, the level or activity of dendritic cell, the level or activity of T cell, the level or activity of CD4+, CD8+ or CD3+ cell, and the level or activity of immunocytokines.

22. A method of increasing the level or activity of NFkB in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cellular disassembly in an amount sufficient to increase the level or activity of NFkB relative to a cell, tissue or subject not treated with the agent that induces iron-dependent cellular disassembly.

23. The method of claim 22, wherein the subject is in need of increased NFkB level or activity.

24. The method of claim 22 or 23, wherein the level or activity of NFkB is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a cell, tissue, or subject not treated with the agent that induces iron-dependent cellular disassembly.

25. A method of increasing the level or activity of an Interferon Regulatory Factor (IRF) or an interferon gene Stimulator (STING) in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of IRF or STING relative to a cell, tissue or subject not treated with the agent that induces iron-dependent cell disassembly.

26. The method of claim 25, wherein the subject is in need of increased IRF or STING levels or activity.

27. The method of claim 25 or 26, wherein the level or activity of IRF or STING is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a cell, tissue, or subject not treated with the agent that induces iron-dependent cell disassembly.

28. A method of increasing the level or activity of macrophages, monocytes, dendritic cells or T cells in a tissue or subject, the method comprising administering to the tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of macrophages, monocytes, dendritic cells or T cells relative to a tissue or subject not treated with the agent that induces iron-dependent cell disassembly.

29. The method of claim 28, wherein the subject is in need of increased macrophage, monocyte, dendritic cell or T cell levels or activity.

30. The method of claim 28, wherein the level or activity of macrophages, monocytes, dendritic cells or T cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a tissue or subject not treated with the agent that induces iron-dependent cell disassembly.

31. A method of increasing the level or activity of CD4+, CD8+, or CD3+ cells in a tissue or subject, the method comprising administering to the tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of CD4+, CD8+, or CD3+ cells relative to a tissue or subject not treated with the agent that induces iron-dependent cell disassembly.

32. The method of claim 31, wherein the subject is in need of increased CD4+, CD8+, or CD3+ cell levels or activity.

33. The method of claim 31 or 32, wherein the level or activity of CD4+, CD8+, or CD3+ cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a tissue or subject not treated with the agent that induces iron-dependent cell disassembly.

34. A method of increasing the level or activity of a proammunocytokine in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of the proammunocytokine relative to a cell, tissue or subject not treated with the agent that induces iron-dependent cell disassembly.

35. The method of claim 34, wherein the subject is in need of increased levels or activity of a pro-immune cytokine.

36. The method of claim 34 or 35, wherein the level or activity of a pro-immune cytokine is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a cell, tissue, or subject not treated with the agent that induces iron-dependent cell disassembly.

37. The method of any one of claims 1 to 36, further comprising, prior to said administering, assessing one or more of the following in said cell, tissue or subject: the level or activity of NFkB; the level or activity of macrophages; the level or activity of monocytes; the level or activity of dendritic cells; the level or activity of a CD4+ cell, CD8+ cell, or CD3+ cell; the level or activity of T cells; and the level or activity of a pro-immunocytokine.

38. The method of any one of claims 1 to 36, further comprising, following said administering, assessing one or more of the following in said cell, tissue or subject: the level or activity of NFkB; the level or activity of macrophages; the level or activity of monocytes; the level or activity of dendritic cells; the level or activity of a CD4+ cell, CD8+ cell, or CD3+ cell; the level or activity of T cells; and the level or activity of a pro-immunocytokine.

39. The method of any one of claims 4 and 34 to 38, wherein the immunocytokine is selected from the group consisting of IFN-a, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF-a, IL-17 and GMCSF.

40. A method of treating a subject in need of increased immune activity, the method comprising administering to the subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the immune activity of the subject.

41. The method of any one of claims 19 to 40, wherein the subject has an infection.

42. The method of claim 41, wherein the infection is a chronic infection.

43. The method of claim 42, wherein the chronic infection is selected from the group consisting of HIV infection, HCV infection, HBV infection, HPV infection, hepatitis B infection, hepatitis C infection, EBV infection, CMV infection, TB infection, and parasitic infection.

44. The method of any one of claims 1 to 39, wherein the cell or tissue is a cancer cell or a cancer tissue.

45. The method of any one of claims 19 to 40, wherein the subject has cancer.

46. The method of claim 45, wherein the cancer is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, non-squamous cell lung cancer, urothelial cancer, Hodgkin's lymphoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, colorectal cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and Merckel cell carcinoma.

47. The method of any one of claims 1 to 46, wherein said iron-dependent cell disassembly is iron death.

48. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a combination of (a) an immunotherapy and (b) an agent that induces iron-dependent cell disassembly, thereby treating the cancer in the subject.

49. The method of claim 48, wherein the agent that induces iron-dependent cell disassembly is administered to the subject in an amount effective to increase the subject's immune response.

50. The method of claim 48, wherein the immunotherapy is selected from the group consisting of: toll-like receptor (TLR) agonists, cell-based therapies, cytokines, cancer vaccines, and immune checkpoint modulators of immune checkpoint molecules.

51. The method of claim 50, wherein the TLR agonist is selected from the group consisting of Coriolis toxin and BCG (BCG).

52. The method of claim 50, wherein the immune checkpoint molecule is selected from the group consisting of CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, ADORA2A, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, and VISTA.

53. The method of claim 50, wherein the immune checkpoint molecule is a stimulatory immune checkpoint molecule and the immune checkpoint modulator is an agonist of the stimulatory immune checkpoint molecule.

54. The method of claim 50, wherein the immune checkpoint molecule is an inhibitory immune checkpoint molecule and the immune checkpoint modulator is an antagonist of the inhibitory immune checkpoint molecule.

55. The method of any one of claims 50 to 54, wherein the immune checkpoint modulator is selected from the group consisting of a small molecule, an inhibitory RNA, an antisense molecule, and an immune checkpoint molecule binding protein.

56. The method of claim 50, wherein the immune checkpoint molecule is PD-1 and the immune checkpoint modulator is a PD-1 inhibitor.

57. The method of claim 56, wherein the PD-1 inhibitor is selected from pembrolizumab, nivolumab, pidilizumab, SHR-1210, MEDI0680R01, BBg-A317, TSR-042, REGN2810, and PF-06801591.

58. The method of claim 50, wherein the immune checkpoint molecule is PD-L1 and the immune checkpoint modulator is a PD-L1 inhibitor.

59. The method of claim 58, wherein the PD-L1 inhibitor is selected from the group consisting of Duvaluzumab, Attributizumab, Avermezumab, MDX-1105, AMP-224, and LY 3300054.

60. The method of claim 50, wherein the immune checkpoint molecule is CTLA-4 and the immune checkpoint modulator is a CTLA-4 inhibitor.

61. The method of claim 60, wherein the CTLA-4 inhibitor is selected from epilimumab, tremelimumab, JMW-3B3, and AGEN 1884.

62. The method of any one of claims 48 to 61, wherein the agent that induces iron-dependent cell disassembly is administered prior to or concurrently with administration of the immunotherapy.

63. The method of any one of claims 48 to 61, wherein the agent that induces iron-dependent cell disassembly is administered after administration of the immunotherapy.

64. The method of any one of claims 48 to 63, wherein the response of the cancer to treatment is improved relative to treatment with the immunotherapy alone.

65. The method of claim 64, wherein the response is improved by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% relative to treatment with the immunotherapy alone.

66. The method of claim 64 or 65, wherein the reply comprises any one or more of: a reduction in tumor burden, a reduction in tumor size, an inhibition of tumor growth, reaching stable cancer in a subject with a progressive cancer prior to treatment, an increase in time to progression of the cancer, and an increase in survival time.

67. The method of any one of claims 48 to 66, wherein the agent that induces iron-dependent cell disassembly and the immunotherapy act synergistically.

68. The method of any one of claims 48 to 67, wherein the cancer is a cancer responsive to immune checkpoint therapy.

69. The method of any one of claims 48 to 68, wherein the cancer is selected from the group consisting of an epithelial cancer, a sarcoma, a lymphoma, a melanoma, and a leukemia.

70. The method of any one of claims 48 to 68, wherein the cancer is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, non-squamous cell lung cancer, urothelial cancer, Hodgkin's lymphoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, colorectal cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and Mercker cell carcinoma.

71. The method of any one of claims 48 to 68, wherein the cancer is renal cell carcinoma.

72. The method of any one of claims 19-71, wherein the subject is a human.

73. The method of any one of claims 1 to 72, wherein the agent that induces iron-dependent cellular disassembly is selected from the group consisting of: antiporter system Xc^-An inhibitor of GPX4, and a statin.

74. The method of claim 73, where the antiporter system Xc^-The inhibitor of (a) is elastine or a derivative or analogue thereof.

75. The method of claim 74, wherein the elastine or derivative or analog thereof has the formula:

or a pharmaceutically acceptable salt or ester thereof, wherein

R₁Selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, hydroxy and halogen;

R₂selected from the group consisting of: H. halo and C_1-4An alkyl group;

R₃selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, 5-7 membered heterocycloalkyl, and 5-6 membered heteroaryl;

R₄selected from the group consisting of: h and C_1-4An alkyl group;

R₅is halo;

optionally substituted with ═ O; and is

n is an integer of 0 to 4.

76. The method of claim 74, wherein the analogue of elastine is PE or IKE.

77. The method of claim 73, wherein said inhibitor of GPX4 is selected from the group consisting of: (1S,3R) -RSL3 or a derivative or analog thereof, ML162, DPI compound 7, DPI compound 10, DPI compound 12, DPI compound 13, DPI compound 17, DPI compound 18, DPI compound 19, FIN56, and FINO 2.

78. The method of claim 77, wherein the RSL3 derivative or analog is a compound represented by structural formula (I):

or an enantiomer, optical isomer, diastereoisomer, N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof, wherein

R₄and R₅Independently selected from H₁ C_1-8Alkyl radical, C_1-8Alkoxy, 3-to 8-membered carbocyclic ring, 3-to 8-membered heterocyclic ring, 3-to 8-membered aryl or 3-to 8-membered heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid, acyl, alkoxy-substituted acyl, sugar alcohol, NR ⁷R⁸、OC(R⁷)₂COOH、SC(R⁷)₂COOH、NHCHR⁷COOH、COR⁸、CO₂R⁸Sulfate, sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether groups, wherein each alkyl, alkoxy, or thioether group is presentRadicals, carbocycles, heterocycles, aryls, heteroaryls, carboxylates, esters, amides, carbohydrates, amino acids, acyl, alkoxy-substituted acyl, sugar alcohols, NR⁷R⁸、OC(R⁷)₂COOH、SC(R⁷)₂COOH、NHCHR⁷COOH、COR⁸、CO₂R⁸Sulfate, sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether are optionally substituted with at least one substituent;

R⁸selected from H, C_1-8Alkyl radical, C_1-8Alkenyl radical, C_1-8Alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle and heteroaromatic, wherein each alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle and heteroaromatic may be optionally substituted with at least one substituent; and X is 0 to 4 substituents on the ring to which it is attached.

79. The method of claim 77, wherein the RSL3 derivative or analog is a compound represented by structural formula (II):

or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; wherein:

R₁selected from the group consisting of: H. OH and- (OCH)₂CH₂)_xOH；

X is an integer from 1 to 6; and is

80. The method of claim 77, wherein the RSL3 derivative or analog is a compound represented by structural formula (III):

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; wherein:

n is 2, 3 or 4; and R is substituted or unsubstituted C₁-C₆Alkyl radical, substituted or unsubstituted C₃-C₁₀Cycloalkyl radical, substituted or unsubstituted C₂-C₈Heterocycloalkyl radical, substituted or unsubstituted C₆-C₁₀Aromatic ring group or substituted or unsubstituted C₃-C₈A heteroaryl ring group; wherein said substitution means that one or more hydrogen atoms in each group are substituted by a group selected from the group consisting of: halogen, cyano, nitro, hydroxy, C ₁-C₆Alkyl, halo C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halo C₁-C₆Alkoxy, COOH (carboxyl), COOC₁-C₆Alkyl, OCOC₁-C₆An alkyl group.

81. The method of claim 73, wherein said statin is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, cerivastatin and simvastatin.

82. The method of any one of claims 1 to 72, wherein the agent that induces iron-dependent cellular disassembly is selected from the group consisting of: sorafenib or a derivative or analogue thereof, sulfasalazine, glutamate, BSO, DPI2, cisplatin, cysteinase, silica-based nanoparticles, CCI4, ferric ammonium citrate, trigonelline, and brucea javanica alcohol.

83. The method of any one of claims 1 to 72, wherein the agent that induces iron-dependent cell disassembly has one or more of the following characteristics:

(a) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of an immune response in co-cultured cells;

(b) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured macrophages, such as RAW264.7 macrophages;

(c) Inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured monocytes, e.g. THP-1 monocytes;

(d) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured bone marrow-derived dendritic cells (BMDCs);

(e) inducing in vitro iron-dependent cell disassembly of target cells and an increase in the level or activity of NFkB, IRF and/or STING in subsequently co-cultured cells;

(f) inducing in vitro iron-dependent cell disassembly of target cells and subsequent increase in the level or activity of a proammunocytokine in the co-cultured cells; and

(g) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured CD4+ cells, CD8+ cells and/or CD3+ cells.

84. The method of any one of claims 1-72, wherein the agent that induces iron-dependent cell disassembly targets cancer cells.

85. A method of screening for an immunostimulant, said method comprising:

(a) providing a plurality of test agents (e.g., a library of test agents);

(b) assessing the ability of each of the plurality of test agents to induce iron-dependent cell disassembly;

(d) Assessing the ability of the candidate immunostimulant to stimulate an immune response.

86. The method of claim 85, wherein the evaluating step (b) comprises contacting a cell or tissue with each of the plurality of test agents.

87. The method of claim 85, wherein the evaluating step (b) comprises administering each of the plurality of test agents to an animal.

88. The method of claim 86, wherein the assessing step (b) further comprises measuring the level or activity of a marker selected from the group consisting of: lipid peroxidation, Reactive Oxygen Species (ROS), isoprostaglandin, Malondialdehyde (MDA), iron, glutathione peroxidase 4(GPX4), prostaglandin-endoperoxide synthase 2(PTGS2), cyclooxygenase 2(COX-2), and Glutathione (GSH).

89. The method of claim 85, wherein the evaluating step (b) further comprises comparing the level or activity of the marker in a cell or tissue contacted with the test agent to the level or activity of the marker in a control cell or tissue not contacted with the test agent.

90. The method of claim 85, wherein the assessing step (d) comprises assessing the in vitro immunostimulatory activity of the test agent that induces iron-dependent cell disassembly.

91. The method of claim 85, wherein the evaluating step (d) comprises measuring an immune response in the animal.

92. The method of any one of claims 85 to 91, wherein an increase in the level or activity of a marker selected from the group consisting of lipid peroxidation, isoprostane, Reactive Oxygen Species (ROS), iron, PTGS2, and COX-2 or a decrease in the level or activity of a marker selected from the group consisting of GPX4, MDA, and GSH indicates that the test agent is an agent that induces iron-dependent cellular disassembly.

93. The method of claim 85, wherein evaluating the candidate immunostimulant comprises culturing an immune cell with a cell contacted with the selected candidate immunostimulant or exposing an immune cell to post-cellular signaling factors produced by a cell contacted with the selected candidate immunostimulant, and measuring the level or activity of NF κ B, IRF or STING in the immune cell.

94. The method of claim 85, wherein evaluating the candidate immunostimulant comprises culturing T cells with cells contacted with the selected candidate immunostimulant or exposing T cells to post-cellular signaling factors produced by cells contacted with the selected candidate immunostimulant, and measuring activation and proliferation of the T cells.

95. A method of identifying an immunostimulant, said method comprising:

(a) contacting a cell with an agent that induces iron-dependent cell disassembly in an amount sufficient to induce iron-dependent cell disassembly in the cell;

(b) isolating one or more post-cellular signaling factors produced by the cells following contact with the agent that induces iron-dependent cell disassembly; and is

96. The method of claim 95, wherein the method further comprises selecting a test agent that stimulates an immune response.

97. The method of claim 95, wherein the method further comprises detecting a marker of iron-dependent cell disassembly in the cell.

98. The method of claim 95, wherein the method further comprises:

i) measuring the level of one or more post-cellular signaling factors produced by the cells following contact with the agent that induces iron-dependent cell disassembly;

ii) comparing the level of one or more post-cellular signaling factors produced by the cells following contact with the agent that induces iron-dependent cell disassembly with the level of one or more test agents in control cells that are not treated with the agent that induces iron-dependent cell disassembly; and is

iii) selecting cells that exhibit an elevated level of a post-cell signaling factor in contact with the agent that induces iron-dependent cell disassembly relative to control cells to produce one or more post-cell signaling factors for use in the assay of step (c).

99. The method of claim 98, wherein the control cells are treated with an agent that induces cell death that is not iron-dependent cell disassembly.

100. The method of claim 95, wherein said assaying comprises administering said one or more post-cellular signaling factors to an animal and measuring an immune response in said animal.

101. The method of claim 95, wherein the assaying comprises treating an immune cell with the one or more post-cellular signaling factors and measuring the level or activity of nfkb activity in the immune cell.

102. The method of claim 95, wherein said assaying comprises treating T cells with said one or more post-cellular signaling factors and measuring activation or proliferation of said T cells.

103. The method of claim 95, wherein said assaying comprises contacting an immune cell with said one or more post-cellular signaling factors and measuring the level or activity of nfk B, IRF or STING in said immune cell.

104. The method of claim 93 or 103, wherein the immune cell is a THP-1 cell.

Background

In multicellular organisms, cell death is a critical and active process that is thought to maintain tissue homeostasis and eliminate potentially harmful cells.

Disclosure of Invention

In certain aspects, the disclosure relates to methods of increasing immune activity in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces disassembly of iron-dependent cells and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase immune activity in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent, wherein the agent that induces disassembly of iron-dependent cells is selected from the group consisting of: antiporter system Xc ^-An inhibitor of GPX4, and a statin.

In certain aspects, the disclosure relates to methods of increasing the level or activity of NFkB in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cellular disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of NFkB in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent, wherein the agent that induces iron-dependent cellular disassembly is selected from the group consisting of: antiporter system Xc^-An inhibitor of GPX4, and a statin.

In certain aspects, the disclosure relates to methods of increasing the level or activity of an Interferon Regulatory Factor (IRF) or an interferon gene Stimulator (STING) in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cell disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of IRF or STING in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent, wherein the agent that induces iron-dependent cell disassembly is selected from the group consisting of: antiporter system Xc ^-An inhibitor of GPX4, and a statin.

In certain aspects, the disclosure relates to methods of increasing the level or activity of a pro-immune cytokine in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cellular disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of a proammunocytokine in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent, wherein the agent that induces iron-dependent cellular disassembly is selected from the group consisting of: antiporter system Xc^-An inhibitor of GPX4, and a statin.

In certain embodiments, the iron-dependent cell disassembly is iron death (ferroptosis). In certain embodiments, antiport system Xc^-The inhibitor of (a) is elastin or a derivative or analogue thereof.

In certain embodiments, elastine or a derivative or analog thereof has the formula:

or a pharmaceutically acceptable salt or ester thereof, wherein

R₁Selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, hydroxy and halogen;

R₂selected from the group consisting of: H. halo and C_1-4An alkyl group;

R₃selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, 5-7 membered heterocycloalkyl, and 5-6 membered heteroaryl;

R₄selected from the group consisting of: h and C_1-4An alkyl group;

R₅is halo;

optionally substituted with ═ O; and is

n is an integer of 0 to 4.

In certain embodiments, the analog of elapsin is PE or IKE.

In certain embodiments, the inhibitor of GPX4 is selected from the group consisting of: (1S,3R) -RSL3 or a derivative or analog thereof, ML162, DPI compound 7, DPI compound 10, DPI compound 12, DPI compound 13, DPI compound 17, DPI compound 18, DPI compound 19, FIN56, and FINO 2.

In certain embodiments, the RSL3 derivative or analog is a compound represented by structural formula (I):

or an enantiomer, optical isomer, diastereoisomer, N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof, wherein

R₄And R₅Independently selected from H₁ C_1-8Alkyl radical, C_1-8Alkoxy, 3-to 8-membered carbocyclic ring, 3-to 8-membered heterocyclic ring, 3-to 8-membered aryl or 3-to 8-membered heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid, acyl, alkoxy-substituted acyl, sugar alcohol, NR⁷R⁸、OC(R⁷)₂COOH、SC(R⁷)₂COOH、NHCHR⁷COOH、COR⁸、CO₂R⁸Sulfate, sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether, wherein each alkyl, alkoxy, carbocycle, heterocycle, aryl, heteroaryl, carboxylate, ester, amide, carbohydrate, amino acid, acyl, alkoxy-substituted acyl, sugar alcohol, NR⁷R⁸、OC(R⁷)₂COOH、SC(R⁷)₂COOH、NHCHR⁷COOH、COR⁸、CO₂R⁸Sulfate, sulfonamide, sulfoxide, sulfonate, sulfone, thioalkyl, thioester, and thioether are optionally substituted with at least one substituent;

R⁸selected from H, C_1-8Alkyl radical, C_1-8Alkenyl radical, C_1-8Alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, alkylheterocycle, and heteroaromatic, wherein each alkyl, alkenyl, alkynyl, aryl, carbocycle, heteroaryl, heterocycle, alkylaryl, alkylheteroaryl, alkyl heterocycle, and heteroaromatic is The heterocyclic and heteroaromatic may be optionally substituted with at least one substituent; and is

X is 0 to 4 substituents on the ring to which it is attached.

In certain embodiments, the RSL3 derivative or analog is a compound represented by structural formula (II):

or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; wherein:

R₁selected from the group consisting of: H. OH and- (OCH)₂CH₂)_xOH；

X is an integer from 1 to 6; and is

In certain embodiments, the RSL3 derivative or analog is a compound represented by structural formula (III):

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; wherein: n is 2, 3 or 4; and R is substituted or unsubstituted C₁-C₆Alkyl radical, substituted or unsubstituted C₃-C₁₀Cycloalkyl radical, substituted or unsubstituted C₂-C₈Heterocycloalkyl radical, substituted or unsubstituted C₆-C₁₀Aromatic ring group or substituted or unsubstituted C₃-C₈A heteroaryl ring group; wherein said substitution means one or more of each group The hydrogen atom is substituted with a group selected from the group consisting of: halogen, cyano, nitro, hydroxy, C₁-C₆Alkyl, halo C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halo C₁-C₆Alkoxy, COOH (carboxyl), COOC₁-C₆Alkyl, OCOC₁-C₆An alkyl group.

In certain embodiments, the statin is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, cerivastatin and simvastatin. In certain embodiments, the immune cell is a macrophage, monocyte, dendritic cell, T cell, CD4+ cell, CD8+ cell, or CD3+ cell. In certain embodiments, the immune cell is a THP-1 cell.

In certain embodiments, the method is performed in vitro or ex vivo. In certain embodiments, the method is performed in vivo. In certain embodiments, step (i) is performed in vitro, and step (ii) is performed in vivo.

In certain aspects, the disclosure relates to a method of increasing immune activity in a cell, tissue, or subject, the method comprising administering to the cell, tissue, or subject an agent that induces disassembly of iron-dependent cells in an amount sufficient to increase immune activity relative to a cell, tissue, or subject that has not been treated with an agent that induces disassembly of iron-dependent cells.

In one embodiment, the subject is in need of increased immune activity.

In one embodiment, the agent that induces iron-dependent cell disassembly is administered in an amount sufficient to increase one or more of the following in a cell, tissue, or subject: the level or activity of NFkB, the level or activity of IRF or STING, the level or activity of macrophage, the level or activity of monocyte, the level or activity of dendritic cell, the level or activity of T cell, the level or activity of CD4+, CD8+ or CD3+ cell, and the level or activity of pro-immune cytokine.

In certain aspects, the disclosure relates to a method of increasing the level or activity of NFkB in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cellular disassembly in an amount sufficient to increase the level or activity of NFkB relative to a cell, tissue or subject not treated with an agent that induces iron-dependent cellular disassembly.

In one embodiment, the subject is in need of increased NFkB level or activity.

In one embodiment, the level or activity of NFkB is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a cell, tissue, or subject not treated with an agent that induces iron-dependent cell disassembly.

In certain aspects, the disclosure relates to a method of increasing the level or activity of IRF or STING in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of IRF or STING relative to a cell, tissue or subject that is not treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the subject is in need of increased level or activity of IRF or STING.

In one embodiment, the level or activity of IRF or STING is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a cell, tissue, or subject that has not been treated with an agent that induces iron-dependent cell disassembly.

In certain aspects, the disclosure relates to a method of increasing the level or activity of macrophages, monocytes, dendritic cells or T cells in a tissue or subject, the method comprising administering to the tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of macrophages, monocytes, dendritic cells or T cells relative to a tissue or subject that is not treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the subject is in need of increased macrophage, monocyte, dendritic cell or T cell levels or activity.

In one embodiment, the level or activity of macrophages, monocytes, dendritic cells or T cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a tissue or subject not treated with an agent that induces iron-dependent cell disassembly.

In certain aspects, the disclosure relates to a method of increasing the level or activity of CD4+, CD8+, or CD3+ cells in a tissue or subject, the method comprising administering to the subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of CD4+, CD8+, or CD3+ cells relative to a tissue or subject not treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the subject is in need of increased CD4+, CD8+, or CD3+ cell levels or activity.

In one embodiment, the level or activity of CD4+, CD8+, or CD3+ cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a tissue or subject not treated with an agent that induces iron-dependent cell disassembly.

In certain aspects, the disclosure relates to a method of increasing the level or activity of a proammunocytokine in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cellular disassembly in an amount sufficient to increase the level or activity of the proammunocytokine relative to a cell, tissue or subject that has not been treated with an agent that induces iron-dependent cellular disassembly.

In one embodiment, the subject is in need of increased levels or activity of a proammunocytokine.

In one embodiment, the level or activity of the pro-immune cytokine is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a cell, tissue, or subject that has not been treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the immunocytokines are selected from IFN- α, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF- α, IL-17, and GMCSF.

In one embodiment, the method further comprises, prior to administration, assessing one or more of the following in the cell, tissue or subject: the level or activity of NFkB; the level or activity of macrophages; the level or activity of monocytes; the level or activity of dendritic cells; the level or activity of a CD4+ cell, CD8+ cell, or CD3+ cell; the level or activity of T cells; and the level or activity of a pro-immunocytokine.

In one embodiment, the method further comprises, after administration, assessing one or more of the following in the cell, tissue, or subject: the level or activity of NFkB; the level or activity of macrophages; the level or activity of monocytes; the level or activity of dendritic cells; the level or activity of a CD4+ cell, CD8+ cell, or CD3+ cell; the level or activity of T cells; and the level or activity of a pro-immunocytokine.

In embodiments, the immunocytokines are selected from IFN- α, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF- α, IL-17, and GMCSF.

In one embodiment, the subject has an infection.

In one embodiment, the infection is a chronic infection.

In embodiments, the chronic infection is selected from HIV infection, HCV infection, HBV infection, HPV infection, hepatitis b infection, hepatitis c infection, EBV infection, CMV infection, TB infection, and parasitic infection.

In one embodiment, the cell or tissue is a cancer cell or tissue.

In one embodiment, the subject has been diagnosed with cancer.

In certain aspects, the disclosure relates to methods of treating a subject in need of increased immune activity, the method comprising administering to the subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the immune activity of the subject.

In one embodiment, the subject has a chronic infection.

In one embodiment, the subject has cancer.

In embodiments, the cancer is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, non-squamous cell lung cancer, urothelial cancer, hodgkin's lymphoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, colorectal cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and Merkel (Merkel) cell carcinoma.

In one embodiment, the iron-dependent cell disassembly is iron death.

In certain aspects, the disclosure relates to a method of treating a subject diagnosed with cancer, the method comprising administering to the subject a combination of (a) an immunotherapy and (b) an agent that induces iron-dependent cell disassembly, thereby treating the cancer in the subject.

In one embodiment, the agent that induces iron-dependent cell disassembly is administered to the subject in an amount effective to increase the immune response of the subject.

In one embodiment, the immunotherapy is selected from the group consisting of: toll-like receptor (TLR) agonists, cell-based therapies, cytokines, cancer vaccines, and immune checkpoint modulators of immune checkpoint molecules.

In one embodiment, the TLR agonist is selected from the group consisting of Coley's toxin (Coley's toxin) and bacillus Calmette-guerin (BCG).

In one embodiment, the immune checkpoint molecule is selected from the group consisting of CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, ADORA2A, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, and VISTA.

In one embodiment, the immune checkpoint molecule is a stimulatory immune checkpoint molecule and the immune checkpoint modulator is an agonist of the stimulatory immune checkpoint molecule.

In one embodiment, the immune checkpoint molecule is an inhibitory immune checkpoint molecule and the immune checkpoint modulator is an antagonist of the inhibitory immune checkpoint molecule.

In one embodiment, the immune checkpoint modulator is selected from the group consisting of a small molecule, an inhibitory RNA, an antisense molecule, and an immune checkpoint molecule binding protein.

In one embodiment, the immune checkpoint molecule is PD-1 and the immune checkpoint modulator is a PD-1 inhibitor.

In one embodiment, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, pidilizumab, SHR-1210, MEDI0680R01, BBg-A317, TSR-042, REGN2810, and PF-06801591.

In one embodiment, the immune checkpoint molecule is PD-L1 and the immune checkpoint modulator is a PD-L1 inhibitor.

In one embodiment, the PD-L1 inhibitor is selected from the group consisting of Duvaluzumab, Attributumab, Ablutumab, MDX-1105, AMP-224, and LY 3300054.

In one embodiment, the immune checkpoint molecule is CTLA-4 and the immune checkpoint modulator is a CTLA-4 inhibitor.

In one embodiment, the CTLA-4 inhibitor is selected from epilimumab, tremelimumab, JMW-3B3, and age 1884.

In embodiments, the agent that induces iron-dependent cell disassembly is administered before, after, or simultaneously with the administration of the immune checkpoint modulator.

In one embodiment, the response of the cancer to the treatment is improved relative to treatment with the immune checkpoint modulator alone.

In embodiments, the response is improved by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% relative to treatment with the immune checkpoint modulator alone, e.g., in a population of subjects.

In one embodiment, the response includes any one or more of: a reduction in tumor burden, a reduction in tumor size, an inhibition of tumor growth, reaching stable cancer in a subject with a progressive cancer prior to treatment, an increase in time to progression of the cancer, and an increase in survival time.

In one embodiment, the agent that induces iron-dependent cell disassembly and the immune checkpoint modulator act synergistically.

In one embodiment, the cancer is a cancer responsive to immune checkpoint therapy.

In embodiments, the cancer is selected from the group consisting of an epithelial cancer, a sarcoma, a lymphoma, a melanoma, and a leukemia.

In various embodiments, the cancer is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, non-squamous cell lung cancer, urothelial cancer, hodgkin's lymphoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, colorectal cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and merkel cell carcinoma.

In particular embodiments, the cancer is renal cell carcinoma.

In one embodiment, the subject is a human.

In one embodiment, the agent that induces iron-dependent cell disassembly is selected from the group consisting of: antiporter system Xc^-An inhibitor of GPX4, and a statin.

In one embodiment, antiport system Xc^-The inhibitor of (a) is elastine or a derivative or analogue thereof.

In one embodiment, the analogue of elapsin is PE or IKE.

In one embodiment, the inhibitor of GPX4 is selected from the group consisting of: (1S,3R) -RSL3 or a derivative or analog thereof, ML162, DPI compound 7, DPI compound 10, DPI compound 12, DPI compound 13, DPI compound 17, DPI compound 18, DPI compound 19, FIN56, and FINO 2.

In one embodiment, the statin is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, cerivastatin and simvastatin.

In one embodiment, the agent that induces iron-dependent cell disassembly is selected from the group consisting of: sorafenib or a derivative or analogue thereof, sulfasalazine, glutamate, BSO, DPI2, cisplatin, cysteinase, silica-based nanoparticles, CCI4, ferric ammonium citrate, trigonelline, and brucea javanica alcohol.

In one embodiment, the agent that induces iron-dependent cell disassembly has one or more of the following characteristics:

(a) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of an immune response in co-cultured cells;

(b) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured macrophages, such as RAW264.7 macrophages;

(c) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured monocytes, e.g. THP-1 monocytes;

(d) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured bone marrow-derived dendritic cells (BMDCs);

(e) Inducing in vitro iron-dependent cell disassembly of target cells and an increase in the level or activity of NFkB, IRF and/or STING in subsequently co-cultured cells;

(f) inducing in vitro iron-dependent cell disassembly of target cells and subsequent increase in the level or activity of a proammunocytokine in the co-cultured cells; and

(g) inducing in vitro iron-dependent cell disassembly of target cells and subsequent activation of co-cultured CD4+, CD8+ and/or CD3+ cells;

in embodiments, the agent that induces iron-dependent cell disassembly targets cancer cells.

In certain aspects, the disclosure relates to methods of screening for an immunostimulatory agent, the method comprising:

(a) providing a plurality of test agents (e.g., a library of test agents);

(b) assessing the ability of each of the plurality of test agents to induce iron-dependent cell disassembly;

(d) Assessing the ability of the candidate immunostimulant to stimulate an immune response.

In one embodiment, the evaluating step (b) comprises contacting the cell or tissue with each of the plurality of test agents.

In one embodiment, the evaluating step (b) comprises administering each of the plurality of test agents to the animal.

In one embodiment, assessing step (b) further comprises measuring the level or activity of a marker selected from the group consisting of: lipid peroxidation, Reactive Oxygen Species (ROS), isoprostane (isoprostane), Malondialdehyde (MDA), iron, glutathione peroxidase 4(GPX4), prostaglandin-endoperoxide synthase 2(PTGS2), cyclooxygenase 2(COX-2), and Glutathione (GSH).

In one embodiment, the evaluating step (b) further comprises comparing the level or activity of the marker in the cell or tissue contacted with the test agent to the level or activity of the marker in a control cell or tissue not contacted with the test agent.

In one embodiment, the assessing step (d) comprises assessing the immunostimulatory activity of a test agent that induces iron-dependent cell disassembly.

In one embodiment, the evaluating step (d) comprises measuring an immune response in the animal.

In one embodiment, an increase in the level or activity of a marker selected from the group consisting of lipid peroxidation, isoprostane, Reactive Oxygen Species (ROS), iron, PTGS2, and COX-2, or a decrease in the level or activity of a marker selected from the group consisting of GPX4, MDA, and GSH, indicates that the test agent is an agent that induces iron-dependent cell disassembly.

In one embodiment, evaluating the candidate immunostimulant comprises culturing the immune cell with a cell contacted with the selected candidate immunostimulant or exposing the immune cell to a post-cellular signaling factor produced by a cell contacted with the selected candidate immunostimulant, and measuring the level or activity of NF κ B, IRF or STING in the immune cell.

In one embodiment, the immune cell is a THP-1 cell.

In one embodiment, evaluating the candidate immunostimulant comprises culturing T cells with cells contacted with the selected candidate immunostimulant or exposing T cells to post-cellular signaling factors produced by cells contacted with the selected candidate immunostimulant, and measuring activation and proliferation of the T cells.

In certain aspects, the disclosure relates to methods of identifying an immunostimulatory agent, the method comprising:

(a) contacting a cell with an agent that induces iron-dependent cell disassembly in an amount sufficient to induce iron-dependent cell disassembly in the cell;

(b) isolating one or more post-cellular signaling factors produced by the cells following contact with the agent that induces iron-dependent cell disassembly; and is

In one embodiment, the method further comprises selecting a test agent that stimulates an immune response.

In one embodiment, the method further comprises detecting a marker of iron-dependent cell disassembly in the cell.

In one embodiment, the method further comprises:

i) measuring the level of one or more post-cellular signaling factors produced by the cells following contact with the agent that induces iron-dependent cell disassembly;

In one example, control cells are treated with an agent that induces cell death (which is not iron-dependent cell disassembly).

In one embodiment, the assay comprises administering one or more cell signaling factors to the animal and measuring an immune response in the animal.

In one embodiment, the assay comprises treating the immune cells with one or more post-cellular signaling factors and measuring the level or activity of nfkb activity in the immune cells.

In one embodiment, the assay comprises treating T cells with one or more post-cellular signaling factors and measuring the activation or proliferation of the T cells.

In one embodiment, the assay comprises contacting an immune cell with one or more post-cellular signaling factors and measuring the level or activity of nfk B, IRF or STING in the immune cell.

In one embodiment, the immune cell is a THP-1 cell.

Drawings

FIG. 1A shows HT1080 fibrosarcoma cells treated with various concentrations of elastin. Figure 1B shows NFkB activity in THP1 monocytes co-cultured with HT1080 cells treated with elastin. Error bars indicate the standard deviation between the three replicates.

Figure 1C shows HT1080 fibrosarcoma cells treated with DMSO or various concentrations of Elastine (ERAS) or elastine analogs Piperazine Elastine (PE) or imidazolone elastine (IKE). The DMSO control is located at the far left. The concentrations of elastin or elastin analogs increase from left to right and are the same as those shown in fig. 1A.

Figure 1D shows NFkB activity in THP1 monocytes co-cultured with HT1080 cells treated with Elastine (ERAS) or elastine analogs Piperazine Elastine (PE) or imidazolone elastine (IKE). The DMSO control is located at the far left. The concentration of elastin or elastin analogs increases from left to right and is the same as shown in fig. 1B. Error bars indicate the standard deviation between the three replicates.

Fig. 2A shows pancreatic cancer cells (PANC1) treated with various concentrations of elastin. Figure 2B shows NFkB activity in THP1 monocytes co-cultured with PANC1 cells treated with elastin.

FIG. 3A shows renal cell carcinoma cells (Caki-1) treated with various concentrations of Alascetin. FIG. 3B shows NFkB activity in THP1 monocytes co-cultured with Caki-1 cells treated with Alastin.

FIG. 4A shows renal cell carcinoma cells (Caki-1) treated with various concentrations of RSL 3. FIG. 4B shows NFkB activity in THP1 monocytes co-cultured with Caki-1 cells treated with RSL 3.

Fig. 5A shows Jurkat T cell leukemia cells treated with various concentrations of RSL 3. Figure 5B shows NFkB activity in THP1 monocytes co-cultured with Jurkat cells treated with RSL 3.

Fig. 6A shows a 20B cell leukemia cells treated with various concentrations of RSL 3. Figure 6B shows NFkB activity in THP1 monocytes co-cultured with a20 cells treated with RSL 3. Figure 6C shows IRF activity in THP1 monocytes co-cultured with a20 cells treated with RSL 3.

Figure 7A shows the viability of HT1080 fibrosarcoma cells treated with various concentrations of elastin alone or in combination with iron death inhibitors (iron-based statins (Ferrostatin) -1, lipstatin (Liproxstatin) -1 or Trolox).

Figure 7B shows NFkB activity in THP1 monocytes co-cultured with HT1080 fibrosarcoma cells treated with elastin alone or in combination with iron death inhibitors (iron-based statin-1, lipstatin-1 or Trolox).

Figure 8A shows the viability of HT1080 fibrosarcoma cells treated with various concentrations of elastin alone or in combination with iron death inhibitors (iron-based statin-1, β -mercaptoethanol or deferoxamine).

Figure 8B shows NFkB activity in THP1 monocytes co-cultured with HT1080 fibrosarcoma cells treated with elastin alone or in combination with iron death inhibitors (iron-based statin-1, β -mercaptoethanol or deferoxamine).

Figure 9A shows the viability of HT1080 fibrosarcoma cells treated with various concentrations of elastin in combination with siRNA control (si control) or siRNA against the ACSL4 gene (siACSL 4).

Figure 9B shows the viability of H1080 fibrosarcoma cells treated with DMSO or elastin in combination with siRNA control (si control), siRNA against the ACSL4 gene (siACSL4), or siRNA against the CARS gene (sicars).

Figure 9C shows fold change in NFkB activity in THP1 monocytes co-cultured with HT1080 fibrosarcoma cells treated with DMSO or elastin in combination with siRNA control (si control), siRNA against ACSL4 gene (siACSL4) or siRNA against CARS gene (sicars).

Figure 10A shows the viability of a20 lymphoma cells treated with DMSO or various concentrations of RSL3 alone or in combination with iron-based statin-1.

Figure 10B shows NFkB activity in THP1 monocytes co-cultured with a20 lymphoma cells treated with DMSO or various concentrations of RSL3 alone or in combination with iron-based statin-1.

Figure 11A shows viability of a20 lymphoma cells treated with DMSO or various concentrations of ML162 alone or in combination with iron-based statin-1.

Figure 11B shows NFkB activity in THP1 monocytes co-cultured with a20 lymphoma cells treated with DMSO or various concentrations of ML162 alone or in combination with iron-based statin-1.

Figure 12A shows viability of a20 lymphoma cells treated with DMSO or various concentrations of ML210 alone or in combination with iron-based statin-1.

Figure 12B shows NFkB activity in THP1 monocytes co-cultured with a20 lymphoma cells treated with DMSO or various concentrations of ML210 alone or in combination with iron-based statin-1.

FIG. 13A shows viability of Caki-1 renal cancer cells treated with DMSO or various concentrations of RSL3 alone or in combination with iron-based statin-1.

Figure 13B shows NFkB activity in THP1 monocytes co-cultured with Caki-1 renal cancer cells treated with DMSO or various concentrations of RSL3 alone or in combination with iron-based statin-1.

Fig. 14A shows viability of Caki-1 renal cancer cells treated with DMSO or various concentrations of ML162 alone or in combination with iron-based statin-1.

Figure 14B shows NFkB activity in THP1 monocytes co-cultured with THP1 monocytes treated with DMSO or ML162 at various concentrations alone or in combination with iron-based statin-1.

Detailed Description

The present disclosure relates to methods of increasing immune activity in a cell, tissue, or subject, comprising administering to the cell, tissue, or subject an agent that induces iron-dependent cell disassembly. Applicants have surprisingly shown that induction of iron-dependent cell disassembly (e.g., iron death) increases the immune response as evidenced by increased NFKB and IRF activity in immune cells. Thus, agents that induce iron-dependent cell disassembly may be administered to treat diseases that would benefit from increased immune activity, such as cancer or infection.

I. Definition of

The term "administering" includes any method of delivering a pharmaceutical composition or agent to the system of a subject or to a specific area within or on a subject.

As used herein, "combined administration," "co-administration," or "combination therapy" is understood to mean the administration of two or more active agents using separate formulations or a single pharmaceutical formulation, or sequential administration in any order, such that there are time periods when the two (or all) active agents overlap in exerting their biological activities. It is contemplated herein that one agent (e.g., an agent that induces iron-dependent cell disassembly) can improve the activity of a second agent, e.g., can sensitize a target cell, e.g., a cancer cell, to the activity of the second agent. "Combined administration" does not require that the agents be administered simultaneously, at the same frequency, or by the same route of administration. As used herein, "combined administration," "co-administration," or "combination therapy" includes administration of an agent that induces iron-dependent cell disassembly with one or more additional anti-cancer agents, such as immune checkpoint modulators. Examples of immune checkpoint modulators are provided herein.

As used herein, "iron death" refers to a cell death process that is subject to regulation, which is iron-dependent and involves the production of reactive oxygen species.

"cellular disassembly" refers to a dynamic process of rearranging and spreading the intracellular material and ultimately leading to cell death. The process of cell disassembly involves the production and release of cell signaling factors by the cells.

As used herein, the terms "increase" (or "activation") and "decrease" refer to modulation resulting in greater or lesser amounts, functions or activities, respectively, of a parameter relative to a reference. For example, after administration of a formulation described herein, a parameter (e.g., activation of NFkB, activation of macrophages, size or growth of a tumor) can be increased or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more in a subject relative to the amount of the parameter prior to administration. Typically, the post-administration indicator is measured at the time the administration has reached said effect, e.g. at least one day, one week, one month, 3 months, 6 months after the start of the treatment regimen. Similarly, the preclinical parameters (e.g., NFkB activation of cells in vitro and/or reduction in tumor burden in a test mammal by a formulation described herein) can be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more relative to the amount of the pre-administration parameters.

As used herein, "antineoplastic agent" refers to a drug used to treat cancer. Antineoplastic agents include chemotherapeutic agents (e.g., alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors corticosteroids, and enzymes), biological anticancer agents, and immune checkpoint modulators.

A "cancer treatment regimen" or "anti-tumor regimen" is a clinically acceptable dosing regimen for treating cancer that includes administering one or more anti-tumor agents to a subject in a specific amount on a specific schedule.

As used herein, an "immune checkpoint" or "immune checkpoint molecule" is a molecule that modulates a signal in the immune system. The immune checkpoint molecule may be a stimulatory checkpoint molecule, i.e. increasing the signal, or an inhibitory checkpoint molecule, i.e. decreasing the signal. As used herein, a "stimulatory checkpoint molecule" is a molecule in the immune system that increases signaling or is co-stimulatory. As used herein, an "inhibitory checkpoint molecule" is a molecule in the immune system that reduces signal or is co-inhibitory.

As used herein, an "immune checkpoint modulator" is an agent capable of altering the activity of an immune checkpoint in a subject. In certain embodiments, the immune checkpoint modulator alters the function of one or more immune checkpoint molecules (which include, but are not limited to, CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, ADORA2A, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, and VISTA). The immune checkpoint modulator may be an agonist or antagonist of an immune checkpoint. In some embodiments, the immune checkpoint modulator is an immune checkpoint binding protein (e.g., an antibody Fab fragment, a bivalent antibody, an antibody drug conjugate, an scFv, a fusion protein, a bivalent antibody, or a tetravalent antibody). In other embodiments, the immune checkpoint modulator is a small molecule. In particular embodiments, the immune checkpoint modulator is anti-PD 1, anti-PD-L1, or anti-CTLA-4 binding protein, e.g., an antibody or antibody fragment.

As used herein, "immunotherapy" refers to a pharmaceutically acceptable compound, composition or therapy that induces or enhances an immune response. Immunotherapy includes, but is not limited to, immune checkpoint modulators, Toll-like receptor (TLR) agonists, cell-based therapies, cytokines, and cancer vaccines.

As used herein, "neoplastic disorder" or "cancer" or "neoplasm" refers to all types of cancer or neoplasm found in humans, including but not limited to: leukemia, lymphoma, melanoma, epithelial carcinoma, and sarcoma. As used herein, the terms "neoplastic disorder," "cancer," and "neoplasm" are used interchangeably and refer in the singular or plural to a cell that has undergone malignant transformation that renders the cell pathological to a host organism. Primary cancer cells (i.e., cells obtained from the vicinity of the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also cancer stem cells, as well as cancer progenitor cells or any cell derived from a cancer cell progenitor. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells.

The particular criteria for cancer staging depend on the particular cancer type, based on tumor size, histological characteristics, tumor markers, and other criteria known to those skilled in the art. In general, the stage of cancer can be described as follows: (i) stage 0, carcinoma in situ; (ii) stage I, II and III, where higher numbers indicate more widespread disease, including larger tumor size and/or spread of the cancer beyond its original developing organ to nearby lymph nodes and/or tissues or organs adjacent to the primary tumor site; and (iii) stage IV, in which the cancer has spread to distant tissues or organs.

"post-cellular signaling factors" are molecules and cell fragments produced by cells undergoing cell disassembly (e.g., iron-dependent cell disassembly), which are ultimately released from the cell and affect the biological activity of other cells. Post-cellular signaling factors may include proteins, peptides, carbohydrates, lipids, nucleic acids, small molecules, and cell fragments (e.g., vesicles and cell membrane fragments).

A "solid tumor" is a tumor that is detectable from a tumor mass, e.g., by procedures such as CAT scanning, MR imaging, X-ray, ultrasound, or palpation, and/or is detectable due to expression of one or more cancer-specific antigens in a sample that may be obtained from a patient. The tumor need not have a measurable size.

A "subject" treated by the methods of the invention may refer to a human or non-human animal, preferably a mammal, more preferably a human. In certain embodiments, the subject has a detectable or diagnosed cancer prior to initiation of treatment using the methods of the invention. In certain embodiments, the subject has a detectable or diagnosed infection, e.g., a chronic infection, prior to initiation of treatment using the methods of the invention.

"therapeutically effective amount" refers to the amount of a compound that, when administered to a patient to treat a disease, is sufficient to effect such treatment for the disease. When administered for prophylaxis of a disease, the amount is sufficient to avoid or delay the onset of the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the patient to be treated. The therapeutically effective amount need not be curative. A therapeutically effective amount need not prevent the disease or condition from ever occurring. Conversely, a therapeutically effective amount is an amount that will at least delay or reduce the onset, severity, or progression of a disease or disorder.

As used herein, "treatment" and "treating" and their cognates refer to the medical management of a subject with the intent to ameliorate, improve, stabilize, prevent or cure a disease, pathological condition or disorder. The term includes active therapy (therapy directed to ameliorating a disease, pathological condition, or disorder), causal therapy (therapy directed to the cause of the associated disease, pathological condition, or disorder), palliative therapy (therapy directed to alleviating symptoms), prophylactic therapy (therapy directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive therapy (therapy to supplement another therapy).

Iron-dependent cell disassembly

Cell disassembly is a dynamic process that rearranges and distributes intracellular material and results in the production and release of post-cellular signaling factors or "effectors" that may have profound effects on the biological activity of other cells. Cell disassembly occurs during regulated cell death and is controlled by a variety of molecular mechanisms. The disassembly of different types of cells results in the production of different post-cellular signaling factors, thereby mediating different biological effects. For example, applicants have surprisingly shown that induction of iron-dependent cell disassembly can increase the immune response as evidenced by an increase in NFKB and IRF activity in immune cells.

In some embodiments, the iron-dependent cell disassembly is iron death. Iron death is a regulated cell death process that involves the production of iron-dependent Reactive Oxygen Species (ROS). In some embodiments, iron death involves iron-dependent accumulation of lipid hydroperoxides to lethal levels. The susceptibility to iron death is closely related to many biological processes, including amino acid, iron and polyunsaturated fatty acid metabolism, and biosynthesis of glutathione, phospholipids, NADPH and coenzyme Q10. Iron death involves metabolic dysfunction that results in the production of both cytosolic ROS and lipid ROS independent of mitochondria, but dependent on NADPH oxidase in certain cellular environments (Dixon et al, 2012, Cell [ Cell ]149(5): 1060-72).

Agent for inducing iron-dependent cell disassembly

Provided herein are agents that induce iron-dependent cell disassembly. Such agents are capable of inducing a process of iron-dependent cell disassembly when present in sufficient amounts and for sufficient time. In certain embodiments, the agent that induces iron-dependent cell disassembly induces a process of iron-dependent cell disassembly in a cell such that the cell produces a post-cellular signaling factor, e.g., a post-immunostimulatory cellular signaling factor, but does not result in cell death. In other embodiments, the agent that induces iron-dependent cell disassembly induces a process of iron-dependent cell disassembly in a portion of the cell population, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more cells of the population, such that the portion of cells in the cell population produce a post-cellular signaling factor, e.g., a post-immunostimulatory cellular signaling factor. Cell death may occur in all or only a portion of the portion of cells in the cell population.

A wide range of agents that induce iron-dependent cell disassembly (e.g., iron death) are known in the art and can be used in the various methods provided by the present invention. For example, two oncogenic RAS Selective Lethal (RSL) small molecules, named eradicants for RAS and ST (elastin) and RAS selective lethal 3(RSL3), were initially identified as small molecules with a selective lethal effect on cells expressing oncogenic mutant RAS proteins, a family of small gtpases that are commonly mutated in cancer. (see Cao et al, 2016, Cell Mol Life Sci [ Cell and molecular Life sciences ]73:2195-2209, incorporated herein in its entirety.) in particular, in engineered human fibroblast Cell lines, small molecules of elastin were found to induce preferential lethality in cells overexpressing oncogenic HRAS (see Dolma et al, 2003, Cancer cells [ Cancer Cell ]3:285-296, incorporated herein in its entirety). Elastin functionally inhibits the cystine-glutamate antiporter system Xc-. System Xc-is a heterodimeric cell surface amino acid antiporter consisting of: a twelve transmembrane transporter SLC7A11(xCT) linked by a disulfide bond to a single transmembrane regulator SLC3A2(4F2hc, CD98 hc). The antiport system Xc-introduces extracellular cystine (the oxidized form of cysteine) which exchanges intracellular glutamate. (see Cao et al, 2016, Cell Mol Life Sci 73:2195-2209, incorporated herein in its entirety.) cells treated with elastin are deprived of cysteine and are unable to synthesize the antioxidant glutathione. Depletion of glutathione eventually leads to excessive lipid peroxidation and ROS elevation, which triggers iron-dependent cell disassembly. Based on morphological, biochemical and genetic criteria, elastin-induced iron-dead cell death is distinct from apoptosis, necrosis and autophagy. (see Yang et al, 2014, Cell 156:317-

In some embodiments, the agent that induces iron-dependent cell disassembly, e.g., iron death, and is useful in the methods provided herein is an inhibitor of the antiporter system Xc-. Inhibitors of the antiporter system Xc-include antiporter system Xc-binding proteins (e.g.antibodies or antibody fragments), nucleic acid inhibitors (e.g.antisense oligonucleotides or siRNAs) and small molecules which specifically inhibit the antiporter system Xc-. For example, in some embodiments, the inhibitor of the antiporter system Xc "is a binding protein (e.g., an antibody or antibody fragment) that specifically inhibits SLC7a11 or SLC3a 2. In some embodiments, the inhibitor of the antiporter system Xc "is a nucleic acid inhibitor that specifically inhibits SLC7a11 or SLC3a 2. In some embodiments, the inhibitor of the antiporter system Xc "is a small molecule that specifically inhibits SLC7a11 or SLC3a 2. Antibodies and nucleic acid inhibitors are well known in the art and are described in detail herein. Small molecule inhibitors of the antiporter system Xc-include, but are not limited to, elastine, sulfasalazine, sorafenib and analogs or derivatives thereof. (see Cao et al, 2016, Cell Mol Life Sci [ Cell and molecular Life sciences ]73:2195-2209, e.g., FIG. 2, incorporated herein in its entirety).

In a particular embodiment, the agent that induces iron-dependent cell disassembly, e.g., iron death, is elastine or an analog or derivative thereof. Analogs of elastin include, but are not limited to, the compounds listed in table 1 below. Each of the references listed in table 1 is incorporated by reference herein in its entirety.

TABLE 1 Alastatin analogs

As used herein, unless otherwise specified, the term "elapsin" includes any pharmaceutically acceptable form of elapsin, including but not limited to N-oxides, crystalline forms, hydrates, salts, esters and prodrugs thereof. As used herein, the term "elafin derivative or elafin analog" refers to a compound having a similar structure and function to elafin. In some embodiments, the elafin derivative/elafin analog includes those having the formula:

or a pharmaceutically acceptable salt or ester thereof, wherein

R₁Selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, hydroxy and halogen;

R₂selected from the group consisting of: H. halo and C_1-4An alkyl group;

R₃selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, 5-7 membered heterocycloalkyl, and 5-6 membered heteroaryl;

R₄Selected from the group consisting of: h and C_1-4An alkyl group;

R₅is halo;

optionally substituted with ═ O; and is

n is an integer of 0 to 4.

In one embodiment, the elastin derivative or analog is a compound represented by structural formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

ra is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl-O-, substituted or unsubstituted alkyl-O-, substituted or unsubstituted alkenyl-O-, or substituted or unsubstituted alkynyl-O-, wherein alkyl, alkenyl and alkynyl are optionally substituted by NR, O or S (O)_nInterrupting;

each R₂Independently selected from the group consisting ofThe following components: halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted non-aromatic heterocycle, -CN, -COOR', -CON (R)₂、-NRC(O)R、-SO₂N(R)₂、-N(R)₂-NO2, -OH and-OR';

each R₃Independently selected from the group consisting of: halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted non-aromatic heterocycle, - (CO) R, -CN, -COOR', -CON (R) ₂、-NRC(O)R、-SO₂N(R)₂、-N(R)₂-NO2, -OH and-OR';

R₄and R₅Independently selected from the group consisting of: -H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted non-aromatic heterocycle and substituted or unsubstituted aryl, wherein alkyl, alkenyl and alkynyl are optionally substituted with NR, O or S (O)_nInterrupting; or R₄And R₅Together form a carbocyclic or heterocyclic group;

v is-NH-L-A-Q or

Wherein ring C is a substituted or unsubstituted heterocyclic aromatic or non-aromatic ring;

a is NR or O; or A is a covalent bond;

l is a substituted or unsubstituted hydrocarbyl group optionally interrupted by one or more heteroatoms selected from N, O and S;

q is selected from the group consisting of: -R, - - - - -C (O) R', - -C (O) N (R)₂-C (O) OR', and-S (O)₂R’；

Each R is independently-H, alkyl, alkenyl, alkynyl, aryl or non-aromatic heterocycle, wherein the alkyl, alkenyl, alkynyl, aryl or non-aromatic heterocycle group is substituted or unsubstituted;

each R' is independently an alkyl, alkenyl, alkynyl group, non-aromatic heterocyclic group, or aryl group, wherein the alkyl, alkenyl, alkynyl, non-aromatic heterocyclic group, or aryl group is substituted or unsubstituted;

j is an integer from 0 to 4;

k is an integer from 0 to 4, provided that at least one of j and k is an integer from 1 to 4;

and each n is independently 0, 1 or 2.

In another embodiment, the elastin derivative is a compound represented by structural formula (I) disclosed in the above embodiments; wherein V is

Suitable examples of V encompassed by the above structure include:

and wherein all other variables are as disclosed in the above examples.

In one embodiment, the elastin derivative or analog is a compound represented by structural formula (II):

wherein R is₁Selected from the group consisting of: H. c_1-6Alkyl and CF₃Wherein each C_1-6The alkyl group may be optionally substituted with an atom or group selected from the group consisting of: halogen atom, saturated or unsaturated C_3-6-heterocycles and amines, each heterocycle being optionally selected from the group consisting of C_1-4Aliphatic, said C_1-4Aliphatic may optionally be via C_1-4alkyl-aryl-O-C_1-4Alkyl substitution;

R₂selected from the group consisting of: H. halo and C_1-6Aliphatic; and is

R₃Is a halogen atom;

or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

In another embodiment, the elafin derivative or analog is a compound represented by structural formula (III):

Wherein

R₁Selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy, hydroxy and halogen;

R₂selected from the group consisting of: H. c_1-4Alkyl radical, C_1-4Alkoxy radical, C_3-8Cycloalkyl radical, C_3-8Heterocycloalkyl, aryl, heteroaryl and C_1-4Aralkyl group;

R₃absent, or selected from the group consisting of: c_1-4Alkyl radical, C_1-4Alkoxy, carbonyl, C_3-8Cycloalkyl and C_3-8A heterocycloalkyl group;

x is selected from the group consisting of: C. n and O; and is

n is an integer of 0 to 6,

provided that when X is C, n is 0 and R₃Is absent when R₂Is CH₃When R is₁Cannot be H;

or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

In a particular embodiment, the elastin derivative is a compound represented by structural formula (IV):

or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof,

wherein all variables are as defined in the above examples disclosing compounds of formula (III).

In another embodiment, the elastin derivative or analog is a compound represented by structural formula (V):

or a pharmaceutically acceptable salt thereof,

wherein R is¹Selected from H, -Z-Q-Z, -C_1-8alkyl-N (R)²)(R⁴)、-C_1-8alkyl-OR³3-to 8-membered carbocyclic or heterocyclic ring, aryl, heteroaryl, and C _1-4Aralkyl group;

for each occurrence, R²And R⁴Each independently selected from H, C_1-4Alkyl radical, C_1-4Aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl and arylsulfonyl groups, with the proviso that when R is²And R⁴When both are on the same N atom and are not both H, they are different, and when R is²And R⁴Both on the same N and R²Or R⁴When it is acyl, alkylsulfonyl or arylsulfonyl, the other is selected from H, C_1-8Alkyl radical, C_1-4Aralkyl, aryl and heteroaryl;

R³selected from H, C_1-4Alkyl radical, C_1-4Aralkyl, aryl and heteroaryl;

w is selected from

Q is selected from O and NR²(ii) a And is

For each occurrence, Z is independently selected from C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl. When Z is alkenyl orAlkynyl groups, one or more double or triple bonds are preferably not at the end of the group (thus excluding, for example, enol ethers, alkynol ethers, enamines and/or alkynylamines).

In particular embodiments, the compounds are represented by structural formula (V) of the embodiments disclosed above; wherein

For each occurrence, R²And R⁴Each independently selected from H, C_1-4Alkyl radical, C_1-4Aralkyl, aryl, heteroaryl, acyl, alkylsulfonyl and arylsulfonyl groups, with the proviso that when R is²And R⁴They are different when both are on the same N atom and not both H;

R³Selected from H, C_1-4Alkyl, aryl and heteroaryl;

for each occurrence, Z is independently selected from C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6An alkynyl group;

wherein each heterocyclic group is a 3 to 10 membered non-aromatic ring including one to four heteroatoms selected from nitrogen, oxygen and sulfur;

wherein each aryl group is phenyl;

wherein each heteroaryl is a 5-to 7-membered aromatic ring comprising one to four heteroatoms selected from nitrogen, oxygen, and sulfur; and is

Wherein each heterocyclic, aryl and heteroaryl group is optionally substituted with one or more moieties selected from the group consisting of: halogen, hydroxyl, carboxyl, alkoxycarbonyl, formyl, acyl, thioester, thioacetate, thiocarbamate, alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidino, imino, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, and sulfonamide.

The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbon atoms of the backbone. Substituents may include, for example, halogen, hydroxyl, carbonyl (e.g., carboxy, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thiocarbamate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. The skilled person will appreciate that the moiety substituted on the hydrocarbon chain may itself be substituted, if appropriate.

In particular embodiments, antiport system Xc^-The inhibitor is

Or a pharmaceutically acceptable salt thereof.

In particular embodiments, antiport system Xc^-The inhibitor is

Or a pharmaceutically acceptable salt thereof.

In particular embodiments, antiport system Xc^-The inhibitor is

Or a pharmaceutically acceptable salt thereof.

Further derivatives or analogues of elastin are described in, for example, WO 2015/109009, US 9695133, US 8535897, WO 2015/051149, US 2008/0299076, US2007/0161644, WO 2008/013987, US 8575143, US 8518959, WO2007/076085, Bioorganic & Medicinal Chemistry Letters [ Rapid biochemicals and Medicinal Chemistry ] (2015),25(21), 4787. minus 4792, eLife [ electronic Life ] (2014),3, Letters in Organic Chemistry [ Rapid Organic Chemistry ] (2015),12(6), 393, Pharmacia Letters [ quick pharmaceutical Letters ] (2012),4(5), 1344. minus 1351, PLoS Pathologens [ Pathogens of Medinics et al ] (2014),10(6), Bioorganic & icying Chemistry Letters [ biochemicals Letters ] (5239), 2011. Biochemical & Medicinal Chemistry Letters (5239, 5243), including pharmaceutical Chemistry [ Indian Journal of Chemistry, Section B: Organic Chemistry incorporated Medicinal Chemistry ] (2010),49B (7), 923-: organic Chemistry, Including Pharmaceutical Chemistry [ Indian Journal of Chemistry ] (1994),33B (3),260-5, Journal of Heterocyclic Chemistry [ Journal of Heterocyclic Chemistry ] (1983),20(5),1339-49, Chemical & Pharmaceutical Bulletin [ Chemical and Pharmaceutical Bulletin ] (1979),27(11),2675-87, Journal of Pharmaceutical Chemistry [ Journal of Pharmaceutical Chemistry ] (1977),20(3),379-86, Indian Journal of Chemistry [ Journal of Indian Chemistry ] (1971),9(3),201-6, and Journal of Pharmaceutical Chemistry ] (8), 11 (1962), 392-5, each of which is incorporated herein by reference in its entirety.

In some embodiments, the agent that induces iron-dependent cell disassembly (e.g., iron death) and is useful in the methods provided herein is an inhibitor of glutathione peroxidase 4(GPX 4). GPX4 is a phospholipid hydroperoxide enzyme that catalyzes the reduction of hydrogen peroxide and organic peroxides, thereby protecting cells from membrane lipid peroxidation or oxidative stress. Thus, GPX4 contributes to the ability of cells to survive in an oxidative environment. Inhibition of GPX4 can induce Cell death due to iron death (see Yang, W.S., et al Regulation of iron-dead cancer Cell death by GPX4[ GPX4 cells ] Cell [ Cell ]156, 317-. Inhibitors of GPX4 include GPX4 binding proteins (e.g., antibodies or antibody fragments), nucleic acid inhibitors (e.g., antisense oligonucleotides or sirnas), and small molecules that specifically inhibit GPX 4. Small molecule inhibitors of GPX4 include, but are not limited to, the compounds listed in table 2 below. Each of the references listed in table 2 is incorporated by reference herein in its entirety.

TABLE 2 inhibitors of GPX4

In particular embodiments, the GPX4 inhibitor is

Or a pharmaceutically acceptable salt thereof.

RSL3 is a known inhibitor of GPX 4. In knock-down studies, RSL3 selectively mediated death of RAS-expressing cells and was identified as an increase in lipid ROS accumulation. See U.S. patent No. 8,546,421.

In some embodiments, the inhibitor of GPX4 is a diastereomer of RSL 3.

In a particular embodiment, the diastereomer of RSL3 is

Or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the diastereomer of RSL3 is

Or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the diastereomer of RSL3 is

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of GPX4 is a pharmaceutically acceptable form of RSL3, including but not limited to N-oxides, crystalline forms, hydrates, salts, esters, and prodrugs thereof.

In some embodiments, the inhibitor of GPX4 is RSL3 or a derivative or analog thereof. Derivatives and analogs of RSL3 are known in the art and are described, for example, in WO2008/103470, WO 2017/120445, WO 2018118711, US 8546421, and CN108409737, each of which is incorporated herein by reference in its entirety.

In some embodiments, the RSL3 derivative or analog is a compound represented by structural formula (I):

or an enantiomer, optical isomer, diastereoisomer, N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof, wherein

X is 0 to 4 substituents on the ring to which it is attached.

In one embodiment, the RSL3 derivative or analog is a compound represented by structural formula (II):

or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; wherein:

R₁selected from the group consisting of: H. OH and- (OCH)₂CH₂)_xOH；

X is an integer from 1 to 6; and is

R₂、R₂'、R₃And R₃' is independently selected from the group consisting of: H. c_3-8Cycloalkyl and combinations thereof, or R₂And R₂' may be joined together to form a pyridyl or pyranyl group, and R₃And R₃' mayTo join together to form a pyridyl or pyranyl group.

In one embodiment, the RSL3 derivative or analog is a compound represented by structural formula (III):

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; wherein: n is 2, 3 or 4; and R is substituted or unsubstituted C ₁-C₆Alkyl radical, substituted or unsubstituted C₃-C₁₀Cycloalkyl radical, substituted or unsubstituted C₂-C₈Heterocycloalkyl radical, substituted or unsubstituted C₆-C₁₀Aromatic ring group or substituted or unsubstituted C₃-C₈A heteroaryl ring group; wherein said substitution means that one or more hydrogen atoms in each group are substituted by a group selected from the group consisting of: halogen, cyano, nitro, hydroxy, C₁-C₆Alkyl, halo C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halo C₁-C₆Alkoxy, COOH (carboxyl), COOC₁-C₆Alkyl, OCOC₁-C₆An alkyl group.

In some embodiments, the GPX4 inhibitor is

Or a pharmaceutically acceptable salt thereof.

ML162 has been identified as a direct inhibitor of GPX4, which induces iron death (see Dixon et al, 2015, ACS chem.bio. [ ACS chemical biology ]10, 1604-.

In some embodiments, the GPX4 inhibitor is a pharmaceutically acceptable form of ML162, including but not limited to N-oxides, crystalline forms, hydrates, salts, esters, and prodrugs thereof.

In some embodiments, the inhibitor of GPX4 is ML162 or a derivative or analog thereof.

In some embodiments, the GPX4 inhibitor is

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the GPX4 inhibitor is a pharmaceutically acceptable form of ML210, including but not limited to N-oxides, crystalline forms, hydrates, salts, esters, and prodrugs thereof.

In some embodiments, the inhibitor of GPX4 is ML210 or a derivative or analog thereof.

In some embodiments, the inhibitor of GPX4 is FIN56 or a derivative or analog thereof.

In one embodiment, FIN56 or a derivative or analog thereof is represented by the formula:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, geometric isomer, solvate or prodrug thereof, wherein

n-0-2, and wherein when n-1, X is selected from CH₂、O、NR_ACO and C ═ NOR_AAnd wherein when n is 2, X is CH₂，

Y is O, S, NOR_AOr NR_A，

Wherein R is_ASelected from H, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, -C (═ O) R_B、-C(＝O)OR_B、-C(＝O)NR_BR_C、-C(＝NR_B)R_C、-NR_BRc, heterocycloalkyl, aryl or polyaromatic, heteroaryl, arylalkyl and alkylaryl,

wherein said R_BAnd Rc are each independently H, alkyl or heteroalkyl,

u and V are each independently selected from C ═ O and O ═ S ═ O, and wherein when U is C ═ O, V is not C ═ O,

R₁、R₂、R₃and R₄Each independently selected from H, alkyl, heteroalkyl, cycloalkyl, arylcycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl and said NR ₁R₂And NR₃R₄Each of which may be independently combined to form a heterocycloalkyl group,

R₅and R₆Each independently selected from H, OH, SH, alkoxy, thioalkoxy, alkyl, halogen, CN, CF₃、NO₂、COOR_D、CONR_DR_E、NR_DR_E、NR_DCOR_E、NR_DSO₂R_EAnd NR_FCONR_DR_E；

Wherein RD, RE and RF are independently H, alkyl, heteroalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl or heterocycloalkyl;

provided that NR is obtained if X is O, Y is O, and U and V are both O ═ S ═ O₁R₂And NR₃R₄Not the same, then R₁And R₃Each independently selected from H and lower alkyl, and wherein R is₂And R₄Each independently selected from lower alkoxy (lower alkyl), di (lower) alkylamino (lower) alkyl, halobenzyl, morpholino (lower) alkyl, or NR₁R₂And NR₃R₄Independently is piperidino, morpholino, piperazino, N-phenylpiperazino, ethylamino or substituted glycine,

and wherein if X is (CH)₂)2, Y is O or NOH, and U and V are each O ═ S ═ O, then R₁、R₂、R₃And R₄Are not all methyl, but are not methyl,

and wherein if n ═ O, Y is O or NOH, and U and V are each O ═ S ═ O, NR is₁R₂And NR₃R₄Is not identical and R₁、R₂、R₃And R₄Each independently selected from C₁-C₅Alkyl radical, C₁Oalkyl, Ci beta-alkyl, C₁₇Alkyl, phenyl, benzyl, naphthyl, piperazino, pyridinyl, pyrazolyl, benzimidazolyl, triazolyl; or NR ₁R₂And NR₃R₄Independently is piperidino, morpholino, or piperazino,

and wherein if X is CO, Y is O, and U and V are each O ═ S ═ O, NR is₁R₂And NR₃R₄Is different, and wherein R₁、R₂、R₃And R₄Each independently selected from methyl, ethyl, hydroxy-d-Cr alkyl, SH, RO, COOH, SO, NH₂And phenyl, or NR which is not identical therein₁R₂And NR₃R₄One or both of which is unsubstituted piperidino, N-methylpiperazino or N-methylpiperazino,

and wherein when X is C ═ O or C ═ NOH, Y is O or NOH, and U and V are each O ═ S ═ O, and R is₁Or R₂One of and R₃Or R₄When one is phenyl, then R₁Or R₂And R₃Or R₄Is not H or alkyl.

In one embodiment, the FIN56 derivative or analog thereof is represented by the formula:

wherein n is 1-2, and when n is 1, X is selected from CH₂O, CO, and C ═ NOR_A(ii) a And wherein when n is 2, X is CH₂，

Y is O, S, NOR_AOr NR_A，

Wherein U and V are each O ═ S ═ O,

wherein R is_ASelected from H, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl,

—C(＝O)RB、—C(＝O)OR_B、—C(＝O)NR_BR_C、—C(＝NR_B)R_C、—NR_BR_CHeterocycloalkyl, aryl or polyaromatic, heteroaryl, arylalkyl and alkylaryl,

wherein said R_BAnd R_CEach of which is independently H, alkyl or heteroalkyl,

R₁、R₂、R₃And R₄Each independently selected from H, alkyl, heteroalkyl, cycloalkyl, arylcycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl and said NR₁R₂And NR₃R₄Each of which may be independently combined to form a heterocycloalkyl group,

R₅and R₆Each independently selected from H, OH, SH, alkoxy, thioalkoxy, alkyl, halogen, CN, CF₃、NO₂、COOR_D、CONR_DR_E、NR_DR_E、NR_DCOR_E、NR_DSO₂R_EAnd NR_FCONR_DR_E；

Wherein R is_D、R_EAnd R_FIndependently is H, alkyl, heteroalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, or heterocycloalkyl; provided that X is O, Y is O, and U and V are both O ═ S ═ O, then NR₁R₂And NR₃R₄Not the same, then R₁And R₃Each independently selected from H and lower alkyl, and wherein R is₂And R₄Each independently selected from lower alkoxy (lower alkyl), di (lower) alkylamino (lower) alkyl, halobenzyl, morpholino (lower) alkyl, or NR₁R₂And NR₃R₄Independently is piperidino, morpholino, piperazino, N-phenylpiperazino, ethylamino or substituted glycine,

and wherein if X is (CH)₂)₂And Y is O or NOH, then R₁、R₂、R₃And R₄Are not a methyl group, either,

and wherein NR if X is CO and Y is O₁R₂And NR₃R₄Is different, and wherein R₁、R₂、R₃And R₄Each independently selected from methyl, ethyl, hydroxy-C ₁-C₃Alkyl, SH, RO, COOH, SO, NH₂And phenyl, or NR which is not identical therein₁R₂And NR₃R₄One or both of which are unsubstituted piperidino, N-methylpiperazino or N-methylpiperazino, wherein the unsubstituted piperidine, N-methylpiperazino or N-methylpiperazino NR₁R₂And NR₃R₄The parts are not the same as each other,

and wherein when X is C ═ O or C ═ NOH, Y is O or NOH, and R₁Or R₂One of and R₃Or R₄When one is phenyl, then R1 or R₂And R₃Or R₄Is not H or an alkyl group,

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

In one embodiment, the FIN56 derivative or analog thereof is represented by the formula:

wherein R is_AIs hydrogen, R₇And R₈Independently selected from H and SO₂NR₃R₄Wherein R is₇And R₈One of which is hydrogen, and wherein NR is₁R₂And NR₃R₄Independently a 6 to 15 membered heterocycloalkyl containing one nitrogen in the ring, or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

Additional FIN56 derivatives or analogs are described, for example, in WO 2008/140792, WO2010/082912, WO 2017/058716, US 6693136, Nature Chemical Biology [ Nature Chemical Biology ] (2016),12(7),497-503doi:10.1038/nchembio.2079, ACS Chemical Biology [ ACS Chemical Biology ] (2015),10(7),1604-1609doi:10.1021/acschembio.5b00245, discovery Abstract International [ International Abstract ], (2015) volume 76, 8B (E) phase order number AAI3688566.Proquest publications & Theses.

In some embodiments, the agent that induces iron-dependent cell disassembly (e.g., iron death) and is useful in the methods provided herein is a statin. In one embodiment, the statin is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, cerivastatin and simvastatin.

In one embodiment, the agent that induces iron-dependent cell disassembly (e.g., iron death) and is useful in the methods provided herein is selected from the group consisting of: glutamate, BSO, DPI2 (see Yang et al, 2014, Cell [ Cell ]156: 317-; FIGS. 5 and S5, incorporated herein in their entirety), cisplatin, cysteinase, silica-based nanoparticles, CCI4, ferric ammonium citrate, trigonelline, and brucellol.

Additional agents that induce iron-dependent Cell disassembly are known in the art and described, for example, in US 8518959, US 8535897, US 8546421, US 9580398, US 9695133, US 2010/0081654, US 2015/0079035, US 2015/0175558, US 2016/0229836, US 2016/0297748, US 2016/0332974, Cell [ cells ]. 5/25 of 2012; 149(5) 1060-72.doi 10.1016/j. cell.2012.03.042;

Cell [ Cell ]2014 1 month 16 days; 156(1-2) 317-331.doi: 10.1016/j.cell.2013.12.010;

j Am Chem Soc. [ journal of american chemical society ] 26 months 3 2014; 136(12) 4551-6.doi 10.1021/ja411006 a;

eife. [ electronic life ]2014 5 months and 20 days; 3, e02523.doi: 10.7554/eLife.02523;

proc Natl Acad Sci U S A. [ Proc Natl academy of sciences USA ] No. 2014, 11 months and 25 days; 111(47) 16836-41.doi 10.1073/pnas.1415518111;

nat Cell Biol [ natural Cell biology ] 12 months 2014; 16(12) 1180-91.doi 10.1038/ncb 3064;

ACS chem.bio [ ACS chemico-biological ]2015, 7 months, 17 days; 1604-9.doi 10.1021/acschembio.5b00245;

bioorg Med Chem Lett. [ bio-organic chemistry and medicinal chemistry communication ]2015, 11 months and 1 days; 25(21) 4787-92.doi 10.1016/j.bmcl.2015.07.018;

nat Rev Drug Discov. [ natural review: Drug discovery ]2016 month 5; 15(5) 348-66.doi: 10.1038/nrd.2015.6;

cell Chem Biol. [ cytochemical biology ]2016, 2 months and 18 days; 23(2), 225-235.doi, 10.1016/j. chembiol.2015.11.016;

chem Biol. [ natural chemistry biology ]2016 month 7; 12(7) 497-503.doi 10.1038/nchembio.2079;

proc Natl Acad Sci U S A. [ Proc Natl Acad Sci USA ]2016, 8/23; 113(34) E4966-75.doi 10.1073/pnas.1603244113;

ACS Cent Sci. [ ACS core science ]2016, 9 months and 28 days; 653 and 659;

chem Biol. [ natural chemistry biology ]2017 month 1; 13(1) 81-90.doi: 10.1038/nchembio.2238;

biochem biophysis Res Commun. [ biochemical and biophysical research communication ]2017, 1 month 15; 482(3), 419-425.doi: 10.1016/j.bbrc.2016.10.086;

chem Biol. [ natural chemistry biology ]2018 month 5; 507-515, doi 10.1038/s41589-018, 0031-6;

cancer Lett. [ Cancer communication ] 24.4.2018, pii: S0304-3835(18)30288-x.doi: 10.1016/j.canlet.2018.04.021;

j Med Chem. [ journal of pharmaceutical chemistry ] 24.4.2018. doi: 10.1021/acs.jmedchem.8b00315;

eur J Med Chem. [ european journal of pharmaceutical chemistry ]2018, 5 months and 10 days; 434-449.doi:10.1016/j. ejmech.2018.04.005;

neuropharmacology [ Neuropharmacology ]2018, 3/15; 135:242-252.doi:10.1016/j. neuropharmam.2018.03.015;

j Am Chem Soc. [ journal of american chemical society ]2018, 3 months and 14 days; 140(10) 3798-3808.doi 10.1021/jacs.8b00998;

biochem biophysis Res Commun. [ biochemical and biophysical research communication ]2018, 2 months and 26 days; 497(1) 233-240.doi 10.1016/j. bbrc.2018.02.061;

Org Biomol Chem. [ organic and biomolecular chemistry ]2018, 2 months 28 days; 16(9) 1465-1479.doi 10.1039/c7ob03086 j;

arch Toxicol [ toxicology profile ]2018 for 4 months; 92 (1507-1524. doi:10.1007/s 00204-018-2170-7);

int J Oncol. [ journal of international oncology ]2018 for 3 months; 52(3) 1011-1022.doi 10.3892/ijo.2018.4259;

cancer Lett. [ Cancer communication ]2018, month 4, day 28; 420:210-227.doi:10.1016/j.canlet.2018.01.061.Feb 1;

free Radic Biol Med [ Free radical biology and medicine ]2018, 3 months; 117:45-57.doi: 10.1016/j.freeradbiomed.2018.01.019;

sci Rep [ science report ]2018, 1 month, 12 days; 574, doi 10.1038/s 41598-017-18935-1;

cancer Lett. [ Cancer communication ]2018, 3/1; 416:124-137.doi: 10.1016/j.canlet.2017.12.025;

ChemMedChem [ chemistry & chemistry ]2018, 1 month 22 days; 13(2) 164-177.doi: 10.1002/cmdc.201700629;

redox Biol. [ Redox biology ]2018 month 4; 14:535-548.doi: 10.1016/j.redox.2017.11.001;

arch Toxicol [ toxicology profile ]2018 for 2 months; 92(2) 759-775.doi 10.1007/s 00204-017-2066-y;

nat Chem. [ natural chemistry ]2017 for 10 months; 9(10) 1025-1033.doi 10.1038/nchem 2778;

Free Radic Biol Med [ Free radical biology and medicine ]2017 for 11 months; 112:597-607.doi: 10.1016/j.freeradbiomed.2017.09.002;

ACS chem.bio [ ACS chemico-biology ]2017, 10 months and 20 days; 2538-2545.doi 10.1021/acschembio.7b00730.20;

PLoS One. [ public science library, integrated ]2017, 8 months, 21 days; 12(8) e0182921.doi:10.1371/journal. hole.0182921. ecollection 2017;

biochem biophysis Res Commun. [ biochemical and biophysical research communication ]2017, 9 months and 30 days; 491(4) 919-925.doi 10.1016/j. bbrc.2017.07.136;

biochem Pharmacol [ biochemistry ]2017, 9, 15 days; 140:41-52.doi: 10.1016/j.bcp.2017.06.112.23;

cancer Res Treat. [ Cancer study and treatment ]2018 for 4 months; 50(2) 445-460.doi 10.4143/crt.2016.572;

cell Death Discov [ Cell Death finding ]2017, 2 months and 27 days; 3:17013.doi:10.1038/cddiscovery.2017.13. ecocollection 2017; nat Nanotechnol [ natural nanotechnology ]2016 for 11 months; 977-985.doi: 10.1038/nnano.2016.164;

biochem biophysis Res Commun. [ biochemical and biophysical research communication ]2016 month 11 and 25; 480(4) 602-607.doi 10.1016/j. bbrc 2016.10.099;

Oncotarget. [ tumor target ]2016, 11, 15; 7(46) 74630-74647.doi 10.18632/oncotarget.11858;

cancer Lett. [ Cancer communication ]2016, 10 months and 10 days; 381(1) 165-75.doi 10.1016/j. canlet.2016.07.033.29;

cell Death Dis [ Cell Death and disease ]2016, 7/21; 7: e2307.doi: 10.1038/cddis.2016.208;

oncol Rep [ oncology report ]2016 month 8; 36(2) 968-76.doi: 10.3892/or.2016.4867.31;

biochem biophysis Res Commun. [ biochemical and biophysical research communication ]2016 year 5, month 13; 473(4):775-780.doi: 10.1016/j.bbrc.2016.03.052; mol Carcinog [ molecular carcinogenesis ]2017 for 1 month; 56(1) 75-93.doi: 10.1002/mc.22474;

ACS chem.bio [ ACS chemi-biological ]2016, 5 months and 20 days; 11(5) 1305-12.doi 10.1021/acschembio.5b00900;

j Med Chem. [ journal of pharmaceutical chemistry ]2016 month 3 and 10; 59(5) 2041-53.doi: 10.1021/acs.jmedchem.5b01641;

phytomedine [ botanical drug ]2015, 10, 15 days; 22(11) 1045-54.doi 10.1016/j. phymed.2015.08.002;

biol Pharm Bull [ biomedical publication ] 2015; 38(8) 1234-9.doi 10.1248/bpb.b 15-00048;

oncoscience 2015, 5 months and 2 days; 517-32. ecoselect 2015;

Pathol Oncol Res [ pathology and oncology studies ]2015 for 9 months; 21(4) 1115-21.doi 10.1007/s 12253-015-9946-3;

anticancer Res. [ Anticancer studies ] 11 months 2014; 34(11) 6417-22; and

int J Cancer [ journal of international Cancer ]2013, month 10, day 1; 133(7) 1732-42.doi: 10.1002/ijc.28159;

each of which is incorporated by reference herein in its entirety.

In one embodiment, agents that induce iron-dependent cell disassembly (e.g., iron death) and are useful in the compositions and methods provided herein induce one or more desired immune effects in co-cultured cells, e.g., immune cells. For example, in embodiments, the agent that induces iron-dependent cell disassembly has one or more of the following characteristics:

(a) inducing in vitro iron-dependent cell disassembly of target cells and activation of immune responses in co-cultured cells;

(b) inducing in vitro iron-dependent cell disassembly of target cells and activation of co-cultured macrophages, such as RAW264.7 macrophages;

(c) inducing in vitro iron-dependent cell disassembly of target cells and activation of co-cultured monocytes, e.g., THP-1 monocytes;

(d) inducing in vitro iron-dependent cell disassembly of target cells and activation of co-cultured bone marrow-derived dendritic cells (BMDCs);

(e) Inducing in vitro iron-dependent cell disassembly of target cells and an increase in the level or activity of NFkB, IRF and/or STING in co-cultured cells;

(f) inducing in vitro iron-dependent cell disassembly of target cells and an increase in the level or activity of a proammunocytokine in co-cultured cells;

(g) inducing in vitro iron-dependent cell disassembly of target cells and activation of co-cultured CD4+, CD8+ and/or CD3+ cells; and

(h) inducing in vitro iron-dependent cell disassembly of the target cells and an increase in the level or activity of T cells.

Various methods for determining agents that induce such an immune effect in co-cultured cells in addition to inducing iron-dependent cell disassembly of target cells are known in the art and are provided and described in detail herein.

In certain aspects of the invention, it may be desirable to target or direct delivery of agents that induce iron-dependent cell disassembly to specific target cells, such as cancer cells. Thus, in some embodiments, the agent that induces iron-dependent cell disassembly targets cancer cells. Methods of targeting therapeutic agents to cancer cells are known in the art and are described, for example, in US 2017/0151345, which is incorporated herein by reference in its entirety. For example, an agent that induces iron-dependent cell disassembly can be targeted to a cancer cell by binding, e.g., in the form of a complex or conjugate, to a molecule that specifically binds to a cancer cell marker. As used herein, the term "cancer cell marker" refers to a polypeptide that is present on the surface of a cancer cell. For example, a cancer cell marker can be a cancer cell receptor, e.g., a polypeptide that specifically binds to a molecule in the extracellular environment. A cancer cell marker (e.g., receptor) can be a polypeptide displayed only on cancer cells, at a higher level on cancer cells than on normal cells of the same or a different tissue type, or on both cancer and normal cell types. In some embodiments, a cancer cell marker (e.g., receptor) can be a polypeptide having altered (e.g., higher or lower than normal) expression and/or activity in a cancer cell. In some embodiments, the cancer cell marker (e.g., receptor) can be a polypeptide associated with a disease process of cancer. In some embodiments, a cancer cell marker (e.g., receptor) can be a polypeptide involved in controlling cell death and/or apoptosis. Non-limiting examples of cancer cell markers include, but are not limited to, EGFR, ER, PR, HER2, PDGFR, VEGFR, MET, c-MET, ALK, CD117, RET, DR4, DR5, and FasR. In some embodiments, the molecule that specifically binds to a cancer cell marker (e.g., receptor) is an antibody or a cancer cell marker-binding fragment thereof. In some embodiments, the cancer cell marker is a receptor and the molecule that specifically binds to the cancer cell receptor is a ligand or ligand mimetic of the receptor.

Thus, in some embodiments, it is contemplated that the compositions of the invention comprise a complex or conjugate comprising an agent that induces iron-dependent cellular disassembly and a molecule that specifically binds to a cancer cell marker (e.g., a receptor). In certain embodiments, the complex or conjugate comprises a pharmaceutically acceptable dendrimer, such as a PAMAM dendrimer. In certain embodiments, the complex comprises a liposome. In certain embodiments, the composite comprises microparticles or nanoparticles.

Methods of increasing immune activity

The agents described herein that induce iron-dependent cell disassembly (e.g., iron death) can be used to increase immune activity in a cell, tissue, or subject (e.g., a subject that benefits from increased immune activity). For example, in some aspects, the disclosure relates to a method of increasing immune activity in a cell, tissue, or subject, the method comprising administering to the cell, tissue, or subject an agent that induces iron-dependent cellular disassembly in an amount sufficient to increase immune activity relative to a cell, tissue, or subject that has not been treated with an agent that induces iron-dependent cellular disassembly. In some aspects, the disclosure relates to a method of increasing immune activity in a tissue or subject, the method comprising administering to the tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase immune activity relative to a tissue or subject not treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the subject is in need of increased immune activity.

Administration of agents that induce iron-dependent cell disassembly results in the production of post-cellular signaling factors that modulate immune activity. Immune activity can be modulated by the interaction of post-cellular signaling factors with a wide range of immune cells, including mast cells, Natural Killer (NK) cells, basophils, neutrophils, monocytes, macrophages, dendritic cells, eosinophils, and lymphocytes (e.g., B-lymphocytes (B cells)) and T-lymphocytes (T cells)).

Mast cells are granulocytes comprising granules rich in histamine and the anticoagulant heparin. Upon activation, mast cells release inflammatory compounds from the granules into the local microenvironment. Mast cells play a role in allergy, anaphylaxis, wound healing, angiogenesis, immune tolerance, pathogen defense, and blood brain barrier function.

Natural Killer (NK) cells are cytotoxic lymphocytes that lyse certain tumor cells and virally infected cells without any prior stimulation or immunization. NK cells are also efficient producers of various cytokines, such as IFN-. gamma.IFN-. gamma.TNF-. alpha.TNF-. alpha.GM-CSF and IL-3. Therefore, NK cells are also thought to play a role in the immune system as regulatory cells, affecting other cells and responses. In humans, NK cells are broadly defined as CD56+ CD 3-lymphocytes. The cytotoxic activity of NK cells is tightly controlled by the balance between cell surface receptor activation and inhibition signals. The main group of receptors that inhibit NK cell activation is the inhibitory killer immunoglobulin-like receptor (KIR). Upon recognition of self MHC class I molecules on target cells, these receptors deliver inhibitory signals that stop the activation signaling cascade, thereby protecting cells with normal MHC class I expression from NK cell lysis. Activating receptors include the Natural Cytotoxic Receptor (NCR) and NKG2D, which push the equilibrium towards cytolysis by engaging with different ligands on the surface of target cells. Thus, NK cell recognition of target cells is tightly controlled by the process of signal integration involving multiple activating receptors and inhibitory receptor delivery.

Monocytes are bone marrow-derived mononuclear phagocytes that circulate in the blood for hours/day before being recruited into a tissue. See Wacleche et al, 2018, Virus [ Virus ] (10)2: 65. Expression of various chemokine receptors and cell adhesion molecules on their surfaces enables them to enter the blood from the bone marrow and subsequently be recruited from the blood into tissues. Monocytes belong to the innate arms of the immune system and provide a response to viral, bacterial, fungal or parasitic infections. Their functions include killing pathogens by phagocytosis, production of Reactive Oxygen Species (ROS), Nitric Oxide (NO), myeloperoxidase, and inflammatory cytokines. Under certain conditions, monocytes can stimulate or suppress T cell responses in cancer as well as infectious and autoimmune diseases. They are also involved in tissue repair and neovascularization.

Macrophages engulf and digest materials such as cell debris, foreign materials, microorganisms, and cancer cells in a process known as phagocytosis. In addition to phagocytosis, macrophages play a key role in nonspecific defense (innate immunity) and also help to initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells (e.g., lymphocytes). For example, macrophages are important as antigen presenters for T cells. In addition to increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can reduce the immune response by releasing cytokines. Macrophages that promote inflammation are referred to as M1 macrophages, while macrophages that reduce inflammation and promote tissue repair are referred to as M2 macrophages.

Dendritic Cells (DCs) play a key role in stimulating immune responses to pathogens and maintaining immune homeostasis against innocuous antigens. DCs represent a heterogeneous group of specialized antigen sensing and Antigen Presenting Cells (APCs) that are essential for inducing and modulating immune responses. In peripheral blood, human DCs are characterized by the absence of T cell markers (CD3, CD4, CD8), B cell markers (CD19, CD20) and monocyte markers (CD14, CD16), but high expression of HLA-DR and other DC lineage markers (e.g., CD1a, CD1 c). See Murphy et al, Janeway's immunology, [ janunwei Immunobiology ] encyclopedia, 8 th edition, Garland Science (Garland Science); 2012.868p in New York, N.Y., USA.

The term "lymphocytes" refers to small white blood cells formed in the systemic lymphoid tissues and in normal adults, accounting for approximately 22% -28% of the total white blood cells in circulating blood, and play an important role in protecting the body against disease. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens through recombination of their genetic material (e.g., production of T cell receptors and B cell receptors). This behavior is present before the immune system first contacts a given antigen, expressed by the presence of specific receptors for determinants (epitopes) on the antigen on the surface membrane of lymphocytes. Each lymphocyte has a unique receptor population, and all receptors of the receptor population have the same binding site. One group or clone of lymphocytes differs from another in the structure of the binding region of its receptor, and therefore also in the epitope that it recognizes. Lymphocytes differ from each other not only in the specificity of the receptor, but also in their function. (Paul, W.E., "Chapter 1: The immune system: an introduction [ Chapter 1: introduction ]," Fundamental immunity [ basic Immunology ], 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999), page 102).

Lymphocytes include B lymphocytes (B cells) and T lymphocytes (T cells) which are precursors of antibody-secreting cells.

B lymphocytes (B cells)

B lymphocytes are derived from hematopoietic cells of the bone marrow. Mature B cells can be activated with antigens that express epitopes recognized by their cell surface. The activation process can be direct (which relies on the cross-linking of the antigen to the membrane Ig molecule (cross-linking dependent B cell activation)) or indirect (by interaction with helper T cells in a process known as homo-helper). In many physiological situations, receptor cross-linking stimulation and homeologous help synergistically generate a more intense B cell response (Paul, W.E., "Chapter 1: The immune system: an introduction [ Chapter 1: immune system: introduction ]," Fundamental Immunology [ basic Immunology ], 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers (Lippicott-Raven Publishers), Philadelphia (Philadelphia), (1999)).

Cross-linking dependent B cell activation requires that the antigen express multiple copies of an epitope that is complementary to the binding site of a cell surface receptor, since each B cell expresses an Ig molecule with the same variable regions. Other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins, can meet this requirement. Cross-linking dependent B-cell activation is The main protective immune response against these microorganisms (Paul, W.E., "Chapter 1: The immune system: an introduction [ Chapter 1: immune system: introduction ]," Fundamental Immunology ], 4 th edition, Paul, W.E. ed., Lippicott-Raven Press (Lippicott-Raven publications), Philadelphia (1999)).

Homology assistance allows B cells to respond to antigens that do not cross-link receptors, while providing a costimulatory signal to rescue B cells from inactivation when they are stimulated by a weak cross-linking event. Homology assistance relies on the binding of B-cell membrane immunoglobulins (Ig) to antigens, their endocytosis and their fragmentation into peptides in the endosomal/lysosomal compartment of cells. Some of the peptides produced are loaded into the recesses of a panel of specialized cell surface proteins, known as Major Histocompatibility Complex (MHC) class II molecules. The resulting class II/peptide complexes are expressed on the cell surface and act as a set of T cells (designated CD 4)⁺T cells) of the antigen-specific receptor. CD4⁺T cells have on their surface receptors specific for class II/peptide complexes of B cells. B cell activation is not only dependent on T cell binding through its T Cell Receptor (TCR), but this interaction also allows the binding of an activating ligand on T cells (CD40 ligand) to its receptor on B cells (CD40), signaling B cell activation. Furthermore, T helper cells secrete several cytokines that regulate The growth and differentiation of stimulated B cells by binding to cytokine receptors on B cells (Paul, W.E., "Chapter 1: The immune system: an expression [ Chapter 1: immune system: introduction: A description) ]"Fundamental Immunology]4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

During cognate helper processing for antibody production, CD40 ligand is on activated CD4⁺Transient expression on T helper cells and binding to CD40 on antigen-specific B cells, thereby transducing a second co-cellA stimulus signal. The second costimulatory signal is necessary for the growth and differentiation of B cells and for the generation of memory B cells (by preventing apoptosis of germinal center B cells that have encountered the antigen). Overexpression of CD40 ligand in both B and T cells has been associated with pathogenic autoantibody production in human SLE patients (Desai-Mehta, A. et al, "overexpression of CD40 ligand by B and T cells in human lung and its role in pathogenic autoantibody production [ overexpression of CD40 ligand in B and T cells in human lupus and their role in pathogenic autoantibody production]"J.Clin.Invest. [ J.Clin.Clest. ] [ J.Clin.Clin.]Volume 97(9), 2063-.

T lymphocytes (T cells)

T lymphocytes derived from hematopoietic tissue precursors are differentiated in the thymus and then seeded into peripheral lymphoid tissues and lymphocyte recirculation pools. T lymphocytes or T cells mediate a wide range of immunological functions. These include the ability to assist B cells in developing antibody-producing cells, the ability to enhance monocyte/macrophage microbicidal action, the inhibition of certain types of immune responses, direct killing of target cells, and the mobilization of inflammatory responses. These effects rely on T cells expressing specific cell surface molecules and secreting cytokines (Paul, W.E., "Chapter 1: The immune system: introduction [ Chapter 1: immune system: introduction ]," Fundamental Immunology [ basic Immunology ], 4 th edition, Paul, W.E. ed., Lippicott-Raven Press (Lippicott-Raven Publishers), Philadelphia (1999)).

T cells and B cells differ in their antigen recognition mechanisms. Immunoglobulins (receptors for B cells) bind to unique epitopes on the surface of soluble molecules or particles. The B cell receptor is directed to an epitope expressed on the surface of the native molecule. When antibodies and B cell receptors are evolved to bind and protect against microorganisms in the extracellular fluid, T cells recognize antigens on the surface of other cells and mediate their functions by interacting with and altering the behavior of these Antigen Presenting Cells (APCs). There are three major types of APCs in peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages and B cells. Among the most effective are dendritic cells, whose sole function is to present foreign antigens to T cells. Immature dendritic cells are located in tissues throughout the body, including the skin, intestinal tract, and respiratory tract. When they encounter invading microorganisms at these sites, they phagocytose the pathogens and their products and transport them lymphatically to regional lymph nodes or gut-associated lymphoid organs. Encounter with a pathogen induces dendritic cells to mature from antigen-capturing cells to APCs that can activate T cells. APCs display three types of protein molecules on their surface, which have a role in activating T cells into effector cells: (1) MHC proteins that present foreign antigens to T cell receptors; (2) a costimulatory protein that binds to a T cell surface complementary receptor; and (3) Cell-Cell adhesion molecules that allow T cells to bind to APC for a sufficient period of time to be activated ("Chapter 24: The adaptive immune system [ Chapter 24: adaptive immune system ]," Molecular Biology of The Cell [ Molecular Biology ], Alberts, B. et al, Hua N.C. (2002)).

T cells are divided into two distinct classes according to the cell surface receptor they express. Most T cells express a T Cell Receptor (TCR) consisting of alpha and beta chains. A small group of T cells express receptors consisting of gamma and delta chains. There are two sub-lineages in α/β T cells: those expressing the co-receptor molecule CD4 (CD 4)⁺T cells); and those expressing CD8 (CD 8)⁺T cells). These cells differ in the way they recognize antigens and their effector and regulatory functions.

CD4⁺T cells are the primary regulatory cells of the immune system. Their regulatory function depends on the expression of their cell surface molecules (e.g., CD40 ligand, whose expression is induced when T cells are activated), and the various cytokines they secrete when activated.

T cells also mediate important effector functions, some of which are determined by the pattern of cytokines they secrete. Cytokines may be directly toxic to target cells and may mobilize effective inflammatory mechanisms.

Furthermore, T cells, in particular CD8⁺T cellsCan be developed into Cytotoxic T Lymphocytes (CTL) capable of effectively lysing target cells expressing an antigen recognized by CTL (Paul, W.E., "Chapter 1: The immune system: an expression [ Chapter 1: introduction of immune system: introduction) ]"Fundamental Immunology]4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

T Cell Receptors (TCRs) recognize complexes of the group consisting of: a proteolytically derived peptide of an antigen that binds to a specialized groove of a class II or class I MHC protein. CD4⁺T cells recognize only peptide/class II complexes, whereas CD8⁺T-cell recognition peptide/class I Complex (Paul, W.E., "Chapter 1: The immune system: an expression [ Chapter1: introduction of immune System:. introduction of T-cell recognition peptide/class I Complex]"Fundamental Immunology]4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

The ligand (i.e., peptide/MHC protein complex) of the TCR is generated within the APC. Typically, MHC class II molecules bind peptides derived from proteins taken up by the APC by endocytic processes. These peptide-loaded class II molecules are then expressed on the cell surface where they can be expressed by CD4⁺T cells (which have a TCR capable of recognizing the expressed cell surface complex) bind. Thus, CD4⁺T cells specifically react with antigens derived from extracellular sources (Paul, W.E., "Chapter 1: The immune system: an expression [ Chapter1: immune system: introduction) ]"Fundamental Immunology]4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

In contrast, class I MHC molecules are loaded predominantly with peptides derived from internal synthetic proteins (e.g. viral proteins). These peptides are produced from cytosolic proteins by the proteolytic action of the proteasome and are transported into the rough endoplasmic reticulum. Such peptides, usually consisting of nine amino acids in length, bind to MHC class I molecules and are carried to the cell surface where they can be expressed by CD8 of the appropriate receptor⁺T cell recognition. This enables the T cell system (especially CD 8)⁺T cells) capable ofDetecting cells expressing proteins which are different from or produced in much larger amounts than those of cells of The rest of The organism (e.g. viral antigens) or mutated antigens (e.g. active oncogene products), even if these proteins are neither expressed on The cell surface nor secreted in their intact form (Paul, W.E., "Chapter 1: The immune system: an expression [ Chapter 1: immune system: introduction)]"Fundamental Immunology]4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

T cells can also be classified according to their function as helper T cells; t cells involved in inducing cellular immunity; a suppressor T cell; and cytotoxic T cells.

Helper T cell

Helper T cells are T cells that stimulate B cells to produce antibody responses to proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which unique epitopes appear only once or a limited number of times, so that they cannot cross-link the membrane immunoglobulin (Ig) of B cells or are inefficient. B cells bind antigen through their membrane Ig, and the complex undergoes endocytosis. In the endosomal and lysosomal compartments, the antigen is fragmented by proteolytic enzymes into peptides, and one or more of the produced peptides are loaded into MHC class II molecules that are transported through the vesicular compartment. The resulting peptide/MHC class II complex is then exported to the B cell surface membrane. T cells with a receptor specific for the peptide/class II molecule complex recognize the complex on the surface of B cells. (Paul, W.E., "Chapter 1: The immune system: an introduction [ Chapter 1: introduction ]," Fundamental immunity [ basic Immunology ], 4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

B cell activation depends on both: t cells interact with CD40 on B cells through binding of their TCR and the interaction of T cell CD40 ligand (CD 40L). T cells do not constitutively express CD 40L. Instead, the result of the interaction with the APC expressing the cognate antigen recognized by the TCR of the T cell and either CD80 or CD86 is the induction of CD40L expression. CD80/CD86 is normally expressed by activated B cells rather than resting B cells, and thus helper interactions involving activated B cells and T cells can lead to efficient antibody production. However, in many cases, the initial induction of CD40L on T cells is dependent on their recognition of surface antigens that group the constitutively expressing APCs (e.g., dendritic cells) of CD 80/86. Such activated helper T cells can then efficiently interact with and help B cells. Even with low efficiency, cross-linking of membrane Ig on B cells can be coordinated with CD40L/CD40 interactions to produce strong B cell activation. Subsequent events in B cell responses, including proliferation, Ig secretion and class switching of expressed Ig classes, are dependent on or enhanced by The action of T cell-derived cytokines (Paul, W.E., "Chapter 1: The immune system: an introduction [ Chapter 1: immune system: introduction ]," Fundamental Immunology ], 4 th edition, Paul, W.E. edition, Lippicot-Raven Press (Lippictot-Raven Publishers), Philadelphia (Philadelphia), (1999)).

CD4⁺T cells tend to differentiate into cells that secrete primarily the cytokines IL-4, IL-5, IL-6, and IL-10 (T)_H2 cells) or differentiation to produce mainly IL-2, IFN-gamma and lymphotoxin (T)_H1 cell). T is_H2 cells are very efficient in assisting B cells to develop into antibody-producing cells, while T cells_H1 cells are then potent inducers of cellular immune responses, involving enhancement of the microbicidal activity of monocytes and macrophages, and thus increasing the efficiency of lysis of microorganisms in the intracellular vesicular compartments. Although with T_HCD4 of 2 cell phenotype (i.e., IL-4, IL-5, IL-6, and IL-10)₊T cells are potent helper cells, but T_H1 cells also have The ability to become helper cells (Paul, W.E., "Chapter 1: The immune system: an expression [ Chapter 1: introduction of immune System:. introduction of cell lines)]"Fundamental Immunology]4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

T cells involved in cellular immune induction

T cells may also function to enhance the ability of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon-gamma (IFN- γ) produced by helper T cells enhances several mechanisms by which mononuclear phagocytes destroy intracellular bacteria and parasites, including the production of nitric oxide and the induction of Tumor Necrosis Factor (TNF). T is _H1The cells are effective in enhancing microbicidal action because they produce IFN-gamma. In contrast, T_H2Two of The major cytokines produced by cells, IL-4 and IL-10, block these activities (Paul, W.E., "Chapter 1: The immune system: an expression [ Chapter 1: introduction of The immune System:)]"Fundamental Immunology]4 th edition, Paul, W.E. eds., Lippicott-Raven Publishers, Philadelphia (Philadelphia), (1999)).

Regulatory T (Treg) cells

Immune homeostasis is maintained by a controlled balance between the initiation and downregulation of the immune response. The mechanism of apoptosis and T cell anergy (a tolerance mechanism in which T cells are inherently functionally inactivated upon encountering antigen (Schwartz, r.h., "T cell anergy [, T cell anergy [)]", Annu.Rev.Immunol. [ annual review of immunization ]]Vol 21:305-334(2003)) contribute to the down-regulation of the immune response. The third mechanism is through repressive or regulatory CD4⁺T (Treg) cells actively suppress activated T cells (in Kronenberg, M. et al, "Regulation of immunity by self-reactive T cells Regulation of immunity]", Nature [ Nature]Reviewed in volumes 435: 598-. CD4 constitutively expressing IL-2 receptor alpha (IL-2R alpha) chain ⁺ Treg(CD4⁺CD25⁺) Is a naturally occurring subset of T cells that is anergic and suppressive (Taams, L.S. et al, "Human anergic/supressive CD4⁺CD25⁺T cells a highlyly differentiated and apoptosis-protein pulsing [ human non-reactive/repressible CD4⁺CD25⁺T cell: highly differentiated and apoptotic populations]", eur.j.immunol. [ journal of european immunology ]]Volume 31:1122-1131 (2001)). Human CD4⁺CD25⁺Tregs, like their murine counterparts,produced in the thymus and characterized by the ability to suppress the proliferation of responsive T cells by cell-cell contact-dependent mechanisms, the inability to produce IL-2, and an anergic phenotype in vitro. Human CD4 based on the expression level of CD25⁺CD25⁺T cells can be classified as suppressor (CD 25)^{Height of}) And non-repressive (CD 25)^{Is low in}) A cell. FOXP3, a member of the forked transcription factor family, has been shown in murine and human CD4⁺CD25⁺Tregs and appears to control CD4⁺CD25⁺The major genes for Treg development (Battaglia, M. et al, "Rapamycin proteins expansion of functional CD4⁺CD25⁺Foxp3⁺regulator T cells of bed health subjects and type 1 diabetes patients rapamycin promotes functional CD4 in healthy subjects and in patients with type 1 diabetes⁺CD25⁺Foxp3⁺Expansion of regulatory T cells ]", j.immunol. [ journal of immunology ]]Volume 177:8338-, (2006)). Thus, in some embodiments, an increase in an immune response may be associated with a lack of activation or proliferation of regulatory T cells.

Cytotoxic T lymphocytes

CD8 recognizing peptide derived from protein produced in target cell⁺T cells are cytotoxic because they cause lysis of target cells. The mechanism of CTL-induced lysis involves the production of perforin by CTL, a molecule that can insert into the membrane of the target cell and facilitate lysis of the cell. Perforin-mediated cleavage is enhanced by granzyme, a series of enzymes produced by activated CTLs. Many active CTLs also express large amounts of fas ligand on their surface. The interaction of fas ligand on the surface of CTL with fas on the surface of target cells initiates apoptosis of the target cells, leading to death of these cells. CTL-mediated lysis appears to be the primary mechanism for destroying virus-infected cells.

Lymphocyte activation

The term "activation" or "lymphocyte activation" refers to the stimulation of lymphocytes by specific antigens, non-specific mitogens or allogeneic cells, resulting in the synthesis of RNA, protein and DNA and the production of lymphokines; this is followed by the proliferation and differentiation of various effector and memory cells. T cell activation relies on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide that binds in the groove of an MHC class I or class II molecule. The molecular events that are mobilized by receptor engagement are complex. One of the earliest steps appears to be the activation of tyrosine kinases, leading to tyrosine phosphorylation of a panel of substrates that control several signaling pathways. These include a group of adaptor proteins that link the TCR to the ras pathway, phospholipase C γ 1, whose tyrosine phosphorylation increases its catalytic activity and participates in the inositol phospholipid metabolism pathway, leading to increased intracellular free calcium concentrations and activation of protein kinase C, as well as a series of other enzymatic activations that control cell growth and differentiation. The full responsiveness of T cells, in addition to receptor engagement, requires co-stimulatory activity delivered by helper cells, e.g., engagement of CD28 on T cells with CD80 and/or CD86 on APCs.

T memory cell

Following recognition and eradication of pathogens by an adaptive immune response, the vast majority (90% -95%) of T cells undergo apoptosis, with the remaining cells forming a pool of memory T cells, called central memory T Cells (TCM), effector memory T cells (TEM), and Resident memory T cells (TRM) (Clark, r.a., "memory T cells in human health and disease Resident memory T cells ]", sci.trans.med. [ scientific transformation medicine ],7,269rv1, (2015)).

These memory T cells have a long life span compared to standard T cells, with different phenotypes such as expression of specific surface markers, rapid generation of different cytokine profiles, ability to direct effector cell function, and a unique homing distribution pattern. Memory T cells exhibit a rapid response upon re-exposure to their respective antigens, thereby eliminating reinfection by the offender, and thereby rapidly restoring the balance of the immune system. There is increasing evidence that autoimmune memory T cells hamper most attempts to treat or cure autoimmune diseases (Clark, r.a., "resource memory T cells in human health and disease Resident memory T cells ]", sci.trans.med. [ scientific transformation medicine ], volume 7,269rv1, (2015)).

For example, in one embodiment, an agent that induces iron-dependent cell disassembly is administered in an amount sufficient to increase one or more of the level or activity of macrophages, the level or activity of monocytes, the level or activity of dendritic cells, the level or activity of T cells, and the level or activity of CD4+, CD8+, or CD3+ T cells (e.g., CD4+, CD8+, or CD3+ T cells) in a tissue or subject.

Agents that induce iron-dependent cell disassembly may also increase immune activity in a cell, tissue, or subject by inducing the production of post-cellular signaling factors that increase the level or activity of pro-immune cytokines. For example, in some embodiments, the agent that induces iron-dependent cell disassembly is administered in an amount sufficient to increase the level or activity of a pro-immune cytokine in a cell, tissue, or subject. In one embodiment, the immunocytokines are selected from IFN- α, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF- α, IL-17, and GMCSF.

Agents that induce iron-dependent cell disassembly may also increase immune activity in a cell, tissue, or subject by inducing the production of post-cellular signaling factors that increase the level or activity of positive regulators of the immune response, such as nuclear factor kappa light chain-enhancer of activated B cells (NFkB), Interferon Regulatory Factor (IRF), and interferon gene Stimulator (STING). For example, in some embodiments, the agent that induces iron-dependent cell disassembly is administered in an amount sufficient to increase the level or activity of NFkB, IRF and/or STING in a cell, tissue or subject.

In some embodiments, the disclosure relates to a method of increasing the immune activity of an immune cell, the method comprising: (i) contacting a target cell with an agent that induces disassembly of iron-dependent cells and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase immune activity in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent that induces disassembly of iron-dependent cells.

In some embodiments, the disclosure relates to a method of increasing the level or activity of NFkB in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cell disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of NFkB in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent that induces iron-dependent cell disassembly.

In some embodiments, the disclosure relates to a method of increasing the level or activity of an Interferon Regulatory Factor (IRF) or an interferon gene Stimulator (STING) in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cell disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of IRF or STING in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent that induces iron-dependent cell disassembly.

In some embodiments, the disclosure relates to a method of increasing the level or activity of a pro-immune cytokine in an immune cell, the method comprising: (i) contacting a target cell with an agent that induces iron-dependent cell disassembly and (ii) exposing the immune cell to the target cell that has been contacted with the agent or a post-cellular signaling factor produced by the target cell that has been contacted with the agent in an amount sufficient to increase the level or activity of a proammunocytokine in the immune cell relative to the immune cell in the absence of contacting the target cell with the agent that induces iron-dependent cell disassembly.

In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo. In some embodiments, step (i) is performed in vitro, and step (ii) is performed in vivo.

In some embodiments, the immune cell is a macrophage, monocyte, dendritic cell, T cell, CD4+ cell, CD8+ cell, or CD3+ cell. In some embodiments, the immune cell is a THP-1 cell.

In some embodiments, the disclosure relates to a method of increasing an immune activity of an immune cell, the method comprising contacting the immune cell with a target cell or a post-cellular signaling factor produced by the target cell, wherein the target cell has been previously contacted with an agent that induces disassembly of iron-dependent cells in an amount sufficient to increase the immune activity in the immune cell relative to the immune cell without contacting the target cell with the agent that induces disassembly of iron-dependent cells.

In some embodiments, the disclosure relates to a method of increasing the level or activity of NFkB in an immune cell, the method comprising contacting the immune cell with a target cell or a post-cellular signaling factor produced by the target cell, wherein the target cell has previously been contacted with an agent that induces disassembly of iron-dependent cells in an amount sufficient to increase the level or activity of NFkB in the immune cell relative to the immune cell without contacting the target cell with the agent that induces disassembly of iron-dependent cells.

In some embodiments, the disclosure relates to a method of increasing the level or activity of an Interferon Regulatory Factor (IRF) or an interferon gene Stimulator (STING) in an immune cell, the method comprising contacting the immune cell with a target cell or a post-cellular signaling factor produced by the target cell, wherein the target cell has previously been contacted with an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of the Interferon Regulatory Factor (IRF) or the interferon gene Stimulator (STING) in the immune cell relative to the immune cell without contacting the target cell with the agent that induces iron-dependent cell disassembly.

In some embodiments, the disclosure relates to a method of increasing the level or activity of a proammunocytokine in an immune cell, the method comprising contacting the immune cell with a target cell or a post-cellular signaling factor produced by the target cell, wherein the target cell has been previously contacted with an agent that induces disassembly of iron-dependent cells in an amount sufficient to increase the level or activity of the proammunocytokine in the immune cell relative to the immune cell without contacting the target cell with the agent that induces disassembly of iron-dependent cells.

In some embodiments, the step of contacting the immune cell with the target cell is performed in vitro. In some embodiments, the step of contacting the immune cell with the target cell is performed ex vivo. In some embodiments, the step of contacting the immune cell with the target cell is performed in vivo.

In some embodiments, the target cell is previously contacted with the agent in vitro. In some embodiments, the target cell is previously contacted with the agent ex vivo. In some embodiments, the target cell is previously contacted with the agent in vivo.

In some embodiments, the disclosure relates to a method of increasing the immune activity of an immune cell in a tissue or subject, the method comprising contacting a target cell in the tissue or subject with an agent that induces disassembly of iron-dependent cells in an amount sufficient to increase the immune activity of the immune cell relative to an immune cell in a tissue or subject in which the target cell is not contacted with an agent that induces disassembly of iron-dependent cells.

In some embodiments, the disclosure relates to a method of increasing the level or activity of NFkB in an immune cell in a tissue or subject, the method comprising contacting a target cell in the tissue or subject with an agent that induces disassembly of iron-dependent cells in an amount sufficient to increase the level or activity of NFkB in an immune cell, relative to an immune cell in a tissue or subject in which the target cell is not contacted with an agent that induces disassembly of iron-dependent cells.

In some embodiments, the disclosure relates to a method of increasing the level or activity of IRF or STING in an immune cell in a tissue or subject, the method comprising contacting a target cell in the tissue or subject with an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of IRF or STING in the immune cell relative to an immune cell in a tissue or subject in which the target cell is not contacted with the agent that induces iron-dependent cell disassembly.

In some embodiments, the disclosure relates to a method of increasing the level or activity of a proammunocytokine in an immune cell in a tissue or subject, the method comprising contacting a target cell in the tissue or subject with an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of the proammunocytokine in the immune cell relative to an immune cell in a tissue or subject in which the target cell is not contacted with the agent that induces iron-dependent cell disassembly. In some embodiments, the target cell and the immune cell are in close proximity or physical contact in the tissue or subject. In some embodiments, the target cell and the immune cell are present in the same tissue or organ of the subject.

In some aspects, the disclosure relates to a method of increasing the level or activity of NFkB in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cellular disassembly in an amount sufficient to increase the level or activity of NFkB relative to a cell, tissue or subject not treated with an agent that induces iron-dependent cellular disassembly.

In one embodiment, the subject is in need of increased NFkB level or activity.

In some aspects, the disclosure relates to a method of increasing the level or activity of IRF or STING in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of IRF or STING relative to a cell, tissue or subject that is not treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the subject is in need of increased level or activity of IRF or STING.

In some aspects, the disclosure relates to a method of increasing the level or activity of macrophages, monocytes, T cells or dendritic cells in a tissue or subject, the method comprising administering to the tissue or subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of macrophages, monocytes, T cells or dendritic cells relative to a tissue or subject that is not treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the subject is in need of an increase in macrophage, monocyte or dendritic cell levels or activity.

In one embodiment, the level or activity of macrophages, monocytes, T cells or dendritic cells is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or at least 2-fold, 4-fold, 6-fold, 8-fold, or 10-fold relative to a tissue or subject not treated with an agent that induces iron-dependent cell disassembly.

In some aspects, the disclosure relates to a method of increasing the level or activity of CD4+, CD8+, or CD3+ cells in a tissue or subject, the method comprising administering to the subject an agent that induces iron-dependent cell disassembly in an amount sufficient to increase the level or activity of CD4+, CD8+, or CD3+ cells relative to a tissue or subject not treated with an agent that induces iron-dependent cell disassembly.

In one embodiment, the subject is in need of increased CD4+, CD8+, or CD3+ cell levels or activity.

In some aspects, the disclosure relates to a method of increasing the level or activity of a proammunocytokine in a cell, tissue or subject, the method comprising administering to the cell, tissue or subject an agent that induces iron-dependent cellular disassembly in an amount sufficient to increase the level or activity of the proammunocytokine relative to a cell, tissue or subject that has not been treated with an agent that induces iron-dependent cellular disassembly.

In one embodiment, the subject is in need of increased levels or activity of a proammunocytokine.

In one embodiment, the immunocytokines are selected from IFN- α, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF- α, IL-17, and GMCSF.

In some embodiments, the methods of the invention further comprise, prior to administering the agent that induces iron-dependent cell disassembly, evaluating the cell, tissue, or subject for one or more of: the level or activity of NFkB; the level or activity of macrophages; the level or activity of monocytes; the level or activity of dendritic cells; the level or activity of a CD4+ cell, CD8+ cell, or CD3+ cell; the level or activity of T cells; and the level or activity of a pro-immunocytokine.

In one embodiment, the method of the invention further comprises, after administering the agent that induces iron-dependent cell disassembly, evaluating the cell, tissue or subject for one or more of: the level or activity of NFkB, IRF or STING; the level or activity of macrophages; the level or activity of monocytes; the level or activity of dendritic cells; the level or activity of a CD4+ cell, CD8+ cell, or CD3+ cell; the level or activity of T cells; and the level or activity of a pro-immunocytokine.

Measuring the level or activity of NFkB, IRF or STING; the level or activity of macrophages; the level or activity of monocytes; the level or activity of dendritic cells; the level or activity of a CD4+ cell, CD8+ cell, or CD3+ cell; the level or activity of T cells; and the level or activity of immunocytokines are known in the art.

For example, the protein levels or activities of NFkB, IRF or STING may be measured by suitable techniques known in the art, including ELISA, western blot or in situ hybridization. The level of NFkB, IRF or STING encoding nucleic acids (e.g., mRNA) can be measured using suitable techniques known in the art, including Polymerase Chain Reaction (PCR) amplification reactions, reverse transcriptase PCR analysis, quantitative real-time PCR, single-strand conformation polymorphism analysis (SSCP), mismatch cleavage detection, heteroduplex analysis, northern blot analysis, in situ hybridization, array analysis, deoxyribonucleic acid sequencing, restriction fragment length polymorphism analysis, and combinations or subcombinations thereof.

Methods for measuring macrophage levels and activity are described, for example, in Chitu et al, 2011, Curr Protoc Immunol [ Current protocols of immunology ]14: 1-33. The level and activity of monocytes can be measured by flow cytometry, as described, for example, in Henning et al, 2015, Journal of Immunological Methods 423: 78-84. The level and activity of dendritic cells can be measured by flow cytometry, as described, for example, in Dixon et al, 2001, infection Immun. [ infection and immunization ]69(7): 4351-4357. Each of these references is incorporated herein by reference in its entirety.

The level or activity of T cells can be assessed using a proliferation assay based on human CD4+ T cells. For example, cells are labeled with the fluorescent dye 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE). Those proliferating cells showed a decrease in CFSE fluorescence intensity, which can be directly measured by flow cytometry. Alternatively, radioactive thymidine incorporation can be used to assess the growth rate of T cells.

In some embodiments, the increase in immune response may be associated with decreased activation of regulatory T cells (tregs). Functionally active tregs can be assessed using an in vitro Treg suppression assay. Such assays are described in Collinson and Vignali (Methods Mol Biol. [ molecular biology Methods ] 2011; 707: 21-37, incorporated herein by reference in its entirety).

The level or activity of the immunocytokine may be quantified, for example, in CD8+ T cells. In embodiments, the immunocytokines are selected from interferon alpha (IFN-alpha), interleukin-1 (IL-1), IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, tumor necrosis factor alpha (TNF-alpha), IL-17, and granulocyte-macrophage colony stimulating factor (GMCSF). Quantitation can be performed using ELISPOT (enzyme linked immunospot) technology, which can detect T cells that secrete a given cytokine (e.g., IFN- α) in response to an antigenic stimulus. T cells are cultured with antigen presenting cells in wells that have been coated with, for example, anti-IFN-alpha antibodies. Secreted IFN- α was captured by the coated antibody and then visualized with a second antibody coupled to a chromogenic substrate. Thus, locally secreted cytokine molecules form spots, each spot corresponding to one IFN- α secreting cell. The number of spots allows the determination of the frequency of IFN-. alpha.secreting cells in the sample which are specific for a given antigen. Also described are ELISPOT assays for detecting TNF- α, interleukin 4(IL-4), IL-6, IL-12, and GMCSF.

Methods of treating disorders

A. Infectious diseases

As provided herein, an agent that induces iron-dependent cell disassembly (e.g., iron death) can activate immune cells (e.g., T cells, B cells, NK cells, etc.) and thus can enhance immune cell function, e.g., inhibit bacterial and/or viral infection, and/or restore immune surveillance and immune memory function to treat the infection. Thus, in some embodiments, compositions of the invention, e.g., comprising an agent that induces iron-dependent cell disassembly (e.g., iron death), are used to treat an infection or infectious disease, e.g., a chronic infection, in a subject.

As used herein, the term "infection" refers to any state in which a cell or tissue of an organism (i.e., a subject) is infected with an infectious agent (e.g., the subject has an intracellular pathogen infection, such as a chronic intracellular pathogen infection). As used herein, the term "infectious agent" refers to a foreign biological entity (i.e., a pathogen) in at least one cell of an infected organism. For example, infectious agents include, but are not limited to, bacteria, viruses, protozoa, and fungi. Intracellular pathogens are of particular interest. Infectious diseases are disorders caused by infectious agents. Under certain conditions, some infectious agents do not cause identifiable symptoms or diseases, but may cause symptoms or diseases under varying conditions. The subject methods can be used to treat chronic pathogen infections, including but not limited to viral infections, such as retroviruses, lentiviruses, hepatitis viruses, herpes viruses, poxviruses, or human papilloma viruses; intracellular bacterial infections, such as mycobacteria (Mycobacterium), Chlamydophila (Chlamydophila), Elekia (Ehrlichia), Rickettsia (Rickettsia), Brucella (Brucella), Legionella (Legionella), Francisella (Francisella), Listeria (Listeria), Cornus (Coxiella), Neisseria (Neisseria), Salmonella (Salmonella), Yersinia species (Yersinia sp) or Helicobacter pylori (Helicobacter pylori); and intracellular protozoan pathogens such as Plasmodium species (Plasmodium sp), Trypanosoma species (Trypanosoma sp.), Giardia species (Giardia sp.), Toxoplasma species (Toxoplasma sp.) or Leishmania species (Leishmania sp.).

Infectious diseases that can be treated using the compositions described herein include, but are not limited to: HIV, influenza, herpes, giardia, malaria, leishmania, viral hepatitis (type A, B or C), herpes viruses (e.g. VZV, HSV-I, HAV-6, HSV-II and CMV, Epstein Barr virus (Epstein Barr virus)), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, HTparvovirus, vaccinia virus, LV virus, dengue virus, papilloma virus, molluscum virus, polio virus, JC virus, rabies virus, and arbovirus-induced pathogen infections, bacterial chlamydia, rickettsia, mycobacteria, staphylococci, streptococcus, pneumococci, meningococcus, and gonococcus (conocci), Klebsiella, Proteus, Serratia, Pseudomonas, Escherichia, Legionella, diphtheria, Salmonella, Bacteria, cholera, tetanus, botulism, anthrax, plague, Juniperus and Lyme disease caused by bacteria, the fungi Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma, and the parasites Proteus dysenteriae, Taenia colophonium, Nagri-Miyami, Acacia species, Giardia lamblia, Cryptosporidium species, Salmonella species, strains, pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and/or Glynonella brasiliensis.

The term "chronic infection" refers to an infection that persists for about one month or more, e.g., for at least one month, two months, three months, four months, five months, or six months. In some embodiments, the chronic infection is associated with increased production of anti-inflammatory chemokines in and/or around one or more infected areas. Chronic infections include, but are not limited to, infection by HIV, HPV, hepatitis b, hepatitis c, EBV, CMV, mycobacterium tuberculosis (m. In some embodiments, the chronic infection is a bacterial infection. In some embodiments, the chronic infection is a viral infection.

B. Cancer treatment

As provided herein, an agent that induces iron-dependent cell disassembly (e.g., iron death) can activate immune cells (e.g., T cells, B cells, NK cells, etc.) and thus can enhance immune cell function, such as, for example, immune cell function involved in immunotherapy. Thus, in certain aspects, the disclosure relates to a method of treating a subject diagnosed with cancer, the method comprising administering to the subject a combination of (a) an immunotherapeutic anti-neoplastic agent and (b) an agent that induces iron-dependent cell disassembly, thereby treating the cancer of the subject.

The ability of cancer cells to prevent the immune system from distinguishing self from non-self using a complex series of overlapping mechanisms represents a fundamental mechanism by which cancer evades immune surveillance. One or more mechanisms include disruption of antigen presentation, disruption of regulatory pathways that control T cell activation or inhibition (immune checkpoint regulation), recruitment of cells that contribute to immune suppression (tregs, MDSCs), or release of factors that influence immune activity (IDO, PGE 2). (see Harris et al, 2013, J immunotherpy Cancer [ journal of Cancer Immunotherapy ]1: 12; Chen et al, 2013, Immunity [ immune ]39: 1; Pardol, et al, 2012, Nature Reviews: Cancer [ natural Reviews: Cancer ]12: 252; and Sharma et al, 2015, Cell [ Cell ]161:205, each of which is incorporated herein by reference in its entirety.)

Immune checkpoint modulators

In some embodiments, the immunotherapy is an immune checkpoint modulator of an immune checkpoint molecule. Examples include LAG-3(Triebel et al, 1990, J.exp. Med. [ J.EXPERIMENT MEDICAL ]171: 1393-. Examples of co-stimulatory molecules that improve immune responses include ICOS (Fan et al, 2014, J.Exp.Med. [ journal of Experimental medicine ]211: 715-.

The immune checkpoint may be a stimulatory immune checkpoint (i.e., a molecule that stimulates an immune response) or an inhibitory immune checkpoint (i.e., a molecule that inhibits an immune response). In some embodiments, the immune checkpoint modulator is an antagonist of an inhibitory immune checkpoint. In some embodiments, the immune checkpoint modulator is an agonist of a stimulatory immune checkpoint. In some embodiments, the immune checkpoint modulator is an immune checkpoint binding protein (e.g., an antibody Fab fragment, a bivalent antibody, an antibody drug conjugate, an scFv, a fusion protein, a bivalent antibody, or a tetravalent antibody). In certain embodiments, the immune checkpoint modulator is capable of binding to or modulating the activity of more than one immune checkpoint. Examples of stimulatory and inhibitory immune checkpoints that can be used in the methods of the invention are provided below, as well as molecules that modulate these immune checkpoints.

i. Stimulatory immune checkpoint molecules

CD27 supports antigen-specific expansion of naive T cells and is critical for the generation of T cell memory (see, e.g., Hendriks et al (2000) nat. immunol. [ natural immunology ]171(5): 433-40). CD27 is also a memory marker for B cells (see, e.g., agamatsu et al (2000) histol. histopatho-histology ]15(2): 573-6. CD27 activity is governed by the transient availability of its ligand CD70 on lymphocytes and dendritic cells (see, e.g., Borst et al (2005) curr. opin. immunol. [ recent concept ]17(3): 275-81.) a variety of immune checkpoint modulators specific for CD27 have been developed and may be used as disclosed herein. The immune checkpoint modulator is a CD27 binding protein (e.g., an antibody). In some embodiments, the immune checkpoint modulator is valrubiumab (Celldex Therapeutics) an additional CD27 binding protein (e.g., an antibody) is known in the art and disclosed in, for example, U.S. patent nos. 9,248,183, 9,102,737, 9,169,325, 9,023,999, 8,481,029, U.S. patent application publication nos. 2016/0185870, 2015/0337047, 2015/0299330, 2014/0112942, 2013/0336976, 2013/0243795, 2013/0183316, 2012/0213771, 2012/0093805, 2011/0274685, 2010/0173324, and PCT publication nos. WO 2015/016718, WO2014/140374, WO 2013/138586, WO 2012/004367, WO 2011/130434, WO 2010/001908, and WO 2008/051424, each of which is incorporated herein by reference.

Cluster of differentiation 28(CD28) is one of the proteins expressed on T cells that provide costimulatory signals required for T cell activation and survival. In addition to the T Cell Receptor (TCR), stimulation of T cells by CD28 provides an effective signal to produce various interleukins (particularly IL-6). Binding to its two ligands, CD80 and CD86, expressed on dendritic cells, promotes T cell expansion (see, e.g., Prasad et al (1994) proc.nat' l.acad.sci.usa [ journal of american national academy of sciences ]91(7): 2834-8). A variety of immune checkpoint modulators specific for CD28 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CD28. In some embodiments, the immune checkpoint modulator is an agent that binds to CD28 (e.g., an anti-CD 28 antibody). In some embodiments, the checkpoint modulator is a CD28 agonist. In some embodiments, the checkpoint modulator is a CD28 antagonist. In some embodiments, the immune checkpoint modulator is a CD28 binding protein (e.g., an antibody). In some embodiments, the immune checkpoint modulator is selected from the group consisting of: TAB08(TheraMab LLC), Ullizumab (also known as BMS-931699, Bristol-Myers Squibb) and FR104(OSE immunotherapy). Additional CD28 binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 9,119,840, 8,709,414, 9,085,629, 8,034,585, 7,939,638, 8,389,016, 7,585,960, 8,454,959, 8,168,759, 8,785,604, 7,723,482; U.S. patent application publication nos. 2016/0017039, 2015/0299321, 2015/0150968, 2015/0071916, 2015/0376278, 2013/0078257, 2013/0230540, 2013/0078236, 2013/0109846, 2013/0266577, 2012/0201814, 2012/0082683, 2012/0219553, 2011/0189735, 2011/0097339, 2010/0266605, 2010/0168400, 2009/0246204, 2008/0038273; and PCT publication nos. WO 2015198147, WO2016/05421, WO 2014/1209168, WO 2011/101791, WO 2010/007376, WO 2010/009391, WO 2004/004768, WO 2002/030459, WO2002/051871, and WO 2002/047721, each of which is incorporated herein by reference.

CD40 cluster of differentiation 40(CD40, also known as TNFRSF5) was found on a variety of immune system cells, including antigen presenting cells. CD40L, also known as CD154, is a ligand for CD40 and is in activated CD4⁺Transient expression on the surface of T cells. CD40 signaling is known to ' permit ' dendritic cell maturation and thereby trigger T cell activation and differentiation (see, e.g., O ' Sullivan et al (2003) crit]23(1):83-107. A variety of immune checkpoint modulators specific for CD40 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CD40. In some embodiments, the immune checkpoint modulator is an agent that binds to CD40 (e.g., an anti-CD 40 antibody). In some embodiments, the checkpoint modulator is a CD40 agonist. In some embodiments, the checkpoint modulator is a CD40 antagonist. In some embodiments, the immune checkpoint modulator is selected fromA CD40 binding protein of the group consisting of: dacislizumab (Genetech/Seattle Genetics), CP-870,893 (Pfizer), breluzumab (Astellas Pharma), lucartumab (Novartis), CFZ533 (Novartis; see, e.g., Cordoba et al (2015) am.J.Transplant. [ journal of U.S. transplantation (2015)), Cycizumab ozoga (Seattle Genetics)), Cycizuki-Karituximab (Novartis)), and Cycizuki-Miyabe ]2825-36), RG7876 (Genetech Inc.), FFP104 (GeneGenetics, B.V.), APX005 (Apexigen), BI 655064 (Boehringer Ingelheim), Chi Lob 7/4 (Cancer Research UK), see, e.g., Johnson et al (2015) clean]21(6) 1321-8), ADC-1013 (BioInvent International), SEA-CD40 (Seattle Genetics), XmAb 5485 (Xencor), PG120 (Genetics), tenecteb (Bethes-Meyer); see, e.g., Thompson et al (2011) am.j.transplant]11(5) 947-57), and AKH3 (Biogen; reference is made, for example, to international publication No. WO 2016/028810). Additional CD40 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. patent nos. 9,234,044, 9,266,956, 9,109,011, 9,090,696, 9,023,360, 9,023,361, 9,221,913, 8,945,564, 8,926,979, 8,828,396, 8,637,032, 8,277,810, 8,088,383, 7,820,170, 7,790,166, 7,445,780, 7,361,345, 8,961,991, 8,669,352, 8,957,193, 8,778,345, 8,591,900, 8,551,485, 8,492,531, 8,362,210, 8,388,971; U.S. patent application publication nos. 2016/0045597, 2016/0152713, 2016/0075792, 2015/0299329, 2015/00574372015/0315282, 2015/0307616, 2014/0099317, 2014/0179907, 2014/0349395, 2014/0234344, 2014/0348836, 2014/0193405, 2014/0120103, 2014/0105907, 2014/0248266, 2014/0093497, 2014/0010812, 2013/0024956, 2013/0023047, 2013/0315900, 2012/0087927, 2012/0263732, 2012/0301488, 2011/0027276, 2011/0104182, 2010/0234578, 2009/0304687, 2009/0181015, 2009/0130715, 2009/0311254, 2008/0199471, 2008/0085531, 2016/0152721, 2015/0110783, 2015/0086991, 2015/0086559, 2014/0341898, 2014/0205602, 2014/0004131, 2013/0011405, 2012/0121585, 2011/0033456, 2011/0002934, 2010/0172912, 2009/0081242, 2009/0130095, 2008/0254026, 2008/0075727, 2009/0304706, 2009/0202531, 2009/0117111, 2009/0041773, 2008/0274118, 2008/0057070, 2007/0098717, 2007/0218060, 2007/0098718, 2007/0110754; and PCT publication nos. WO 2016/069919, WO2016/023960, WO 2016/023875, WO 2016/028810, WO 2015/134988, WO 2015/091853, WO 2015/091655, WO 2014/065403, WO2014/070934, WO 2014/065402, WO 2014/207064, WO 2013/034904, WO 2012/125569, WO 2012/149356, WO 2012/111762, WO2012/145673, WO 2011/123489, WO 2010/123012, WO 2010/104761, WO 2009/094391, WO 2008/091954, WO 2007/129895, WO2006/128103, WO 2005/063289, WO 2005/063981, WO 2003/040170, WO 2002/011763, WO 2000/075348, WO 2013/164789, WO2012/075111, WO 2012/065950, WO 2009/062054, WO 2007/124299, WO 2007/053661, WO 2007/053767, WO 2005/044294, WO2005/044304, WO 2005/044306, WO 2005/044855, WO 2005/044854, WO 2005/044305, WO 2003/045978, WO 2003/029296, WO2002/028481, WO 2002/028480, WO 2002/028904, WO 2002/028905, WO 2002/088186, and WO 2001/024823, each of which is incorporated herein by reference.

CD122 is the beta subunit of interleukin-2 receptor and is known to increase CD8⁺Proliferation of effector T cells. See, e.g., Boyman et al (2012) nat. rev. immunol. [ natural review immunology ]]12(3):180-190. A variety of immune checkpoint modulators specific for CD122 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CD122. In some embodiments, the immune checkpoint modulator is an agent that binds to CD122 (e.g., an anti-CD 122 antibody). In some embodiments, the checkpoint modulator is a CD122 agonist. In some embodiments, the checkpoint modulator is a CD22 agonist. In some embodiments, the immune checkpoint modulator is humanized MiK- β -1 (Roche; see, e.g., Morris et al (2006) Proc Nat' l.Acad, sci, usa [ journal of the national academy of sciences of the united states of america]103(2) 401-6, which is incorporated by reference). Additional CD122 binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. patent No. 9,028,830, which is incorporated herein by reference.

Ox40.ox40 receptor (also known as CD134) promotes expansion of effector and memory T cells. OX40 also suppresses differentiation and activity of T regulatory cells and regulates cytokine production (see, e.g., Croft et al (2009) immunol. rev. [ [ immunological review ] ]229(1): 173-91). A variety of immune checkpoint modulators specific for OX40 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of OX40. In some embodiments, the immune checkpoint modulator is an agent that binds to OX40 (e.g., an anti-OX 40 antibody). In some embodiments, the checkpoint modulator is an OX40 agonist. In some embodiments, the checkpoint modulator is an OX40 antagonist. In some embodiments, the immune checkpoint modulator is an OX40 binding protein (e.g., an antibody) selected from the group consisting of: MEDI6469 (AgonOx/medimune), pogalizumab (pogallizumab) (also known as MOXR0916 and RG 7888; genetech Inc.)), tavlizumab (tavolizumab) (also known as MEDI 0562; Medimmune), and GSK3174998 (GlaxoSmithKline). Additional OX-40 binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 9,163,085, 9,040,048, 9,006,396, 8,748,585, 8,614,295, 8,551,477, 8,283,450, 7,550,140; U.S. patent application publication nos. 2016/0068604, 2016/0031974, 2015/0315281, 2015/0132288, 2014/0308276, 2014/0377284, 2014/0044703, 2014/0294824, 2013/0330344, 2013/0280275, 2013/0243772, 2013/0183315, 2012/0269825, 2012/0244076, 2011/0008368, 2011/0123552, 2010/0254978, 2010/0196359, 2006/0281072; and PCT publication nos. WO 2014/148895, WO 2013/068563, WO 2013/038191, WO2013/028231, WO 2010/096418, WO 2007/062245, and WO 2003/106498, each of which is incorporated herein by reference.

Glucocorticoid-induced TNFR family-associated Genes (GITR) are members of the Tumor Necrosis Factor Receptor (TNFR) superfamily, which are constitutively or conditionally expressed on Treg, CD4 and CD 8T cells. GITR is rapidly upregulated on effector T cells following TCR ligation and activation. Human GITR ligand (GITRL) is constitutively expressed on APCs in secondary lymphoid organs and some non-lymphoid tissues. Downstream effects of GITR: GITRL interaction induces attenuation of Treg activity and enhances CD4⁺T cell activity, leading to reversal of Treg-mediated immune suppression and enhanced immune stimulation. A variety of immune checkpoint modulators specific for GITR have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of GITR. In some embodiments, the immune checkpoint modulator is an agent that binds to GITR (e.g., an anti-GITR antibody). In some embodiments, the checkpoint modulator is a GITR agonist. In some embodiments, the checkpoint modulator is a GITR antagonist. In some embodiments, the immune checkpoint modulator is a GITR binding protein (e.g., an antibody) selected from the group consisting of: TRX518 (Leap Therapeutics), MK-4166 (Merck) &Co.)), MEDI-1873 (MedImmune), incagnn 1876 (amdius/generd (Agenus/Incyte)), and FPA154 (Five primary Therapeutics). Additional GITR binding proteins (e.g., antibodies) are known in the art and are described in, for example, U.S. patent nos. 9,309,321, 9,255,152, 9,255,151, 9,228,016, 9,028,823, 8,709,424, 8,388,967; U.S. patent application publication nos. 2016/0145342, 2015/0353637, 2015/0064204, 2014/0348841, 2014/0065152, 2014/0072566, 2014/0072565, 2013/0183321, 2013/0108641, 2012/0189639; and PCT publication nos. WO 2016/054638, WO2016/057841, WO 2016/057846, WO 2015/187835, WO 2015/184099, WO 2015/031667, WO 2011/028683, and WO 2004/107618, each of which is incorporated herein by reference.

Inducible T cell co-stimulator (ICOS, also known as CD278) is expressed on activated T cells. Its ligand is ICOSL expressed primarily on B cells and dendritic cells. ICOS is important in T cell effector function. ICOS expression is upregulated upon T cell activation (see, e.g., Fan et al (2014) j.exp.med. [ journal of experimental medicine ]211(4): 715-25). A variety of immune checkpoint modulators specific for ICOS have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of ICOS. In some embodiments, the immune checkpoint modulator is an agent that binds ICOS (e.g., an anti-ICOS antibody). In some embodiments, the checkpoint modulator is an ICOS agonist. In some embodiments, the checkpoint modulator is an ICOS antagonist. In some embodiments, the immune checkpoint modulator is an ICOS binding protein (e.g., an antibody) selected from the group consisting of: MEDI-570 (also known as JMab-136, Immunol), GSK3359609 (Kulansu Schker/INSERM), and JTX-2011 (Jounce Therapeutics). Additional ICOS binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 9,376,493, 7,998,478, 7,465,445, 7,465,444; U.S. patent application publication nos. 2015/0239978, 2012/0039874, 2008/0199466, 2008/0279851; and PCT publication No. WO 2001/087981, each of which is incorporated herein by reference.

4-1BB.4-1BB (also known as CD137) is a member of the Tumor Necrosis Factor (TNF) receptor superfamily. 4-1BB (CD137) is a type II transmembrane glycoprotein, which triggers CD4⁺And CD8⁺Expression is induced on T cells, activated NK cells, DCs and neutrophils and functions as a T cell co-stimulatory molecule when bound to 4-1BB ligand (4-1BBL) found on activated macrophages, B cells and DCs. Ligation of the 4-1BB receptor results in activation of NF-B, c-Jun and p38 signaling pathways, and has been shown to promote CD8 by up-regulating expression of the anti-apoptotic genes BcL-x (L) and Bfl-1⁺Survival of T cells. In this way, 4-1BB acts to enhance or even rescue the suboptimal immune response. A variety of immune checkpoint modulators specific for 4-1BB have been developed and may be used as disclosed herein. In some casesIn embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of 4-1BB. In some embodiments, the immune checkpoint modulator is an agent that binds to 4-1BB (e.g., an anti-4-1 BB antibody). In some embodiments, the checkpoint modulator is a 4-1BB agonist. In some embodiments, the checkpoint modulator is a 4-1BB antagonist. In some embodiments, the immune checkpoint modulator is a 4-1BB binding protein that is udeluzumab (also known as BMS-663513; behme schnobuba) or utoluzumab (feverfew). In some embodiments, the immune checkpoint modulator is a 4-1BB binding protein (e.g., an antibody). 4-1BB binding proteins (e.g., antibodies) are known in the art and are described in, for example, U.S. patent nos. 9,382,328, 8,716,452, 8,475,790, 8,137,667, 7,829,088, 7,659,384; U.S. patent application publication nos. 2016/0083474, 2016/0152722, 2014/0193422, 2014/0178368, 2013/0149301, 2012/0237498, 2012/0141494, 2012/0076722, 2011/0177104, 2011/0189189, 2010/0183621, 2009/0068192, 2009/0041763, 2008/0305113, 2008/0008716; and PCT publication nos. WO 2016/029073, WO2015/188047, WO 2015/179236, WO 2015/119923, WO 2012/032433, WO 2012/145183, WO 2011/031063, WO 2010/132389, WO2010/042433, WO 2006/126835, WO 2005/035584, WO 2004/010947; and Martinez-Forero et al (2013) j ]190(12) 6694-]59(8) 1223-33, each of which is incorporated herein by reference.

An inhibitory immune checkpoint molecule

The adora2a. adenosine A2A receptor (A2a4) is a member of the G protein-coupled receptor (GPCR) family, which possesses seven transmembrane α helices and is considered to be an important checkpoint in cancer therapy. The A2A receptor can down-regulate over-reacted immune cells (see, e.g., Ohta et al (2001) Nature [ Nature ]414(6866): 916-20). A variety of immune checkpoint modulators specific for ADORA2A have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of ADORA2A. In some embodiments, the immune checkpoint modulator is an agent that binds to ADORA2A (e.g., an anti-ADORA 2A antibody). In some embodiments, the immune checkpoint modulator is an ADORA2A binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is an ADORA2A agonist. In some embodiments, the checkpoint modulator is an ADORA2A antagonist. ADORA2A binding proteins (e.g., antibodies) are known in the art and are disclosed, for example, in U.S. patent application publication No. 2014/0322236, which is incorporated herein by reference.

B7-H3.B7-H3 (also known as CD276) belongs to the B7 superfamily, a group of molecules that co-stimulate or down-regulate T cell responses. B7-H3 effectively and consistently down-regulated human T cell responses (see, e.g., Leitner et al (2009) eur.j. immunol. [ european journal of immunology ]39(7): 1754-64). A variety of immune checkpoint modulators specific for B7-H3 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of B7-H3. In some embodiments, the immune checkpoint modulator is an agent that binds to B7-H3 (e.g., an anti-B7-H3 antibody). In some embodiments, the checkpoint modulator is a B7-H3 agonist. In some embodiments, the checkpoint modulator is a B7-H3 antagonist. In some embodiments, the immune checkpoint modulator is an anti-B7-H3 binding protein selected from the group consisting of: DS-5573 (Daiichi Sankyo, Inc.), enotuzumab (Macro genes, Inc.) and 8H9 (Sloan Kettering Institute for Cancer Research), see, e.g., Ahmed et al (2015) J.biol.Chem. [ J.Biol.290 (50):30018-29), in some embodiments, the immune checkpoint modulator is B7-H3 binding protein (e.g., antibody), B7-H3-binding protein (e.g., antibody) is known in the art, and in, e.g., U.S. Pat. Nos. 9,371,395, 9,150,656, 9,062,110, 8,802,091, 8,501,471, 8,414,892, U.S. patent application publication Nos. 2015/0352224, 2015/0297748, 2015/0259434, 4684, 4642, 58465, 465, 58 2013/0078234, 465, 58465, 573, 2002/0102264, respectively; PCT publication nos. WO 2016/106004, WO 2016/033225, WO 2015/181267, WO2014/057687, WO 2012/147713, WO 2011/109400, WO 2008/116219, WO 2003/075846, WO 2002/032375; and Shi et al (2016) mol.med.rep. [ molecular medical report ]14(1):943-8, each of which is incorporated herein by reference.

B7-H4.B7-H4 (also known as O8E, OV064 and V-set domain-containing inhibitors of T cell activation (VTCN1)) belong to the B7 superfamily. By preventing the cell cycle, the B7-H4 linkage of T cells has profound inhibitory effects on cell growth, cytokine secretion, and development of cytotoxicity. Administration of B7-H4Ig to mice impairs antigen-specific T cell responses, while blocking of endogenous B7-H4 by specific monoclonal antibodies promotes T cell responses (see, e.g., Sica et al (2003) Immunity [ immune ]18(6): 849-61). A variety of immune checkpoint modulators specific for B7-H4 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of B7-H4. In some embodiments, the immune checkpoint modulator is an agent that binds to B7-H4 (e.g., an anti-B7-H4 antibody). In some embodiments, the immune checkpoint modulator is a B7-H4 binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a B7-H4 agonist. In some embodiments, the checkpoint modulator is a B7-H4 antagonist. B7-H4 binding proteins (e.g., antibodies) are known in the art and are described in, for example, U.S. patent nos. 9,296,822, 8,609,816, 8,759,490, 8,323,645; U.S. patent application publication nos. 2016/0159910, 2016/0017040, 2016/0168249, 2015/0315275, 2014/0134180, 2014/0322129, 2014/0356364, 2014/0328751, 2014/0294861, 2014/0308259, 2013/0058864, 2011/0085970, 2009/0074660, 2009/0208489; and PCT publication nos. WO2016/040724, WO 2016/070001, WO 2014/159835, WO 2014/100483, WO 2014/100439, WO 2013/067492, WO 2013/025779, WO2009/073533, WO 2007/067991, and WO 2006/104677, each of which is incorporated herein by reference.

B and T Lymphocyte Attenuator (BTLA), also known as CD272, have HVEM (herpes virus entry mediator) as its ligand. In human CD8⁺During the phenotypic differentiation of T cells from naive to effector cells, the surface expression of BTLA was gradually down-regulated, but tumor-specific human CD8⁺T cells express high levels of BTLA (see, e.g., Derre et al (2010) j]120(1):157-67). A variety of immune checkpoint modulators specific for BTLA have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of BTLA. In some embodiments, the immune checkpoint modulator is an agent that binds BTLA (e.g., an anti-BTLA antibody). In some embodiments, the immune checkpoint modulator is a BTLA binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a BTLA agonist. In some embodiments, the checkpoint modulator is a BTLA antagonist. BTLA binding proteins (e.g., antibodies) are known in the art and are described in, for example, U.S. patent nos. 9,346,882, 8,580,259, 8,563,694, 8,247,537; U.S. patent application publication nos. 2014/0017255, 2012/0288500, 2012/0183565, 2010/0172900; and PCT publication nos. WO 2011/014438 and WO 2008/076560, each of which is incorporated herein by reference.

Cytotoxic T lymphocyte antigen 4(CTLA-4) is a member of the immunoregulatory CD28-B7 immunoglobulin superfamily and acts on naive and resting T lymphocytes to promote immune suppression via B7-dependent and B7-independent pathways (see, e.g., Kim et al (2016) j.immunol.res. [ journal of immunological research ], article ID4683607, page 14). CTLA-4 is also known as CD 152. CTLA-4 regulates the threshold of T cell activation. See, e.g., Gajewski et al (2001) j. immunol. [ journal of immunology ]166(6) 3900-7. A variety of immune checkpoint modulators specific for CTLA-4 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of CTLA-4. In some embodiments, the immune checkpoint modulator is an agent that binds to CTLA-4 (e.g., an anti-CTLA-4 antibody). In some embodiments, the checkpoint modulator is a CTLA-4 agonist. In some embodiments, the checkpoint modulator is a CTLA-4 antagonist. In some embodiments, the immune checkpoint modulator is a CTLA-4 binding protein (e.g., an antibody) selected from the group consisting of: epimemumab (Yervoy; Madary/Bezie McSt., Metare/Bristol-Myers Squibb), tremelimumab (formerly ticilimumab; Pemeride/Astrazeneca), JMW-3B3 (University of Aberdeen), and AGEN1884 (Agenus). Additional CTLA-4 binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 8,697,845; U.S. patent application publication nos. 2014/0105914, 2013/0267688, 2012/0107320, 2009/0123477; and PCT publication nos. WO 2014/207064, WO 2012/120125, WO 2016/015675, WO2010/097597, WO 2006/066568, and WO 2001/054732, each of which is incorporated herein by reference.

Indoleamine 2, 3-dioxygenase (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. Another important molecule is TDO, tryptophan 2, 3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate tregs and suppressor cells derived from myeloid lineage, and promote tumor angiogenesis. Prendergast et al, 2014, Cancer Immunol immunothers [ Cancer immunology and immunotherapy ]63(7):721-35, which are incorporated herein by reference.

A variety of immune checkpoint modulators specific for IDO have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of IDO. In some embodiments, the immune checkpoint modulator is an agent that binds to IDO (e.g., an IDO binding protein, such as an anti-IDO antibody). In some embodiments, the checkpoint modulator is an IDO agonist. In some embodiments, the checkpoint modulator is an IDO antagonist. In some embodiments, the immune checkpoint modulator is selected from the group consisting of: norharman, rosmarinic acid, COX-2 inhibitor, alpha-methyltryptophan, and etodolat. In one embodiment, the modulator is etoposide.

Killer cell immunoglobulin-like receptors (KIRs) comprise a diverse pool of mhc i binding molecules that negatively regulate Natural Killer (NK) cell function to protect cells from NK-mediated cell lysis. KIRs are usually expressed on NK cells, but have also been detected on tumor-specific CTLs. A variety of immune checkpoint modulators specific for KIRs have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of KIR. In some embodiments, the immune checkpoint modulator is an agent that binds to KIR (e.g., an anti-KIR antibody). In some embodiments, the immune checkpoint modulator is a KIR binding protein (e.g., an antibody). In some embodiments, the checkpoint modulator is a KIR agonist. In some embodiments, the checkpoint modulator is a KIR antagonist. In some embodiments, the immune checkpoint modulator is risreulumab (also known as BMS-986015; behcet schnobao). Additional KIR binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 8,981,065, 9,018,366, 9,067,997, 8,709,411, 8,637,258, 8,614,307, 8,551,483, 8,388,970, 8,119,775; U.S. patent application publication nos. 2015/0344576, 2015/0376275, 2016/0046712, 2015/0191547, 2015/0290316, 2015/0283234, 2015/0197569, 2014/0193430, 2013/0143269, 2013/0287770, 2012/0208237, 2011/0293627, 2009/0081240, 2010/0189723; and PCT publication nos. WO 2016/069589, WO 2015/069785, WO 2014/066532, WO 2014/055648, WO 2012/160448, WO2012/071411, WO 2010/065939, WO 2008/084106, WO 2006/072625, WO 2006/072626, and WO 2006/003179, each of which is incorporated herein by reference.

LAG-3, lymphocyte activation gene 3(LAG-3, also known as CD223), is a transmembrane protein associated with CD4 that competitively binds MHC II and serves as a co-inhibitory checkpoint for T cell activation (see, e.g., Goldberg and Drake (2011) curr. top. microbiology. immunol. [ microbiological and immunological recent topics ]344: 269-78). A variety of immune checkpoint modulators specific for LAG-3 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of LAG-3. In some embodiments, the immune checkpoint modulator is an agent that binds to LAG-3 (e.g., an anti-PD-1 antibody). In some embodiments, the checkpoint modulator is a LAG-3 agonist. In some embodiments, the checkpoint modulator is a LAG-3 antagonist. In some embodiments, the immune checkpoint modulator is a LAG-3-binding protein (e.g., an antibody) selected from the group consisting of: pembrolizumab (Keytruda; formerly Lamborzumab (lambrolizumab); Merck & Co., Inc.)), nivolumab (Opdivo; Postmei Shinobao), Pelizumab (CT-011, curative technology Co., Ltd.), SHR-1210 (Incyte/Jiangsu Hengrui Medicine Co., Ltd.), MEDI0680 (also known as AMP-514; applied immunization/medical immunization (amplimene Inc./medimune)), PDR001 (nova), BGB-a317 (BeiGene Ltd.), TSR-042 (also known as ANB 011; amber bio/Tesaro, Inc.), REGN2810 (regen Pharmaceuticals/Sanofi-anntat), and PF-06801591 (bright corporation). Additional PD-1-binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 9,181,342, 8,927,697, 7,488,802, 7,029,674; U.S. patent application publication nos. 2015/0152180, 2011/0171215, 2011/0171220; and PCT publication nos. WO2004/056875, WO 2015/036394, WO 2010/029435, WO 2010/029434, WO 2014/194302, each of which is incorporated herein by reference.

PD-1. programmed cell death protein 1(PD-1, also known as CD279 and PDCD1) is an inhibitory receptor that negatively regulates the immune system. In contrast to CTLA-4, which primarily affects naive T cells, PD-1 is more widely expressed on immune cells and modulates mature T cell activity in the surrounding tissues and tumor microenvironment. PD-1 inhibits T cell responses by interfering with T cell receptor signaling. PD-1 has two ligands, PD-L1 and PD-L2. A variety of immune checkpoint modulators specific for PD-1 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of PD-1. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-1 (e.g., an anti-PD-1 antibody). In some embodiments, the checkpoint modulator is a PD-1 agonist. In some embodiments, the checkpoint modulator is a PD-1 antagonist. In some embodiments, the immune checkpoint modulator is a PD-1-binding protein (e.g., an antibody) selected from the group consisting of: pembrolizumab (Keytruda; formerly Lambda; Merck), nivolumab (Opdivo; Peume Shinobao), Pelizumab (CT-011, curative technologies), SHR-1210 (Nesteru/Hengsu constant drug industries, Inc.), MEDI0680 (also known as AMP-514; applied immunization/medical immunization), PDR001 (Nowa), BGB-A317 (Baiji, Inc.), TSR-042 (also known as ANB 011; Andert Bio-Tesaro Co.), REGN2810 (Renyuan pharmaceuticals/Senofei-Androst), and PF-06801591 (Perey). Additional PD-1-binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 9,181,342, 8,927,697, 7,488,802, 7,029,674; U.S. patent application publication nos. 2015/0152180, 2011/0171215, 2011/0171220; and PCT publication nos. WO 2004/056875, WO 2015/036394, WO 2010/029435, WO2010/029434, WO 2014/194302, each of which is incorporated herein by reference.

PD-L1/PD-L2.PD ligand 1(PD-L1, also known as B7-H1) and PD ligand 2(PD-L2, also known as PDCD1LG2, CD273 and B7-DC) bind to PD-1 receptors. Both ligands belong to the same B7 family of B7-1 and B7-2 proteins that interact with CD28 and CTLA-4. PD-L1 can be expressed on many cell types including, for example, epithelial cells, endothelial cells, and immune cells. PDL-1 ligation reduces IFN, TNF, and IL-2 production and stimulates IL10 production, IL10 is an anti-inflammatory cytokine associated with reduced T cell reactivity and proliferation and antigen-specific T cell anergy. PDL-2 is expressed predominantly on Antigen Presenting Cells (APC). PDL2 ligation also leads to T cell suppression, but in the case of PDL-1-PD-1 interaction inhibiting proliferation by cell cycle arrest at G1/G2 phase, PDL2-PD-1 conjugation has been shown to inhibit TCR-mediated signaling by blocking B7: CD28 signaling at low antigen concentrations and reducing cytokine production at high antigen concentrations. A variety of immune checkpoint modulators specific for PD-L1 and PD-L2 have been developed and may be used as disclosed herein.

In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of PD-L1. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-L1 (e.g., an anti-PD-L1 antibody). In some embodiments, the checkpoint modulator is a PD-L1 agonist. In some embodiments, the checkpoint modulator is a PD-L1 antagonist. In some embodiments, the immune checkpoint modulator is a PD-L1-binding protein (e.g., an antibody or Fc fusion protein) selected from the group consisting of: duvaluzumab (also known as MEDI-4736; AstraZeneca/Celgene Corp.), Alterlizumab (Teentriq; also known as MPDL3280A and RG 7446; Genetech Inc.)), Avermemab (also known as MSB 0010718C; Merck Celanno./AstraZeneca)); MDX-1105 (Madary/Bezilla Shibao Co.), AMP-224 (Utility Immunity, Kulansu Schke Co.), LY3300054 (Li Lai Lilly and Co.). Additional PD-L1 binding proteins are known in the art and are disclosed, for example, in U.S. patent application publication nos. 2016/0084839, 2015/0355184, 2016/0175397, and PCT publication nos. WO 2014/100079, WO 2016/030350, WO 2013181634, each of which is incorporated herein by reference.

In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of PD-L2. In some embodiments, the immune checkpoint modulator is an agent that binds to PD-L2 (e.g., an anti-PD-L2 antibody). In some embodiments, the checkpoint modulator is a PD-L2 agonist. In some embodiments, the checkpoint modulator is a PD-L2 antagonist. PD-L2 binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 9,255,147, 8,188,238; U.S. patent application publication nos. 2016/0122431, 2013/0243752, 2010/0278816, 2016/0137731, 2015/0197571, 2013/0291136, 2011/0271358; and PCT publication nos. WO 2014/022758 and WO 2010/036959; each of which is incorporated herein by reference.

T-cell immunoglobulin mucin 3(TIM-3, also known as hepatitis A virus cell receptor (HAVCR2)) is a type I glycoprotein receptor that binds to the S-type lectin galectin 9 (Gal-9). TIM-3 is a ligand that is widely expressed on lymphocytes, liver, small intestine, thymus, kidney, spleen, lung, muscle, reticulocytes, and brain tissue. Tim-3 was originally identified as being selectively expressed on IFN- γ secreting Th1 and Tc1 cells (Monney et al (2002) Nature 415: 536-41). The binding of TIM-3 receptors to Gal-9 triggers downstream signaling, negatively regulating T cell survival and function. A variety of immune checkpoint modulators specific for TIM-3 have been developed and may be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of TIM-3. In some embodiments, the immune checkpoint modulator is an agent that binds to TIM-3 (e.g., an anti-TIM-3 antibody). In some embodiments, the checkpoint modulator is a TIM-3 agonist. In some embodiments, the checkpoint modulator is a TIM-3 antagonist. In some embodiments, the immune checkpoint modulator is an anti-TIM-3 antibody selected from the group consisting of: TSR-022 (amber Biol., Tesaro Co.) and MGB453 (Nowa Co.). Additional TIM-3 binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent nos. 9,103,832, 8,552,156, 8,647,623, 8,841,418; U.S. patent application publication nos. 2016/0200815, 2015/0284468, 2014/0134639, 2014/0044728, 2012/0189617, 2015/0086574, 2013/0022623; and PCT publication nos. WO 2016/068802, WO 2016/068803, WO 2016/071448, WO 2011/155607, and WO 2013/006490, each of which is incorporated herein by reference.

V-domain Ig repressor of T cell activation (VISTA, also known as platelet receptor Gi24) is an Ig superfamily ligand that negatively regulates T cell responses. See, e.g., Wang et al, 2011, j.exp.med. [ journal of experimental medicine]208:577-92. Direct repression of CD4 by VISTA expressed on APC⁺And CD8⁺T cell proliferation and cytokine production (Wang et al (2010) J Exp Med. [ practice)Medical examination magazine]208(3):577-92). A variety of immune checkpoint modulators specific for VISTA have been developed and can be used as disclosed herein. In some embodiments, the immune checkpoint modulator is an agent that modulates the activity and/or expression of VISTA. In some embodiments, the immune checkpoint modulator is an agent that binds to VISTA (e.g., an anti-VISTA antibody). In some embodiments, the checkpoint modulator is a VISTA agonist. In some embodiments, the checkpoint modulator is a VISTA antagonist. In some embodiments, the immune checkpoint modulator is a VISTA binding protein (e.g., an antibody) selected from the group consisting of: TSR-022 (amber Biol., Tesaro Co.) and MGB453 (Nowa Co.). VISTA binding proteins (e.g., antibodies) are known in the art and described in, for example, U.S. patent application publication nos. 2016/0096891, 2016/0096891; and PCT publication nos. WO 2014/190356, WO 2014/197849, WO 2014/190356, and WO 2016/094837, each of which is incorporated herein by reference.

Additional immunotherapies that may be used in the methods disclosed herein include, but are not limited to, Toll-like receptor (TLR) agonists, cell-based therapies, cytokines, and cancer vaccines.

TLR agonists

TLRs are single transmembrane, non-catalytic receptors that recognize structurally conserved molecules derived from microorganisms. TLRs form together with interleukin-1 receptors a receptor superfamily, referred to as the "interleukin-1 receptor/Toll-like receptor superfamily". Members of this family are structurally characterized by an extracellular leucine-rich repeat (LRR) domain, a conserved pattern of membrane-proximal cysteine residues, and an intracytoplasmic signaling domain that forms a platform for downstream signaling by recruiting adapters containing TIR domains (including MyD88), adapters containing TIR domains (TRAP), and adapters containing TIR domains (induced IFN β) (TRIF) (O' Neill et al, 2007, Nat Rev Immunol [ natural immunology review ]7,353).

TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR 10. TLR2 mediates cellular responses to a variety of microbial products, including peptidoglycans, bacterial lipopeptides, lipoteichoic acids, mycobacterial lipoarabinomannans, and yeast cell wall components. TLR4 is a transmembrane protein that belongs to the family of Pattern Recognition Receptors (PRRs). Its activation leads to the intracellular signaling pathway NF-. kappa.B and inflammatory cytokine production, which are responsible for activating the innate immune system. TLR5 is known to recognize bacterial flagellin from invading mobile bacteria and has been shown to be involved in the onset of many diseases, including inflammatory bowel disease.

TLR agonists are known in the art and are described, for example, in US2014/0030294, which is incorporated herein by reference in its entirety. Exemplary TLR2 agonists include mycobacterial cell wall glycolipids, Lipoarabinomannan (LAM) and mannosylated Phosphatidylinositol (PIIM), MALP-2 and Pam3Cys and synthetic variants thereof. Exemplary TLR4 agonists include lipopolysaccharide or synthetic variants thereof (e.g., MPL and RC529) and lipid a or synthetic variants thereof (e.g., aminoalkyl aminoglycoside 4-phosphate). See, e.g., Cluff et al, 2005, Infection and Immunity [ Infection and Immunity ], p.3044-3052: 73; lembo et al, 2008, The Journal of Immunology 180, 7574-E7581; and Evans et al, 2003, Expert Rev Vaccines [ review by vaccine experts ]2: 219-29. Exemplary TLR5 agonists include flagellin or synthetic variants thereof (e.g., pharmacologically optimized TLR5 agonists (e.g., CBLB502) with reduced immunogenicity made by deleting flagellin moieties that are not essential for TLR5 activation).

Other TLR agonists include colexin and bacillus calmette-guerin (BCG). The Colistin is a mixture of killed bacteria of the species Streptococcus pyogenes and Serratia marcescens. See Taniguchi et al, 2006, Anticancer Res [ Anticancer studies ]26 (6A): 3997-4002. BCG is prepared from live attenuated Mycobacterium bovis, Mycobacterium bovis. See Venkatasswamy et al, 2012, Vaccine 30(6), 1038-.

Cell-based therapies

Cell-based therapies for treating cancer include administering immune cells (e.g., T cells, Tumor Infiltrating Lymphocytes (TILs), natural killer cells, and dendritic cells) to a subject. In autologous cell-based therapies, the immune cells are derived from the same subject to which they are administered. In allogeneic cell-based therapy, immune cells are derived from one subject and administered to a different subject. Prior to administration to a subject, immune cells can be activated, for example, by treatment with cytokines. In some embodiments, e.g., in Chimeric Antigen Receptor (CAR) T cell immunotherapy, the immune cells are genetically modified prior to administration to a subject.

In some embodiments, the cell-based therapy comprises Adoptive Cell Transfer (ACT). ACT generally consists of three parts: lymphocyte depletion, cell administration, and high dose IL-2 therapy. Cell types that may be administered in ACT include Tumor Infiltrating Lymphocytes (TILs), T Cell Receptor (TCR) -transduced T cells, and Chimeric Antigen Receptor (CAR) T cells.

Tumor infiltrating lymphocytes are immune cells observed in many solid tumors including breast cancer. They are cell populations comprising cytotoxic and helper T cells and a mixture of B cells, macrophages, natural killer cells and dendritic cells. The general procedure for autologous TIL therapy is as follows: (1) digesting the excised tumor into fragments; (2) each fragment grows in IL-2 and lymphocyte proliferation destroys tumors; (3) expanding the lymphocytes after the presence of the pure lymphocyte population; and (4) amplification to 10 ¹¹After each cell, lymphocytes are injected into the patient. See Rosenberg et al, 2015, Science [ Science]348(6230) 62-68, which are incorporated herein by reference in their entirety.

TCR-transduced T cells are generated by genetic induction of tumor-specific TCRs. This is typically done by cloning specific antigen-specific TCRs into the retroviral backbone. Blood was drawn from the patient and Peripheral Blood Mononuclear Cells (PBMCs) were extracted. PBMC were stimulated with CD3 in the presence of IL-2 and then transduced with a retrovirus encoding an antigen-specific TCR. These transduced PBMCs were further expanded in vitro and infused back into the patient. See Robbins et al, 2015, Clinical Cancer Research 21(5):1019-1027, which is incorporated herein by reference in its entirety.

Chimeric Antigen Receptors (CARs) are recombinant receptors that comprise an extracellular antigen recognition domain, a transmembrane domain, and a cytoplasmic signaling domain (e.g., CD3 ζ, CD28, and 4-1 BB). CARs have both antigen binding and T cell activation functions. Thus, CAR-expressing T cells can recognize a variety of cell surface antigens, including glycolipids, carbohydrates, and proteins, and can attack malignant cells expressing these antigens by activating cytoplasmic co-stimulation. See Pang et al, 2018, Mol Cancer [ molecular Cancer ]17:91, which is incorporated herein by reference in its entirety.

In some embodiments, the cell-based therapy is a Natural Killer (NK) cell-based therapy. NK cells are large granular lymphocytes that have the ability to kill tumor cells without any prior sensitization or limitation of Major Histocompatibility Complex (MHC) molecules. See Uppentahl et al, 2017, Frontiers in Immunology 8: 1825. Adoptive transfer of autologous Lymphokine Activated Killer (LAK) cells in the context of high dose IL-2 therapy has been evaluated in human clinical trials. Similar to LAK immunotherapy, cytokine-induced killer (CIK) cells were generated from peripheral blood mononuclear cell cultures under stimulation with anti-CD 3 mAb, IFN-. gamma.and IL-2. CIK cells are characterized by a mixed T-NK phenotype (CD3+ CD56+) and show enhanced cytotoxic activity against ovarian and cervical cancers compared to LAK cells. Human clinical trials were also conducted to study adoptive transfer of autologous CIK cells following primary tumor debulking and adjuvant carboplatin/paclitaxel chemotherapy. See Liu et al, 2014, J Immunother [ J. Immunotherapy 37(2): 116-122.

In some embodiments, the cell-based therapy is a dendritic cell-based immunotherapy. Vaccination with tumor lysate treated Dendritic Cells (DCs) has been shown to increase therapeutic anti-tumor immune responses in vitro and in vivo. See Jung et al, 2018, Translational Oncology [ transformation Oncology ]11(3): 686-690. DCs capture and process antigens, migrate into lymphoid organs, express lymphocyte costimulatory molecules, and secrete cytokines that elicit an immune response. They also stimulate immune effector cells (T cells) expressing tumor-associated antigen-specific receptors and reduce the number of immunosuppressive factors (e.g., CD4+ CD25+ Foxp3+ regulatory T (treg) cells). For example, a tumor cell lysate-DC hybrid based Renal Cell Carcinoma (RCC) DC vaccination strategy has shown therapeutic potential in preclinical and clinical trials. See Lim et al, 2007, Cancer Immunol Immunother [ Cancer immunology and immunotherapy ]56: 1817-1829.

Cytokine

Several cytokines including IL-2, IL-12, IL-15, IL-18 and IL-21 have been used in the treatment of cancer to activate immune cells, such as NK cells and T cells. IL-2 is one of the first cytokines used clinically and is expected to induce anti-tumor immunity. As a single agent at high doses, IL-2 induces remission in certain Renal Cell Carcinoma (RCC) and metastatic melanoma patients. Low doses of IL-2 have also been studied and the aim is to selectively link the IL-2 α β γ receptor (IL-2R α β γ) to reduce toxicity while maintaining biological activity. See Romee et al, 2014, scientific, volume 2014, article ID 205796, page 18, which is incorporated herein by reference in its entirety.

Interleukin 15(IL-15) is a cytokine with a similar structure to Interleukin 2 (IL-2). Like IL-2, IL-15 binds and signals through a complex consisting of the IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD 132). Recombinant IL-15 has been evaluated for the treatment of solid tumors (e.g., melanoma, renal cell carcinoma) and support of NK cells following adoptive transfer in cancer patients. See Romee et al, cited above.

IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits (IL-12. alpha. and. beta. chains) and was originally identified as "NK cell stimulating factor (NKSF)" based on its ability to potentiate NK cytotoxicity. Upon encountering a pathogen, IL-12 is released by activated dendritic cells and macrophages and binds to its cognate receptor expressed primarily on activated T and NK cells. A number of preclinical studies have shown that IL-12 has anti-tumor potential. See Romee et al, cited above.

IL-18 is a member of the proinflammatory IL-1 family and, like IL-12, is secreted by activated phagocytes. IL-18 has shown significant anti-tumor activity in preclinical animal models and has been evaluated in human clinical trials. See Robertson et al, 2006, Clinical Cancer Research 12: 4265-4273.

IL-21 has been used in anti-tumor immunotherapy because of its ability to stimulate NK cells and CD8+ T cells. For ex vivo NK cell expansion, membrane-bound IL-21 has been expressed in K562 stimulated cells with potent results. See Denman et al, 2012, PLoS One [ public science library. integrated ]7(1) e 30264. Recombinant human IL-21 was also shown to increase soluble CD25 and induce expression of perforin and granzyme B on CD8+ cells. IL-21 has been evaluated in several clinical trials for the treatment of solid tumors. See Romee et al, cited above.

Cancer vaccine

Therapeutic cancer vaccines eliminate cancer cells by enhancing the patient's own immune response to cancer, particularly a CD8+ T cell-mediated response, with the aid of an appropriate adjuvant. The therapeutic efficacy of cancer vaccines depends on the differential expression of Tumor Associated Antigens (TAAs) by tumor cells relative to normal cells. TAAs are derived from cellular proteins and should be expressed predominantly or selectively on cancer cells to avoid immune tolerance or autoimmune effects. See, Circelli et al, 2015, Vaccines 3(3) 544-. Cancer vaccines include, for example, Dendritic Cell (DC) based vaccines, peptide/protein vaccines, genetic vaccines, and tumor cell vaccines. See Ye et al, 2018, J Cancer J9 (2): 263-268.

Combination therapy comprising an agent inducing iron-dependent cell disassembly and immunotherapy

Methods of treating a neoplastic disorder by administering to a subject an agent that induces iron-dependent cell disassembly in combination with at least one immune checkpoint modulator are provided. In certain embodiments, the immune checkpoint modulator stimulates an immune response in the subject. For example, in some embodiments, the immune checkpoint modulator stimulates or increases the expression or activity of a stimulatory immune checkpoint (e.g., CD27, CD28, CD40, CD122, OX40, GITR, ICOS, or 4-1 BB). In some embodiments, the immune checkpoint modulator inhibits or reduces the expression or activity of an inhibitory immune checkpoint (e.g., A2a4, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, or VISTA).

In certain embodiments, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, A2A4, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3 and VISTA. In certain embodiments, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: CD27, CD28, CD40, CD122, OX40, GITR, ICOS, 4-1BB, A2A4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3 and VISTA. In particular embodiments, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-L1 and PD-1. In another specific embodiment, the immune checkpoint modulator targets an immune checkpoint molecule selected from the group consisting of: PD-L1 and PD-1.

In some embodiments, more than one (e.g., 2, 3, 4, 5 or more) immune checkpoint modulator is administered to the subject. When more than one immune checkpoint modulator is administered, the modulators may each target a stimulatory immune checkpoint molecule, or each target an inhibitory immune checkpoint molecule. In other embodiments, the immune checkpoint modulator comprises at least one modulator targeting a stimulatory immune checkpoint and at least one immune checkpoint modulator targeting an inhibitory immune checkpoint molecule. In certain embodiments, the immune checkpoint modulator is a binding protein, such as an antibody. As used herein, the term "binding protein" refers to a protein or polypeptide that can specifically bind to a target molecule (e.g., an immune checkpoint molecule). In some embodiments, the binding protein is an antibody or antigen-binding portion thereof, and the target molecule is an immune checkpoint molecule. In some embodiments, the binding protein is a protein or polypeptide that specifically binds to a target molecule (e.g., an immune checkpoint molecule). In some embodiments, the binding protein is a ligand. In some embodiments, the binding protein is a fusion protein. In some embodiments, the binding protein is a receptor. Examples of binding proteins that can be used in the methods of the invention include, but are not limited to, humanized antibodies, antibody Fab fragments, bivalent antibodies, antibody drug conjugates, scFv, fusion proteins, bivalent antibodies, and tetravalent antibodies.

The term "antibody" as used herein refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains-two heavy (H) chains and two light (L) chains or any functional fragment, mutant, variant or derivative thereof. Such mutant, variant, or derivative antibody forms are known in the art. In a full-length antibody, each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is a murine antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a chimeric antibody. Chimeric and humanized antibodies can be prepared by methods well known to those skilled in the art, including CDR-grafting methods (see, e.g., U.S. Pat. Nos. 5,843,708, 6,180,370, 5,693,762, 5,585,089, and 5,530,101), chain shuffling strategies (see, e.g., U.S. Pat. No. 5,565,332; Rader et al (1998) PROC. NAT' L. ACAD. SCI.USA [ Proc. Natl. Acad. Sci. USA ]95:8910-8915), molecular modeling strategies (U.S. Pat. No. 5,639,641), and the like.

As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, bispecific or multispecific versions; for example, a multispecific form; specifically binds to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab')2 fragment which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al (1989) NATURE [ Nature ]341: 544-546; and WO 90/05144A 1, the contents of which are incorporated herein by reference) which comprise a single variable domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined using recombinant methods by a synthetic linker that enables them to be formed as a single protein chain, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) SCIENCE [ SCIENCE ]242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also contemplated. Antigen-binding moieties may also be incorporated into single domain antibodies, macroantibodies (maxibodes), minibodies (minibodies), nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology [ Nature Biotechnology ]23: 1126-.

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are three CDRs in each variable region of the heavy and light chains, called CDR1, CDR2, and CDR3, respectively. As used herein, the term "set of CDRs" refers to a set of three CDRs present in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently from system to system. The system described by Kabat (Kabat et al, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST [ immunologically significant protein SEQUENCES ] (National Institutes OF Health, Besseda, Md., 1987 and 1991) provides not only a clear residue numbering system suitable for any variable region OF an antibody, but also precise residue boundaries defining the three CDRs, which may be referred to as Kabat CDR & Chothia and co-workers, some sub-portions within Kabat CDRs adopt almost identical peptide backbone conformations despite the large diversity at the amino acid sequence level (Chothia et al (1987) J. MOL. BIOL. [ journal OF molecular biology ]196:901-917, and Chothia et al (1989) TURE [ natural ]342:877 ] 11. sub-L917, these sub-portions and NAoth L2 & NAH 26, where these sub-portions may be referred to as the heavy chain 8826, 368826 and 3 "H",368826 and 368826 "3" where these sub-chain regions are referred to as CHbat H26 and 368826, the boundaries of which overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in Padlan et al (1995) FASEB J. [ J. Association of Experimental biology ]9: 133-. Still other CDR boundary definitions may not strictly follow one of the above systems but still overlap with the Kabat CDRs, although they may be shortened or lengthened according to the following predictions or experimental findings: a particular residue or group of residues or even the entire CDR does not significantly affect antigen binding. Although the preferred embodiment uses Kabat or Chothia defined CDRs, the methods used herein may utilize CDRs defined according to any of these systems.

As used herein, the term "humanized antibody" refers to a non-human (e.g., murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab ', F (ab')2, or other antigen binding subsequences of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies/antibody fragments may comprise residues that are not found in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further improve and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one (typically two) variable domain, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a substantial portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For more details, see Jones et al (1986) NATURE [ Nature ]321: 522-525; reichmann et al (1988) NATURE [ Nature ]332: 323-329; and Presta (1992) CURR.OP.STRUCT.BIOL. [ Current views of structural biology ]2: 593-.

As used herein, the term "immunoconjugate" or "antibody drug conjugate" refers to the linkage of an antibody or antigen-binding fragment thereof to another agent, e.g., a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, etc. The linkage may be covalent or non-covalent, such as by electrostatic forces. To form an immunoconjugate, various linkers known in the art may be used. In addition, the immunoconjugate may be provided in the form of a fusion protein that is expressible from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to a protein produced by the ligation of two or more genes or gene fragments that originally encode separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each native protein.

"bivalent antibody" refers to an antibody or antigen-binding fragment thereof comprising two antigen-binding sites. Two antigen binding sites may bind to the same antigen, or they may each bind to a different antigen, in which case the antibody or antigen binding fragment is characterized as "bispecific". "tetravalent antibody" refers to an antibody or antigen-binding fragment thereof comprising four antigen-binding sites. In certain embodiments, the tetravalent antibody is bispecific. In certain embodiments, the tetravalent antibody is multispecific, i.e., binds to more than two different antigens.

Fab (fragment antigen binding) antibody fragments are immunoreactive polypeptides comprising a monovalent antigen binding domain of an antibody comprising a heavy chain variable region (V)_H) And heavy chain constant region 1 (C)_H1) Partially composed polypeptides and light chain variable (V)_L) And constant light chain (C)_L) A partially composed polypeptide, wherein C_LAnd C_H1The moieties are preferably held together by disulfide bonds between Cys residues.

Immune checkpoint modulator antibodies include, but are not limited to, at least 4 major classes: i) antibodies that directly block inhibitory pathways on T cells or Natural Killer (NK) cells (e.g., PD-1-targeting antibodies such as nivolumab and pembrolizumab, antibodies that target TIM-3, and antibodies that target LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, and KIR), ii) antibodies that directly activate stimulatory pathways on T cells or NK cells (e.g., antibodies that target OX40, GITR, and 4-1 BB), iii) antibodies that block inhibitory pathways on immune cells or deplete a suppressive population of immune cells by virtue of antibody-dependent cellular cytotoxicity (e.g., CTLA-4-targeting antibodies such as epilimumab, antibodies that target VISTA, and antibodies that target PD-L2, Gr1, and Ly 6G), and iv) antibodies that directly block suppressive pathways on cancer cells or enhance cytotoxicity on cancer cells by virtue of antibody-dependent cellular cytotoxicity (e.g., rituximab, antibodies targeting PD-L1, and antibodies targeting B7-H3, B7-H4, Gal-9, and MUC 1). Examples of checkpoint inhibitors include inhibitors such as CTLA-4, e.g., epilimumab or tremelimumab; inhibitors of the PD-1 pathway, such as anti-PD-1, anti-PD-L1 or anti-PD-L2 antibodies. Exemplary anti-PD-1 antibodies are described in WO 2006/121168, WO 2008/156712, WO2012/145493, WO 2009/014708 and WO 2009/114335. Exemplary anti-PD-L1 antibodies are described in WO 2007/005874, WO 2010/077634 and WO2011/066389, and exemplary anti-PD-L2 antibodies are described in WO 2004/007679.

In particular embodiments, the immune checkpoint modulator is a fusion protein, e.g., a fusion protein that modulates the activity of the immune checkpoint modulator.

In one embodiment, the immune checkpoint modulator is a therapeutic nucleic acid molecule, e.g., a nucleic acid that modulates the expression of an immune checkpoint protein or mRNA. Nucleic acid therapeutics are well known in the art. Nucleic acid therapeutics include single-stranded and double-stranded (i.e., nucleic acid therapeutics having a region of complementarity of at least 15 nucleotides in length) nucleic acids that are complementary to a target sequence in a cell. In certain embodiments, the nucleic acid therapeutic agent targets a nucleic acid sequence encoding an immune checkpoint protein.

Antisense nucleic acid therapeutics are single-stranded nucleic acid therapeutics, typically about 16 to 30 nucleotides in length, and are complementary to a target nucleic acid sequence in a target cell in culture or an organism.

In another aspect, the agent is a single-stranded antisense RNA molecule. The antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation stoichiometrically by base pairing with mRNA and physically impeding the translation machinery, see Dias, N. et al, (2002) Mol Cancer Ther [ molecular Cancer therapy ]1: 347-355. Antisense RNA molecules can have about 15-30 nucleotides that are complementary to the target mRNA. Patents relating to antisense nucleic acids, chemical modifications, and therapeutic uses include, for example: U.S. patent No. 5,898,031 relates to chemically modified RNA-containing therapeutic compounds; U.S. Pat. No. 6,107,094 relates to methods of using these compounds as therapeutic agents; U.S. patent No. 7,432,250 relates to a method of treating a patient by administering a single stranded chemically modified RNA-like compound; and U.S. patent No. 7,432,249 relates to pharmaceutical compositions containing single-stranded chemically modified RNA-like compounds. U.S. patent No. 7,629,321 relates to a method of cleaving a target mRNA using a single stranded oligonucleotide having a plurality of RNA nucleosides and at least one chemical modification. The entire contents of each patent listed in this paragraph are incorporated herein by reference.

Nucleic acid therapeutics useful in the methods of the invention also include double-stranded nucleic acid therapeutics. As used interchangeably herein, "RNAi agent", "double-stranded RNAi agent", double-stranded rna (dsRNA) molecule, also referred to as "dsRNA agent", "dsRNA", "siRNA", "iRNA agent" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary (as defined below) nucleic acid strands. As used herein, RNAi agents can also include dsiRNA (see, e.g., U.S. patent publication 20070104688, which is incorporated herein by reference). Typically, most of the nucleotides of each strand are ribonucleotides, but as described herein, each strand or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and/or modified nucleotides. Furthermore, as used herein, an "RNAi agent" can include a ribonucleotide with a chemical modification; the RNAi agent can include substantial modifications at multiple nucleotides. Such modifications may include all types of modifications disclosed herein or known in the art. For the purposes of the present specification and claims, any such modification, as used in siRNA type molecules, is encompassed by "RNAi agent". RNAi agents for use in the methods of the invention include agents with chemical modifications, as disclosed, for example, in WO/2012/037254 and WO2009/073809, the entire contents of each of which are incorporated herein by reference.

Immune checkpoint modulators may be administered at appropriate doses to treat neoplastic disorders, for example by using standard doses. One skilled in the art will be able to determine by routine experimentation an effective non-toxic amount of an immune checkpoint modulator for the purpose of treating a neoplastic disorder. Standard dosages of immune checkpoint modulators are known to those skilled in the art and can be obtained, for example, from product instructions provided by the manufacturer of the immune checkpoint modulator. Examples of standard doses of immune checkpoint modulators are provided in table 3 below. In other embodiments, the immune checkpoint modulator is administered at a dose that is different (e.g., lower) than the standard dose of the immune checkpoint modulator used to treat the tumor disorder at standard of care for the particular tumor disorder.

TABLE 3 exemplary Standard dosages of immune checkpoint modulators

In certain embodiments, the immune checkpoint modulator is administered at a dose that is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the standard dose of immune checkpoint modulator for the particular tumor disorder. In certain embodiments, the dose of administration of the immune checkpoint modulator is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the standard dose of the immune checkpoint modulator for the particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulators is administered, at least one of the immune checkpoint modulators is administered at a dose that is lower than the standard dose of immune checkpoint modulators for the particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulators is administered, at least two of the immune checkpoint modulators are administered at a dose that is lower than the standard dose of immune checkpoint modulators for the particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulators is administered, at least three of the immune checkpoint modulators are administered at a dose that is lower than the standard dose of immune checkpoint modulators for the particular tumor disorder. In one embodiment, where a combination of immune checkpoint modulators is administered, all of the immune checkpoint modulators are administered at a dose that is lower than the standard dose of immune checkpoint modulators for the particular tumor disorder.

Co-administration of an agent that induces iron-dependent cell disassembly and an immune checkpoint modulator

As used herein, the terms "combined administration", "co-administration" or "co-administration" refer to the administration of an agent that induces iron-dependent cellular disassembly prior to, simultaneously or substantially simultaneously, subsequently or intermittently with the administration of an immune checkpoint modulator. In certain embodiments, the agent that induces iron-dependent cell disassembly is administered prior to administration of the immune checkpoint modulator. In certain embodiments, the agent that induces iron-dependent cell disassembly is administered concurrently with the immune checkpoint modulator. In certain embodiments, the agent that induces iron-dependent cell disassembly is administered after administration of the immune checkpoint modulator.

The agent that induces iron-dependent cell disassembly and the immune checkpoint modulator may act additively or synergistically. In one embodiment, the agent that induces iron-dependent cell disassembly and the immune checkpoint modulator act synergistically. In some embodiments, the synergistic effect is in the treatment of an oncological disorder. For example, in one embodiment, the combination of an agent that induces iron-dependent cell disassembly and an immune checkpoint modulator improves the durability, i.e., prolongs the duration, of the immune response against the cancer targeted by the immune checkpoint modulator. In some embodiments, the agent that induces iron-dependent cell disassembly and the immune checkpoint modulator add up.

The combination therapy of the present invention may be used to treat oncological disorders. In some embodiments, the combination therapy of an agent that induces iron-dependent cell disassembly and an immune checkpoint modulator inhibits tumor cell growth. Accordingly, the present invention further provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject an agent that induces iron-dependent cell disassembly and at least one immune checkpoint modulator, thereby inhibiting tumor cell growth. In certain embodiments, treating cancer comprises prolonging survival or prolonging time to tumor progression as compared to a control. In some embodiments, the control is a subject treated with an immune checkpoint modulator but not with an agent that induces iron-dependent cell disassembly. In some embodiments, the control is a subject treated with an agent that induces iron-dependent cell disassembly but not treated with an immune checkpoint modulator. In some embodiments, the control is a subject that is not treated with an immune checkpoint modulator or an agent that induces iron-dependent cell disassembly. In certain embodiments, the subject is a human subject. In a preferred embodiment, the subject is identified as having a tumor prior to administering the first dose of the agent that induces iron-dependent cell disassembly or the first dose of the immune checkpoint modulator. In certain embodiments, the subject has a tumor at the first administration of an agent that induces iron-dependent cell disassembly or at the first administration of an immune checkpoint modulator.

In certain embodiments, the subject is administered at least 1, 2, 3, 4, or 5 cycles of the combination therapy. Subjects were evaluated for response criteria at the end of each cycle. Adverse events (e.g., blood clotting, anemia, liver and kidney function, etc.) were also monitored throughout each cycle to ensure that the treatment regimen was adequately tolerated.

It should be noted that more than one immune checkpoint modulator, e.g., 2, 3, 4, 5 or more immune checkpoint modulators, may be administered in combination with an agent that induces iron-dependent cell disassembly.

In one embodiment, administration of the agent that induces iron-dependent cell disassembly and the immune checkpoint modulator described herein results in one or more of: reducing tumor size, weight, or volume, increasing time to progression, inhibiting tumor growth, and/or prolonging survival of a subject having a neoplastic disorder. In certain embodiments, administration of an agent that induces iron-dependent cell disassembly and an immune checkpoint modulator reduces tumor size, weight, or volume, increases time to progression, inhibits tumor growth, and/or extends survival of a subject by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% relative to a corresponding control subject administered the agent that induces iron-dependent cell disassembly alone or the immune checkpoint modulator alone. In certain embodiments, administration of an agent that induces iron-dependent cell disassembly and an immune checkpoint modulator reduces tumor size, weight, or volume, increases time to progression, inhibits tumor growth, and/or extends survival by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% in a population of subjects suffering from a neoplastic disorder relative to a corresponding population of control subjects suffering from a neoplastic disorder administered the agent that induces iron-dependent cell disassembly alone or the immune checkpoint modulator alone. In other embodiments, the administration of an agent that induces iron-dependent cell disassembly and an immune checkpoint modulator to a subject having a progressive neoplastic disorder prior to treatment stabilizes the neoplastic disorder.

In certain embodiments, treatment with an agent that induces iron-dependent cell disassembly and at least one immune checkpoint modulator is combined with an additional anti-neoplastic agent (e.g., standard of care for treating the particular cancer to be treated, e.g., by administering a standard dose of one or more anti-neoplastic agents (e.g., chemotherapeutic agents)). Standard of care for a particular cancer type can be determined by one skilled in the art based on, for example, the type and severity of the cancer, the age, weight, sex, and/or medical history of the subject, and the success or failure of prior treatments. In certain embodiments of the invention, standard of care includes any one or combination of surgery, radiation, hormone therapy, antibody therapy, growth factor therapy, cytokines, and chemotherapy. In one embodiment, the additional anti-neoplastic agent is not an iron-dependent cell disassembly inducing agent and/or an immune checkpoint modulator.

Other antineoplastic agents suitable for use in the methods disclosed herein include, but are not limited to, chemotherapeutic agents (e.g., alkylating agents (e.g., alexmmine, busulfan, carboplatin, carmustine, chlorobutyric acid, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxaliplatin, temozolomide, tiatipar)), antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine CytarabineFloxuridine, fludarabine, gemcitabineHydroxyurea, methotrexate, pemetrexedAntitumor antibiotics, e.g. anthracyclines (e.g. daunorubicin, doxorubicin)Epirubicin, idarubicin), actinomycin-D, bleomycin, mitomycin-C, mitoxantrone (also used as topoisomerase II inhibitor); topoisomerase inhibitors, such as topotecan, irinotecan (CPT-11), etoposide (VP-16), teniposide, mitoxantrone (also used as an antitumor antibiotic); mitotic inhibitors such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, vinorelbine; corticosteroids, e.g. prednisone, methylprednisoloneDexamethasoneEnzymes, e.g. L-asparaginase and bortezomibAntineoplastic agents also include biological anticancer agents, such as anti-TNF antibodies, e.g., adalimumab or infliximab; anti-CD 20 antibodies, e.g., rituximab, anti-VEGF antibodies (e.g., bevacizumab); anti-HER 2 antibodies, such as trastuzumab; anti-RSV, e.g. palivizumab. Cancers treated using the methods of the invention include, for example, all types of cancers or neoplasms or malignancies found in mammals, including but not limited to: sarcomas, melanomas, epithelial cancers, leukemias, and lymphomas.

The term "sarcoma" generally refers to a tumor that is composed of a substance, such as embryonic connective tissue, and is generally composed of tightly packed cells embedded in a fibrous or homogeneous substance. Examples of sarcomas that can be treated by the methods of the invention include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, myxosarcoma, osteosarcoma, Abelmoschus's sarcoma, liposarcoma, alveolar soft tissue sarcoma, amelogenic sarcoma, botryoid sarcoma, chloroma, choriocarcinoma, embryosarcoma, Wilms ' sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fasciosarcoma, fibrosarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple-sex-pigment hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Enjensen's sarcoma (Jensen's sarcoma), Kaposi's sarcoma, Kupffer's sarcoma, and Kupffer's cell sarcoma, Angiosarcoma, leukemic sarcoma, malignant stromal tumor sarcoma, paraosteosarcoma, reticulocytic sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, uterine sarcoma, mucoid liposarcoma, leiomyosarcoma, spindle cell sarcoma, desmoplastic sarcoma, and telangiectatic sarcoma.

The term "melanoma" refers to tumors derived from the melanocytic system of the skin and other organs. Melanoma that may be treated by the methods of the present invention include, for example, acromelastoma melanoma (acral-lentiginous melanoma), melanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, malignant lentigo melanoma, malignant melanoma, nodular melanoma, sublingual melanoma, and superficial spreading melanoma.

The term "epithelial cancer" refers to a malignant new growth made up of epithelial cells that tends to infiltrate the surrounding tissue and cause metastasis. As described herein, epithelial cancers that can be treated with the methods of the invention include, for example, acinar cancer, adenocarcinoma, adenocystic cancer, adenoid cystic cancer, adenoma cancer, adrenocortical cancer, alveolar carcinoma, alveolar cell cancer, basal epithelial cell cancer, basal-like cancer, basal squamous cell cancer, bronchoalveolar carcinoma, bronchial cancer, bronchogenic cancer, cerebroid cancer, cholangiocellular cancer, chorioepithelial cancer, jelly-like cancer, colonic adenocarcinoma of the colon, acne-like cancer, uterine corpus cancer, cribriform cancer, armor cancer, skin cancer, columnar cell cancer, ductal cancer, dural cancer, embryonal cancer, medullary cancer, epithelioid cancer, adenoid epithelial cell cancer, explanted cancer, ulcerative gastric cancer (carcinoma ex ulcer), fibroma, jelly-like cancer, giant cell cancer (giantcell carcinoma), giant cell carcinoma (carcinomular), adenocarcinoma, Granulosa cell carcinoma, choriocarcinoma, leukemia, hepatocellular carcinoma, schlempe cell carcinoma (Hurthle cell carcinoma), hyaline carcinoma, adenoid carcinoma of the kidney, immature embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, crohn's carcinoma (kromphecher's carcinoma), Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma (carcinosoma), lenticular carcinoma (carcinosculate), lipoma carcinoma, lymphoepithelial carcinoma, medullary carcinoma (carcinoid medullaria), medullary carcinoma (medullaria carcinosoma), melanosquamous carcinoma, squamous cell carcinoma (carcinoma molle), merkel cell carcinoma (merkel carcinosoma), mucinous carcinoma (mucomucocutaneous carcinoma, mucocutaneous carcinoma (mucoma), papillary carcinoma (mucoma molle carcinoma), mucoid carcinoma (mucosis), mucosis carcinoma, mucoma carcinoma (mucoma), mucosis carcinoma of the phylum, mucoma), mucoma carcinoma of the phylum, and squamous cell carcinoma of the lung Pre-invasive carcinoma, echinocytic carcinoma, brain-like carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcoid carcinoma, schneiderian carcinoma (schneiderian carcinosoma), scleroma, scrotal carcinoma (carcinosa scroti), signet cell carcinoma, simple carcinoma, small cell carcinoma, eggplant carcinoma (solanoid carcinosa), globular cell carcinoma, spindle cell carcinoma, medullary carcinoma (carcinosum), squamous cell carcinoma (squamous carcinosa), squamous cell carcinoma (squamous l carcinosa), tethered carcinosa (stringcarcinosma), vascular carcinoma (carcinosa epidermicutentimatism), angioectatic carcinoma (carcinosa telematodes), transitional cell carcinoma, nodular skin carcinoma (carcinosa tuberositism), dermatocarcinoma (warts carcinosa), nodular skin carcinoma (carcinosa), squamous cell carcinoma of the cervix carcinoma, and squamous cell carcinoma of the cervix. In particular embodiments, the cancer is renal cell carcinoma.

The term "leukemia" refers to a type of cancer of the blood or bone marrow characterized by an abnormal increase in immature white blood cells (referred to as "blast cells"). Leukemia is a broad term covering a wide range of diseases. It is, in turn, part of a broader group of diseases affecting the blood, bone marrow and lymphatic system, all known as blood neoplasms. Leukemias can be divided into four major categories: acute lymphoblastic (lymphoblastic) leukemia (ALL), acute myeloid (or myeloid or non-lymphoid) leukemia (AML), Chronic Lymphocytic Leukemia (CLL) and Chronic Myeloid Leukemia (CML). Other types of leukemia include Hairy Cell Leukemia (HCL), T-cell lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia. In certain embodiments, the leukemia includes acute leukemia. In certain embodiments, the leukemia includes chronic leukemia.

The term "lymphoma" refers to a group of blood cell tumors that develop from lymphocytes. Two major classes of lymphoma are Hodgkin Lymphoma (HL) and non-hodgkin lymphoma (NHL). Lymphoma includes any neoplasm of lymphoid tissue. The main category is cancer of lymphocytes, a kind of white blood cells that belong to both the lymph fluid and the blood and are spread throughout both.

In some embodiments, the compositions are used to treat various types of solid tumors, such as breast cancer (e.g., triple negative breast cancer), bladder cancer, genitourinary tract cancer, colon cancer, rectal cancer, endometrial cancer, kidney (renal cell) cancer, pancreatic cancer, prostate cancer, thyroid cancer (e.g., papillary thyroid cancer), skin cancer, bone cancer, brain cancer, cervical cancer, liver cancer, stomach cancer, oral and oral cancer, esophageal cancer, adenoid cystic cancer, neuroblastoma, testicular cancer, uterine cancer, thyroid cancer, head and neck cancer, kidney cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelioma, ovarian cancer, sarcoma, gastric cancer, uterine cancer, cervical cancer, medulloblastoma, and vulvar cancer. In certain embodiments, the skin cancer comprises melanoma, squamous cell carcinoma, and cutaneous T-cell lymphoma (CTCL).

Additional cancers that may be treated with the compositions of the invention include, for example, multiple myeloma, primary thrombocytosis, primary macroglobulinemia, malignant pancreatic insulinoma, malignant carcinoid, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, and malignant fibrous histiocytoma.

In some embodiments, the combination therapies described herein can be administered to a subject who has previously failed to treat cancer with another anti-tumor (e.g., chemotherapy) regimen. A "subject with failed anti-tumor regimen" is a subject with cancer who has not responded or stopped responding to treatment with an anti-tumor regimen according to RECIST 1.1 criteria, i.e., has not reached a complete response, a partial response, or disease stability in the target lesion; or does not achieve a complete response or non-CR/non-PD of the non-target lesion during or after the completion of an anti-tumor treatment regimen, either alone or in combination with surgery and/or radiation therapy, as may be generally indicated clinically in combination with anti-tumor therapy. RECIST 1.1 criteria are described, for example, in Eisenhauer et al, 2009, eur.j. cancer [ european journal of cancer ]45:228-24 (incorporated herein by reference in its entirety), and discussed in more detail below. Failure of an anti-tumor therapy results in, for example, tumor growth, increased tumor burden, and/or tumor metastasis. A failed anti-tumor regimen as used herein includes a treatment regimen that is terminated by dose-limiting toxicity (e.g., grade III or IV toxicity that cannot be addressed to allow continuation or resumption of treatment with the anti-tumor agent or regimen that caused the toxicity). In one embodiment, the subject has failed treatment with an anti-tumor regimen comprising administration of one or more anti-angiogenic agents.

Failed anti-tumor regimens include treatment regimens that do not result in at least stable disease for an extended period of time for all target and non-target lesions, e.g., at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 18 months, or less than any period of clinically defined cure. Failed anti-tumor regimens include treatment regimens that result in progressive disease of at least one target lesion during treatment with an anti-tumor agent, or that result in progressive disease less than 2 weeks, less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 12 months or less than 18 months, or less than any clinically defined period of cure after the treatment regimen is concluded.

A failed anti-tumor regimen does not include a treatment regimen in which the subject being treated for the cancer reaches a clinically defined cure (e.g., a 5 year complete response) after the treatment regimen is over, and wherein the subject is subsequently diagnosed with a different cancer after the treatment regimen is over, e.g., more than 5 years, more than 6 years, more than 7 years, more than 8 years, more than 9 years, more than 10 years, more than 11 years, more than 12 years, more than 13 years, more than 14 years, or more than 15 years.

RECIST criteria are clinically accepted evaluation criteria for providing standard methods for solid tumor measurement and for providing definitions for objective evaluation of tumor size changes in clinical trials. Such criteria may also be used to monitor the response of an individual undergoing treatment for a solid tumor. RECIST 1.1 criteria are discussed in detail in Eisenhauer et al, 2009, eur.j. cancer [ european journal of cancer ]45:228-24, which is incorporated herein by reference. The response criteria for the target lesion include:

complete Response (CR): all target lesions disappeared. Any pathological lymph node (whether targeted or non-targeted) must reduce the short axis to <10 mm.

Partial Response (PR): the sum of target lesion diameters is reduced by at least 30% with reference to the sum of baseline diameters.

Progressive Disease (PD): the sum of the diameters of the target lesions increased by at least 20%, referenced to the smallest sum in the study (including the baseline sum if the baseline sum in the study was smallest). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered to be progressive.)

Stability Disease (SD): with reference to the smallest sum of diameters at the time of study, there was neither sufficient shrinkage to satisfy PR nor sufficient increase to satisfy PD.

RECIST 1.1 standard also considers non-target lesions, which are defined as measurable but not necessarily measured lesions, that should be assessed only qualitatively at the desired time points. Response criteria for non-target lesions include:

complete Response (CR): all non-target lesions disappeared and tumor marker levels normalized. All lymph node sizes must be non-pathological (minor axis <10 mm).

non-CR/non-PD: the persistence of one or more non-target lesions and/or the tumor marker levels are maintained above normal limits.

Progressive Disease (PD): there is clear progress in non-target lesions. The appearance of one or more new lesions is also considered to be progressing. To achieve "clear progression" on the basis of non-target disease, the overall level of non-target disease must be severely exacerbated so that even in the presence of SD or PR in the target disease, the overall tumor burden has increased sufficiently to merit discontinuation of therapy. A modest "increase" in the size of one or more non-target lesions is often insufficient to justify a definite state of progression. Thus, it is extremely rare to assign overall progression based only on changes in non-target disease facing either SD or PR in the target disease.

In some embodiments, the combination therapies described herein can be administered to a subject having a refractory cancer. "refractory cancer" is a surgically ineffective malignancy that is initially unresponsive to chemotherapy or radiation therapy, or unresponsive to chemotherapy or radiation therapy over time.

Pharmaceutical compositions and modes of administration

The pharmaceutical compositions described herein may be administered to a subject in any suitable formulation. These include, for example, liquid, semi-solid, and solid dosage forms. The preferred form depends on the intended mode of administration and therapeutic application.

In certain embodiments, the composition is suitable for oral administration. In certain embodiments, the formulations are suitable for parenteral administration, including topical administration and intravenous, intraperitoneal, intramuscular, and subcutaneous injection. In particular embodiments, the composition is suitable for intravenous administration.

Pharmaceutical compositions for parenteral administration comprise aqueous solutions of the active compounds in water-soluble form. For intravenous administration, the formulation may be an aqueous solution. The aqueous solution may include Hank's solution, Ringer's solution, Phosphate Buffered Saline (PBS), physiological saline buffer, or other suitable salts or combinations to achieve a suitable pH and osmotic pressure for parenteral delivery formulations. The aqueous solution may be used to dilute the formulation to a desired concentration for application. The aqueous solution may contain substances which increase the viscosity of the solution, such as sodium carboxymethyl cellulose, sorbitol or dextran. In some embodiments, the formulation comprises a phosphate buffered saline solution comprising disodium phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, and water for injection.

Formulations suitable for topical application include liquid or semi-liquid preparations suitable for penetration through the skin, such as liniments, lotions, creams, ointments or pastes, and drops suitable for ocular, otic or nasal administration. Formulations suitable for oral administration include those containing an inert diluent or an ingestible carrier. Formulations for oral administration may be encapsulated in hard or soft shell gelatin capsules, or may be compressed into tablets, or may be combined directly with the food in the diet. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. Pharmaceutical compositions suitable for use in the present invention include compositions in which an effective amount of the active ingredient is included to achieve its intended purpose. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredient, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.

It will be apparent to those skilled in the art that useful in vivo dosages to be administered and the particular mode of administration will vary depending upon the age, body weight, severity of the condition and the mammalian species being treated, the particular compound employed, and the particular use for which it is to be used. One skilled in the art can determine effective dosage levels, i.e., the dosage levels required to achieve a desired result, using routine methods, such as human clinical trials, animal models, and in vitro studies.

In certain embodiments, the composition is delivered orally. In certain embodiments, the composition is administered parenterally. In certain embodiments, the composition is delivered by injection or infusion. In certain embodiments, the composition is delivered topically (including transmucosally). In certain embodiments, the composition is delivered by inhalation. In one embodiment, the compositions provided herein can be administered by direct injection into a tumor. In some embodiments, the composition may be administered by intravenous injection or intravenous infusion. In certain embodiments, the administration is systemic. In certain embodiments, the administration is topical.

Identification method of immunostimulant inducing iron-dependent cell disassembly

In addition to agents known in the art and described herein that induce disassembly of iron-dependent cells, the present disclosure also relates to methods of identifying other compounds that induce disassembly of iron-dependent cells and stimulate immune activity.

For example, in certain aspects, the disclosure relates to methods of screening for an immunostimulatory agent, the method comprising:

(a) providing a plurality of test agents (e.g., a library of test agents);

(b) assessing the ability of each of the plurality of test agents to induce iron-dependent cell disassembly (e.g., iron death);

(c) selecting a test agent that increases iron-dependent cell disassembly (e.g., iron death) as a candidate immunostimulatory agent; and is

(d) Evaluating the ability of the candidate immunostimulant to increase an immune response.

In some embodiments, assessing the ability of a test agent to induce iron-dependent cell disassembly (e.g., iron death) comprises contacting the cell or tissue with each of a plurality of test agents.

Several methods are known in the art and can be used to identify cells that undergo iron-dependent cell disassembly (e.g., iron death) and to distinguish from other types of cell disassembly and/or cell death by detecting specific markers. (see, e.g., Stockwell et al, 2017, Cell [ Cell ]171: 273-. For example, since disassembly of iron-dependent cells may be caused by lethal lipid peroxidation, measuring lipid peroxidation provides a method of identifying cells undergoing iron-dependent cell disassembly. C11-BODIPY and Liperfluor are lipophilic ROS sensors that provide a rapid, indirect means to detect the lipid ROS (Dixon et al, 2012, Cell [ Cell ]149: 1060-1072). Liquid Chromatography (LC)/tandem Mass Spectrometry (MS) analysis can also be used to directly detect specific oxidized lipids (Friedmann Angeli et al, 2014, nat. cell Biol. [ Nature cell biology ]16: 1180-. Isoprostaglandin and Malondialdehyde (MDA) can also be used to measure lipid peroxidation (Milne et al, 2007, nat. Protoc. [ Nature laboratory Manual ]2: 221-. Kits for measuring MDA are commercially available (biyuntian (Beyotime), Haimen (Haimen), china).

Other useful assays for studying iron-dependent cell disassembly include measuring iron abundance and GPX4 activity. Iron abundance can be measured using inductively coupled plasma-MS or calcein AM quenching and other specific iron probes (Hirayama and Nagasawa,2017, J.Clin.biochem.Nutr. [ J.Clin.Biochem.J. ]60: 39-48; Spangler et al, 2016, nat.chem.biol. [ Nature Chem.Biol. ]12:680-685), while GPX4 activity can be detected using LC-MS using phosphatidylcholine hydrogen peroxide reduction in Cell lysates (Yang et al, 2014, Cell [ Cell ]156: 317: 331). In addition, iron-dependent cell disassembly can be assessed by measuring Glutathione (GSH) content. GSH may be measured, for example, by using the commercially available GSH-Glo glutathione assay (Promega, Madison, wisconsin).

Iron-dependent cell disassembly can also be assessed by measuring the expression of one or more marker proteins. Suitable marker proteins include, but are not limited to, glutathione peroxidase 4(GPX4), prostaglandin endoperoxide synthase 2(PTGS2), and cyclooxygenase 2 (COX-2). The level of expression of a marker protein or a nucleic acid encoding a marker protein can be determined using suitable techniques known in the art, including, but not limited to, Polymerase Chain Reaction (PCR) amplification reactions, reverse transcriptase PCR analysis, quantitative real-time PCR, single-strand conformation polymorphism analysis (SSCP), mismatch cleavage detection, heteroduplex analysis, northern blot analysis, western blot analysis, in situ hybridization, array analysis, deoxyribonucleic acid sequencing, restriction fragment length polymorphism analysis, and combinations or subcombinations thereof.

In some embodiments, assessing the ability of a test agent to induce iron-dependent cellular disassembly comprises measuring the level or activity of an iron death marker, e.g., a marker selected from the group consisting of: lipid peroxidation, Reactive Oxygen Species (ROS), isoprostaglandin, Malondialdehyde (MDA), iron, glutathione peroxidase 4(GPX4), prostaglandin-endoperoxide synthase 2(PTGS2), cyclooxygenase 2(COX-2), and Glutathione (GSH).

In some embodiments, assessing the ability of a test agent to induce iron-dependent cellular disassembly comprises comparing the level or activity of the marker in a cell or tissue contacted with the test agent to the level or activity of the marker in a control cell or tissue not contacted with the test agent.

In one embodiment, assessing the ability of a test agent to induce iron-dependent cellular disassembly comprises measuring lipid peroxides in cells or tissues contacted with the test agent.

In one embodiment, an increase in the level of lipid peroxide in a cell or tissue contacted with a test agent indicates that the test agent is an agent that induces iron-dependent cell disassembly.

In one embodiment, assessing the ability of a test agent to induce iron-dependent cell disassembly further comprises assessing whether one or more activities of the test agent (e.g., markers that modulate iron death (e.g., lipid peroxidation) and/or immunostimulatory activity) are inhibited by known iron death inhibitors (e.g., iron-based statins, B-mercaptoethanol, or iron chelators).

In one embodiment, assessing the ability of a candidate immunostimulatory agent to increase an immune response comprises assessing the immunostimulatory activity of a test agent that induces iron-dependent cell disassembly. Any of the methods described herein for assessing an immune response may be used to assess a candidate immune stimulant.

In one embodiment, the immune cell is a THP-1 cell. For example, NF κ B and IRF activity may be measured in commercially available THP1-Dual cells (InviVoGen, San Diego, Calif.). THP1-Dual cells are human monocytes that induce a reporter protein upon activation of the NFKB or IRF pathway. THP-1 cells can be cultured with cells contacted with a selected candidate immunostimulant, or exposed to post-cellular signaling factors produced by cells contacted with a selected candidate immunostimulant, and then mixed with 200 μ l quantilblue (invitrogen, san diego, ca) or 50 μ l QuantiLuc for detection of NFKB and IRF activity. NFKB and IRF activities can be quantified by measuring absorbance or luminescence on a Molecular Device (Molecular Device) reader.

In one embodiment, the immune cell is a macrophage. For example, it can be commercially available in Raw-Dual ^TMAnd J774-Dual^TMNF-. kappa.B and IRF activity were measured in macrophages (Invitrogen, san Diego, Calif.). Raw-Dual^TMAnd J774-Dual^TMThe cells are mouse macrophage cell lines that induce the reporter protein upon activation of the NFKB or IRF pathway. Macrophages can be cultured with cells contacted with a selected candidate immunostimulant, or exposed to post-cellular signaling factors produced by cells contacted with a selected candidate immunostimulant, and then mixed with 200 μ l quantiBlue (Invitrogen, san Diego, Calif.) or 50 μ l quantiLuc for detection of NFKB and IRF activity. NFKB and IRF activity can be quantified by measuring absorbance or luminescence on a molecular device plate reader.

In one embodiment, the immune cell is a dendritic cell. For example, costimulatory markers (e.g., CD80, CD86) or markers of enhanced antigen presentation (e.g., MHCII) can be measured in dendritic cells by flow cytometry. Dendritic cells can be cultured with cells contacted with a selected candidate immunostimulant, or exposed to a compound produced by cells contacted with a selected candidate immunostimulant, and then stained with an antibody specific for a cell surface marker indicative of activation status. The expression levels of these markers were then determined by flow cytometry.

Candidate immunostimulants can also be assessed by measuring the level of pro-immunocytokines in macrophages and/or dendritic cells. For example, in some embodiments, evaluating a candidate immunostimulant comprises culturing macrophages and/or dendritic cells with cells contacted with the selected candidate immunostimulant, or contacting macrophages and/or dendritic cells with a post-cellular signaling factor produced by cells contacted with the selected candidate immunostimulant, and measuring the level of an immunopotentiating factor (e.g., IFN- α, IL-1, IL-12, IL-18, IL-2, IL-15, IL-4, IL-6, TNF- α, IL-17, and GMCSF). The level of the immunocytokine may be determined by methods known in the art, such as ELISA.

Method for identifying post-cellular immunostimulants produced by iron-dependent cell disassembly

Applicants have shown that treatment of cells with agents that induce iron-dependent cell disassembly results in the production and release of post-cellular signaling factors that increase immune activity. Thus, agents that induce iron-dependent cell disassembly and increase immune activity may be useful in treating disorders that can benefit from increased immune activity, such as cancer and infections. In an alternative approach, post-cellular signaling factors produced by the disassembled cells can be isolated and screened for immune activity. In this way, post-cellular signaling factors or "effectors" that increase immune activity can be identified for use in treating disorders.

For example, in certain aspects, the disclosure relates to methods of identifying an immunostimulatory agent, the method comprising:

(a) contacting a cell with an agent that induces iron-dependent cell disassembly in an amount sufficient to induce iron-dependent cell disassembly in the cell;

(b) isolating one or more post-cellular signaling factors produced by the cells following contact with the agent that induces iron-dependent cell disassembly; and is

(c) Determining the ability of one or more post-cellular signaling factors to modulate, e.g., increase or induce, an immune response.

One or more post-cellular signaling factors produced by the cells can be isolated, for example, by separating the cells from the medium in which they are grown (e.g., by centrifugation) and subjecting the conditioned medium to further analysis. For example, in some embodiments, the conditioned media is extracted with an organic solvent and then subjected to HPLC fractionation. In other embodiments, the conditioned media is subjected to size exclusion chromatography and the different fractions are collected. For example, conditioned media may be applied to a size exclusion column and fractionated on FPLC.

The ability of a post-cellular signaling factor to modulate an immune response can be determined by contacting the post-cellular signaling factor with an immune cell and assessing immune activity. Any of the methods described herein for measuring an immune response, e.g., measuring the level or activity of NFkB, IRF and/or STING, the level or activity of macrophages, the level or activity of monocytes, the level or activity of dendritic cells, the level or activity of CD4+, CD8+ or CD3+ cells, the level or activity of T cells, and the level or activity of pro-immune cytokines can be used to measure the ability of post-cellular signaling factors to modulate an immune response. For example, in some embodiments, fractions containing post-cellular signaling factors are collected for application to THP-1Dual cells and NFKB and/or IRF1 reporter activity is assessed. The positive hit fraction was confirmed by its ability to induce NFKB or IRF activity in THP 1Dual cells. The positive hits can be further characterized by mass spectrometry (macromolecules) or NMR (small molecules) to identify specific compounds with immunological activity. The immunological activity of each compound or species can be tested by: such compounds or species are added in synthetic or recombinant form to THP 1Dual cells, and then NFKB or IRF activity is measured as described above.

The immunological activity of the post-cellular signaling factor may be determined by applying the post-cellular signaling factor to macrophages, monocytes, dendritic cells, CD4+, CD8+ or CD3+ cells and/or T cells and measuring the level or activity of the cells. For example, in one embodiment, the assay comprises treating the immune cells with one or more post-cellular signaling factors and measuring the level or activity of nfkb activity in the immune cells. In one embodiment, the assay comprises treating T cells with one or more post-cellular signaling factors and measuring the activation or proliferation of the T cells. In one embodiment, the assay comprises contacting an immune cell with one or more post-cellular signaling factors and measuring the level or activity of nfk B, IRF or STING in the immune cell. In one embodiment, the immune cell is a THP-1 cell.

The immunostimulatory activity of the post-cellular signaling factor can also be assessed in animal models (e.g., animal cancer models). For example, in some embodiments, the immune response is measured in the animal after administration of the post-cellular signaling factor to the animal and after administration of the post-cellular signaling factor, e.g., by measuring changes in the level or activity of NFkB, IRF and/or STING, the level or activity of macrophages, the level or activity of monocytes, the level or activity of dendritic cells, the level or activity of CD4+, CD8+ or CD3+ cells, the level or activity of T cells, and the level or activity of pro-immune cytokines.

In one embodiment, the method further comprises selecting a post-cellular signaling factor that stimulates the immune response.

In one embodiment, the method further comprises detecting a marker of iron-dependent cell disassembly (e.g., iron death) in the cell.

Post-cell signaling factors that can be produced at higher levels in iron-dependent cell disassembly (e.g., iron death) relative to cells that have not undergone cell disassembly can be identified by comparing the levels of post-cell signaling factors in treated and untreated cells. For example, in one embodiment, the method further comprises:

i) measuring the level of one or more post-cellular signaling factors produced by the cells following contact with the agent that induces iron-dependent cell disassembly;

ii) comparing the level of one or more post-cellular signaling factors produced by the cells after contact with the agent that induces iron-dependent cell disassembly with the level of one or more post-cellular signaling factors in control cells that are not treated with the agent that induces iron-dependent cell disassembly; and is

Post-cell signaling factors that are specific for iron-dependent cell disassembly (e.g., iron death) or are produced at higher levels in iron-dependent cell disassembly (e.g., iron death) relative to other cell death processes can also be identified by comparing the levels of post-cell signaling factors in cells undergoing different cell death processes.

For example, in one embodiment of the above method, the control cells are treated with an agent that induces disassembly of cells that are not iron-dependent cell disassembly, such as a cell death process that is not iron-dependent, such as apoptosis, necroptosis, or apoptosis.

Examples of the invention

Example 1: activation/stimulation of human monocytes by Epstein-treated HT1080 fibrosarcoma cells

Agent/treatment design:

HT1080 fibrosarcoma cells were treated with control (DMSO) or different doses of elastine, Piperazine Elastine (PE) or imidazolone elastine (IKE) for 24 hours and then co-cultured with THP1-Dual cells for another 24 hours. Elastin was purchased from selekchem (seleckchem) (Houston, texas) and dissolved in DMSO. Subsequently, THP1 supernatant was evaluated for nuclear factor μ light chain enhancer (NFKB) or Interferon Regulatory Factor (IRF) reporter activity of activated B cells.

Materials/methods:

HT1080 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). THP1-Dual cells are human monocytes that induce a reporter protein upon activation of the NFKB or IRF pathway. During the assay, both cell types were cultured in 96-well plates. HT1080 cells were cultured in DMEM containing 10% FBS, and THP1-Dual cells were cultured in RPMI containing 10% FBS. 7,500 HT1080 cells were plated 24 hours prior to elastine, PE or IKE administration, with a final DMSO concentration of 0.5%. 24 hours after treatment, THP1-Dual cells (25,000 cells/well) were added to HT1080 cells. After 24 hours, 30 μ l of the supernatant was mixed with 200 μ l quantilblue (invitrogen, san diego, ca) (for NFKB reporter activity) or 50 μ l QuantiLuc (for IRF reporter activity) and absorbance or luminescence recorded on a molecular device reader.

And (4) conclusion:

as shown in fig. 1A, the ilastin treatment negatively affected the viability of HT1080 cells. Figure 1B shows that HT1080 cells treated with elastin (but not vehicle control DMSO) triggered NFKB signaling in THP1 monocytes. The elastin analogues PE and IKE also negatively affected the viability of HT1080 cells (fig. 1C) and triggered NFKB signaling in THP1 monocytes (fig. 1D).

Example 2: activation/stimulation of human monocytes by elastin-treated PANC1 pancreatic cancer cells.

Agent/treatment design:

PANC1 pancreatic cancer cells were treated with control (DMSO) or different doses of elastin for 24 hours and then co-cultured with THP1-Dual cells for another 24 hours. Elastin was purchased from selektimm (houston, texas) and dissolved in DMSO. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity.

Materials/methods:

PANCl cells were obtained from ATCC, and THP1-Dual cells were obtained from invitrogen (san diego, ca). During the assay, both cell types were cultured in 96-well plates. 24 hours prior to the administration of the elastin, 7,500 PANC-1 cells were plated with a final DMSO concentration of 0.5%. 24 hours after treatment, THP1-Dual cells (25,000 cells/well) were added to PANC-1 cells. After 24 hours, 30 μ l of the supernatant was mixed with 200 μ l of quantilblue (invitrogen, san diego, ca) or 50 μ l of QuantiLuc and the absorbance or luminescence was recorded on a molecular device plate reader.

And (4) conclusion:

as shown in fig. 2A, elastin treatment negatively affected the viability of PANC1 cells. Figure 1B shows that PANC1 cells treated with elastin (but not vehicle control DMSO) triggered NFKB signaling in THP1 monocytes.

Example 3: activation/stimulation of human monocytes by Epstein-treated Caki-1 renal cell carcinoma cells

Agent/treatment design:

caki-1 renal cell carcinoma cells were treated with control (DMSO) or different doses of elastin for 24 hours and then co-cultured with THP1-Dual cells for another 24 hours. Elastin was purchased from selektimm (houston, texas) and dissolved in DMSO. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity.

Materials/methods:

caki-1 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. 24 hours prior to the administration of the elastin, 7,500 Caki-1 cells were plated out with a final DMSO concentration of 0.5%. 24 hours after treatment, THP1-Dual cells (25,000 cells/well) were added to Caki-1 cells. After 24 hours, 30. mu.l of the supernatant was mixed with 200. mu.l of QuantiBlue (Invitrogen) or 50. mu.l of QuantiLuc, and the absorbance or luminescence was recorded on a molecular device plate reader.

And (4) conclusion:

as shown in FIG. 3A, the activity of Caki-1 cells was negatively affected by the treatment with elastin. Fig. 3B shows that Caki-1 cells treated with elastine (but not vehicle control DMSO) triggered NFKB signaling in THP1 monocytes.

Example 4: RSL 3-treated Caki-1 renal cell carcinoma cells activation/stimulation agents/treatment design for human monocytes:

caki-1 renal cell carcinoma cells were treated with control (DMSO) or different doses of RSL3 for 24 hours and then co-cultured with THP1-Dual cells for another 24 hours. RSL3 was purchased from selekchem and dissolved in DMSO. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity.

Materials/methods:

caki-1 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. 24 hours prior to administration of RSL3, 7,500 Caki-1 cells were plated out with a final DMSO concentration of 0.5%. 24 hours after treatment, THP1-Dual cells (25,000 cells/well) were added to Caki-1 cells. After 24 hours, 30 μ l of the supernatant was mixed with 200 μ l of quantilblue (invitrogen, san diego, ca) or 50 μ l of QuantiLuc and the absorbance or luminescence was recorded on a molecular device plate reader. And (4) conclusion:

as shown in FIG. 4A, RSL3 treatment negatively affected the viability of Caki-1 cells. Fig. 4B shows that Caki-1 cells treated with RSL3 (but not vehicle control DMSO) triggered NFKB signaling in THP1 monocytes.

Example 5: activation/stimulation of human monocytes by RSL 3-treated Jurkat T cell leukemia cells

Agent/treatment design:

jurkat T cell leukemia cells were treated with control (DMSO) or different doses of RSL3 for 24 hours and then co-cultured with THP1-Dual cells for another 24 hours. RSL3 was purchased from Seller chem corporation (Houston, Tex.) and dissolved in DMSO. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity.

Materials/methods:

jurkat cells were obtained from ATCC and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. 24 hours prior to RSL3 administration, 100,000 Jurkat cells were plated with a final DMSO concentration of 0.5%. 24 hours after treatment, THP1-Dual cells (25,000 cells/well) were added to Jurkat cells. After 24 hours, 30 μ l of the supernatant was mixed with 200 μ l of quantilblue (invitrogen, san diego, ca) or 50 μ l of QuantiLuc and the absorbance or luminescence was recorded on a molecular device plate reader.

And (4) conclusion:

as shown in fig. 5A, RSL3 treatment negatively affected the viability of Jurkat T cell leukemia cells. Figure 5B shows that Jurkat cells treated with RSL3 (but not vehicle control DMSO) triggered NFKB signaling in THP1 monocytes.

Example 6: activation/stimulation of human monocytes by RSL 3-treated A20B cell leukemia cells

Agent/treatment design:

A20B cell leukemia cells were treated with control (DMSO) or different doses of RSL3 for 24 hours and then co-cultured with THP1-Dual cells for another 24 hours. RSL3 was purchased from Seller chem corporation (Houston, Tex.) and dissolved in DMSO. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity.

Materials/methods:

a20 cells were obtained from ATCC and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. 50,000A 20 cells were plated 24 hours prior to administration of RSL3, with a final DMSO concentration of 0.5%. 24 hours after treatment, THP1-Dual cells (25,000 cells/well) were added to A20 cells. After 24 hours, 30 μ l of the supernatant was mixed with 200 μ l of quantilblue (invitrogen, san diego, ca) or 50 μ l of QuantiLuc and the absorbance or luminescence was recorded on a molecular device plate reader.

And (4) conclusion:

as shown in fig. 6A, RSL3 treatment negatively affected the viability of a 20B cell leukemia cells. A20 cells treated with RSL3 (but not vehicle control DMSO) triggered NFKB (fig. 6B) and IRF (fig. 6C) signaling in THP1 monocytes.

Example 7: specificity of pro-inflammatory signaling elicited by HT1080 fibrosarcoma cells treated with elastin.

Agent/treatment design:

HT1080 fibrosarcoma cells were treated with control (DMSO) or different doses of elastine (e.g., 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, and 25 μ M) in the presence or absence of 1 μ M iron-based statin for 24 hours, and then co-cultured with THP1-Dual cells for an additional 24 hours. Iron-based statins were purchased from selektimm (houston, texas) and dissolved in DMSO. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity. HT1080 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. The specificity of induction of NFKB signaling elicited by elastin-treated HT1080 cells was assessed by its reversal (by concurrent iron-based statin treatment of HT1080 cells).

Example 8: specificity of Caki-1 cell-initiated proinflammatory signaling by treatment with Alascetin

Agent/treatment design:

caki-1 renal carcinoma cells were treated with control (DMSO) or different doses of elastine (e.g., 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, and 25 μ M) in the presence or absence of 1 μ M iron-based statin (Seiller chem, Houston, Tex.) for 24 hours, and then co-cultured with THP1-Dual cells for an additional 24 hours. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity. Caki-1 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. Specificity of induction of NFKB signaling by elastin-treated Caki-1 cells was assessed by their reversal (by concurrent iron-based statin treatment of Caki-1 cells).

Example 9: specificity of proinflammatory Signaling triggered by Caki-1 renal carcinoma cells treated with RSL3

Agent/treatment design:

caki-1 renal carcinoma cells were treated with control (DMSO) or different doses of RSL3 (e.g., 0.002, 0.005, 0.014, 0.041, 0.123, 0.370, 1.111, 3.333, and 10 μ M) in the presence or absence of 1 μ M iron-based statin for 24 hours, and then co-cultured with THP1-Dual cells for an additional 24 hours. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity. Caki-1 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. The specificity of induction of NFKB signaling triggered by Caki-1 cells treated with RSL3 was assessed by its reversal (by concurrent iron-based statin treatment of Caki-1 cells).

Example 10: specificity of pro-inflammatory signaling elicited by A20 cells treated with RSL3

Agent/treatment design:

a20 lymphoma cells were treated with control (DMSO) or different doses of RSL3 (e.g., 0.002, 0.005, 0.014, 0.041, 0.123, 0.370, 1.111, 3.333, and 10 μ M) for 24 hours in the presence or absence of 1 μ M iron-based statin, and then co-cultured with THP1-Dual cells for an additional 24 hours. Subsequently, THP1 supernatants were evaluated for NFKB or IRF reporter activity. A20 cells were obtained from ATCC and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates. The specificity of induction of NFKB signaling triggered by RSL 3-treated a20 cells was assessed by their reversal (by concurrent iron-based statin treatment of a20 cells).

Example 11: treatment of A20 lymphoma tumor xenografts by RSL3 induces anti-tumor/pro-inflammatory responses in vivo

Agent/treatment design:

BALB/C mice were injected subcutaneously with 5x10⁶And a20 lymphoma cells. When the median size of the tumor reaches 150-³In this case, intratumoral administration was performed by injecting mice with vehicle or RSL 3. After 48 hours, immunophenotyping of tumor infiltrating cells, lymphocytes and splenocytes was performed to characterize the recruitment and activation status of myeloid and lymphoid cells. Immunophenotyping was performed by immunohistochemical/immunofluorescent staining of tumor sections or by first dissociating tumors into single cell suspensions and then flow cytometry of cells (J Vis Exp. [ journal of visualization experiments ]],2015, (98): 52657; j Natl Cancer Inst. [ J national Cancer institute J. ]]2015, 2 months and 3 days; 107 (3); cancer Discov]7 months 2012; 2(7): 608-23.). The pro-inflammatory response induced by RSL3 treatment was assessed by the increase in monocyte, macrophage and T cell recruitment into the tumor microenvironment, as compared to vehicle. In addition, anti-tumor immune responses were assessed by determining an increase in activation markers in macrophages (MHCII and CD80), CD11+ CD103+ dendritic cells (MHCII), and CD4 and CD 8T cells (Ki67 and CD69), without simultaneously activating CD4+ FoxP3+ T regulatory cells. In addition, tumor size was measured over a three week period or until the tumor reached a maximum size of 2000mm ³To evaluate the inhibition of tumor growth by RSL 3.

Example 12: treatment of A20 lymphoma tumor xenografts by local RSL3 induces systemic anti-tumor/pro-inflammatory responses in vivo

Agent/treatment design:

BALB/C mice were injected subcutaneously with 5x10 at two different sites in vivo⁶And a20 lymphoma cells. When the median size of the tumor reaches 150-³At one tumor site, the mouse was intratumorally administered with vehicle or RSL 3. By measuring the size of the treated and untreated (contralateral) tumors over a three week period or until the maximum tumor size reaches 2000mm³To evaluate the inhibition of tumor growth by RSL 3. In addition, the involvement of a systemic adaptive immune response was assessed by analyzing Tumor Infiltrating Lymphocytes (TILs) in contralateral tumor sites. The adaptive immune response associated with treatment in contralateral tumors can be assessed by quantitative increase in the number of T effector cells (FoxP3-CD4+ T cells, CD8+ T cells) or by increase in the activation state of T cells (CD69, ki67), macrophages (MHCII and CD80) or CD11+ CD103+ dendritic cells (MHCII).

Example 13: use of RSL3 in human clinical trials for the treatment of renal cell carcinoma

Agent/treatment design:

RSL3 induces cell death of renal cell carcinoma cells in a manner that causes proinflammatory signaling. Using the methods of the present disclosure involving the use of RSL3, it is expected that this example will provide evidence for the therapeutic effects of RSL 3.

A Randomized Control Trial (RCT) was performed to assess the safety and efficacy of RSL3 in treating renal cancer patients who failed 1 or 2 anti-angiogenic regimens compared to multiple infusions of RSL3 alone or in combination with nivolumab.

One hundred patients with advanced RCC who failed anti-angiogenic therapy were randomized to receive RSL3 alone (10 mg per day), nivolumab alone (3 mg/kg every 2 weeks), or RSL3 in combination with nivolumab. Patients are required to have a carnososky Performance Score (KPS) of at least 70%, no evidence of brain metastases, no prior treatment with nivolumab, and no active autoimmune disease or medical condition requiring systemic immunosuppression. KPS ranks from 100 to 0, where 100 indicates no signs of disease and 0 indicates death, for evaluation of chemotherapy survival of patients.

Tumor assessment began 8 weeks after initiation of therapy, thereafter the first year continued once every 8 weeks, and once every 12 weeks until progression or discontinuation of treatment. RSL3 efficacy was assessed by assessing overall survival.

Example 14: treatment of b16.bl6 melanoma tumor xenografts with piperazine elastine in combination with anti-CTLA 4 antibody (9D9) induced anti-tumor immune responses in vivo.

Agent/treatment design:

C57/BL6 mice subcutaneous injection 1x10⁵B16.bl6 melanoma cells. Intratumoral administration of vehicle, piperazine elastine (40mg/kg, i.p.), anti-CTLA 4 antibody 9D9(10mg/kg, i.p.), or a combination of piperazine elastine and 9D9 in mice. When the median size of the tumor reaches 150-³When administered to mice. After 48 hours, immunophenotyping of tumor infiltrating cells, lymphocytes and splenocytes was performed to characterize the recruitment and activation status of myeloid and lymphoid cells. The induction of the maximal pro-inflammatory response by the combination treatment of elastine piperazine and 9D9 compared to vehicle was assessed by: recruitment of CD3+ T cells into the tumor microenvironment was increased compared to either treatment alone. In addition, by measuring tumor size over a three week period or until the tumor reaches a maximum size of 2000mm³To assess the maximal inhibition of tumor growth by the combination therapy compared to either treatment alone.

Example 15: method for screening compounds inducing proinflammatory iron death

Agent/treatment design:

caki-1 renal cancer cells in 384-well format were exposed to test compounds from the chemical screening library for 24-48 hours in the presence or absence of iron death inhibitors (iron-based statins, B-mercaptoethanol, or iron chelators). Subsequently, THP1 dual cells were co-cultured with the treated Caki-1 cells. 24 hours after conditioning THP1-Dual cells, supernatants were evaluated for NFKB or IRF reporter activity. A compound that induces NFKB or IRF reporter activity in the absence of an iron death inhibitor, but does not induce NFKB or IRF reporter activity in the presence of an iron death inhibitor, is selected as the pro-inflammatory compound.

Example 16: method for testing the inflammatory properties of substances derived from cells treated with an iron death inducer

Agent/treatment design:

caki-1 renal carcinoma cells were exposed to elastine or RSL3 (or other iron death inducers) and conditioned medium was collected after 24-48 hours. Subsequently, the conditioned medium was extracted with an organic solvent and then subjected to HPLC fractionation. Specifically, the conditioned medium was extracted with ethyl acetate, concentrated, and fractionated by polarity. Alternatively, the conditioned medium is subjected to size exclusion chromatography and fractions are collected. Specifically, conditioned media was applied to a size exclusion column and fractionated on FPLC. The collected fractions were applied to THP1-Dual cells for 24 hours, followed by evaluation of reporter activity. The positive hit fraction was confirmed by its ability to induce NFKB or IRF activity in THP1Dual cells. The positive hits were further characterized by mass spectrometry (macromolecules) or NMR (small molecules) to identify specific compounds with inflammatory activity. The inflammatory properties of each compound or class were tested by: such compounds or species are added to THP1Dual cells in synthetic or recombinant form and then NFKB or IRF activity is measured as described above.

Example 17: specificity of pro-inflammatory signaling elicited by HT1080 fibrosarcoma cells treated with elastin.

Agent/treatment design:

HT1080 fibrosarcoma cells were treated with different doses of elastine (e.g., 0.8, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, or 20 μ M) alone or in combination with iron death inhibitors (1 μ M iron-based statin-1, 1 μ M lipstatin-1, 100 μ M Trolox, 25 μ M β -mercaptoethanol, or 100 μ M deferoxamine) for 24 hours, followed by co-culture with THP1-Dual cells for an additional 24 hours. Iron-based statin-1 and lipstatin-1 were purchased from selektimm, houston, texas, and dissolved in DMSO. Trolox was purchased from Kalman Chemical Company (Cayman Chemical Company Inc) and resuspended in DMSO. Deferoxamine mesylate was purchased from Sigma Aldrich (Sigma-Aldrich) and resuspended in water. Beta-mercaptoethanol was purchased from Life Technologies, USA. Subsequently, THP1 supernatant was evaluated for NFKB activity. HT1080 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates.

As a result:

As shown in fig. 7A and 8A, elafin treatment of HT1080 fibrosarcoma cells reduced the viability of the cells in a dose-dependent manner, and this reduced viability was attenuated by each of the iron death inhibitors. As shown in fig. 7B and 8B, elafin treatment of HT1080 fibrosarcoma cells increased NFKB activity in THP1 cells in a dose-dependent manner, and this increased NFKB activity was abolished by each of the iron death inhibitors. These results demonstrate that cell death plays a role in the induction of NFKB signaling triggered by elastin-treated HT1080 cells.

Example 18: knockdown of ACSL4 and CARS genes inhibits elapsin-mediated cell death in HT1080 fibrosarcoma cells

Agent/treatment design:

HT1080 cells (5,000 cells/well) were reverse transfected in a 96-well format using the following: DharmaFECT I transfection reagent (catalog No. T-2001) and control siRNA (catalog No. D-001810-10-05) or siRNA targeting ACSL4 [37.5nM ] (FIG. 9A, catalog No. L-009364-00-005) or pool of siRNAs to ACSL4 (Seamer Feishol science, catalog No.: S5001, S5001, S5002) or to CARS (Seamer Feishol science, catalog No.: S2404, S2405, S2406) (FIG. 9B/C). 48 hours after transfection, the cell culture medium was changed to fresh medium containing various concentrations of elapsin (FIG. 9A) or fixed concentrations of elapsin (10. mu.M, FIG. 9B/C). In addition, 50,000 reported THP1-dual cells were added to some plates (fig. 9C). After 24 hours, HT1080 cell viability was measured (fig. 9A/B) or THP1 supernatant was assessed for NFKB activity (fig. 9C). HT1080 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.).

As a result:

the ACSL4 gene encodes a long-chain fatty acid-CoA ligase 4, which is an acyl-CoA synthetase, controls levels of arachidonic acid in cells, and is involved in the regulation of cell death. The CARS gene encodes cysteinyl-tRNA synthetase. CARS knockdown has been shown to inhibit elastin-induced iron death by preventing the induction of lipid reactive oxygen. See Hayano et al, 2016, Cell Death Differ [ Cell Death and differentiation ]23 (2): 270-278. As shown in fig. 9A and 9B, the gene knock-out portion of ACLS4 or CARS in HT1080 cells rescued the viability of HT1080 cells cultured in the presence of elastin. Furthermore, gene knock-down of ACLS4 or CARS in HT1080 cells abolished NFKB activity in monocytes co-cultured with elastin-treated HT1080 cells (fig. 9C). These results demonstrate that cell death plays a role in the induction of NFKB signaling triggered by elastin-treated HT1080 cells and that the absence of specific intracellular proteins reduces the pro-inflammatory properties of iron death.

Example 19: specificity of pro-inflammatory signaling triggered by A20 cells treated with GPX4 inhibitor (RSL3, ML162, or ML210)

Agent/treatment design:

a20 lymphoma cells were treated with different doses (e.g., 0.002, 0.005, 0.014, 0.041, 0.123, 0.370, 1.111, 3.333, and 10 μ M) of GPX4 inhibitor (RSL3, ML162, or ML210) in the presence or absence of 1 μ M iron-based statin-1 for 24 hours. ML162 was purchased from kahmann chemical company and resuspended in DMSO. ML210 was purchased from sigma aldrich and resuspended in DMSO. A20 lymphoma cells were also treated with DMSO as a negative control. After 24 hours of treatment with DMSO or GPX4 inhibitor, a20 lymphoma cells were co-cultured with THP1-Dual cells for an additional 24 hours. Subsequently, THP1 supernatants were evaluated for NFKB reporter activity. A20 cells were obtained from ATCC and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates.

As a result:

as shown in figures 10A, 11A, and 12A, treatment of a20 lymphoma cells with each of the GPX4 inhibitors (RSL3, ML162, or ML210) reduced the viability of the cells in a dose-dependent manner, and this reduced viability was attenuated by the iron death inhibitor, iron-based statin-1. As shown in figures 10B, 11B, and 12B, treatment of a20 lymphoma cells with a GPX4 inhibitor increased NFKB activity in THP1 cells in a dose-dependent manner, and this increased NFKB activity was attenuated by the iron death inhibitor, iron-based statin-1. These results indicate that cell death plays a role in the induction of NFKB signaling initiated by GPX-4 inhibitor treated a20 lymphoma cells.

Example 20: specificity of proinflammatory Signaling triggered by Caki-1 renal carcinoma cells treated with GPX4 inhibitor (RSL3 or ML162)

Agent/treatment design:

caki-1 renal cancer cells were treated with control (DMSO) or different doses (e.g., 0.002, 0.005, 0.014, 0.041, 0.123, 0.370, 1.111, 3.333, and 10 μ M) of GPX4 inhibitor (RSL3 or ML162) in the presence or absence of 1 μ M iron-based statin for 24 hours and then co-cultured with THP1-Dual cells for another 24 hours. Subsequently, THP1 supernatants were evaluated for NFKB reporter activity. Caki-1 cells were obtained from ATCC, and THP1-Dual cells were obtained from Invitrogen (san Diego, Calif.). During the assay, both cell types were cultured in 96-well plates.

As a result:

as shown in fig. 13A and 14A, treatment of Caki-1 renal cancer cells with GPX4 inhibitor (RSL3 or ML162) reduced the viability of the cells in a dose-dependent manner, and this reduced viability was attenuated by the iron death inhibitor iron-based statin-1. As shown in fig. 13B and 14B, treatment of Caki-1 renal cancer cells with RSL3 or ML162 increased NFKB activity in THP1 cells in a dose-dependent manner, and this increased NFKB activity was attenuated by the iron death inhibitor iron-based statin-1. These results indicate that cell death plays a role in the induction of NFKB signaling triggered by GPX-4 inhibitor-treated Caki-1 renal cancer cells.

Equivalents of

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Is incorporated by reference

Each reference, patent, and patent application cited in this application is hereby incorporated by reference in its entirety as if each reference were individually indicated to be incorporated.

116页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：取代的四氢环戊二烯并[c]吡咯、取代的二氢吡咯嗪、其类似物和其使用方法

Increasing immune activity through modulation of post-cellular signaling factors

相关技术

网友询问留言